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VASCULAR ENDOTHELIAL GROWTH FACTOR ISOFORM A
LATEST LITERATURE
1: The Analyst, 2010 Sep 6, 102(4)
Capillary electrophoretic development of aptamers for a glycosylated VEGF peptide fragment.
[Abstract]The emergence of functional genomics and proteomics has added to the growing need for improved analysis methods that can detect and distinguish between protein variants resulting from allelic variation, mutation, or post-translational modification. Aptamers, single-stranded DNA or RNA molecules that fold into three-dimensional structures conducive to binding targets, have become an attractive alternative to antibodies for this type of analysis. Although aptamers have been developed for a wide range of target species, very few sequences have been identified that bind selectively to proteins with specific post-translational modifications. Using capillary electrophoresis-based selection, we have developed DNA aptamer sequences that selectively bind an N-glycosylated peptide fragment of vascular endothelial growth factor (VEGF). The selection method incorporates alternating positive- and counter-selection steps in free solution in order to obtain aptamers with both high affinity toward the glycosylated target and high selectivity versus a non-glycosylated variant. Affinity capillary electrophoresis and surface plasmon resonance binding assays indicate these sequences have low-microM dissociation constants and preferentially bind the glycosylated peptide with as much as 50-fold specificity. Such aptamers could serve as tools for rapid and simple monitoring of disease-linked functional changes in proteins, with potential applications in drug screening and disease diagnosis.
2: The British journal of ophthalmology, 2010 Sep 6, 102(4)
Serum vascular endothelial growth factor (VEGF) levels correlate with number and location of micrometastases in a murine model of uveal melanoma.
[Abstract]Background A preliminary animal study was performed to determine if hepatic micrometastases from uveal melanoma secrete vascular endothelial growth factor (VEGF) that is measurable in serum. Methods We analysed the serum of a C57Bl/6 mouse model of uveal melanoma (n=10) at days 4, 7, 14 and 21 post-inoculation for VEGF levels. We compared the serum VEGF levels with the number and location of hepatic micrometastases and their respective expression of VEGF mRNA. Results Serum VEGF levels rose after inoculation of C57Bl/6 mice eyes with B16LS9 cutaneous melanoma cells. Beginning on day 14 there was a statistically significant (p<0.05) increase in VEGF levels, rising to an average peak level of 37.985 pg/ml at day 21. Peak serum VEGF levels correlated with the total number of hepatic micrometastases (R=0.444) and there was moderate correlation of peak VEGF serum levels with micrometastases in more hypoxic locations (R=0.572). VEGF mRNA expression by micrometastases was highest in the most hypoxic regions of the hepatic lobule. Conclusions Hepatic micrometastastic melanoma arising in a mouse model of ocular melanoma secretes VEGF. The number and location of the micrometastases correlate with serum VEGF levels.
3: Cancer biology & therapy, 2010 Nov 3, 10(9)
A fusion protein with the receptor binding domain of vascular endothelial growth factor-A (VEGF-A) is an antagonist of angiogenesis in cancer treatment: Simultaneous blocking of VEGF receptor-1 and 2.
[Abstract]Vascular endothelial growth factor (VEGF) is a mainly angiogenic factor through VEGFR-1 and VEGFR-2, which are expressed preferentially in proliferating endothelial cells. Thus, simultaneous blockage of both VEGF receptors may provide a more efficient therapeutic effect in cancer treatment. A recombinant fusion protein (RBDV-IgG1 Fc), which was composed of a receptor binding domain of human VEGF-A (residues 8-109) and a Fc region of human IgG1 immunoglobulin, could bind to both mouse VEGFR-1 and VEGFR-2 to decrease VEGF-induced proliferation and tube formation of endothelial cells in vitro. In parallel unpublished data also indicate similar binding anti-tumor activity towards the human receptors to block angiogenesis in vitro. In this study, RBDV-IgG1 Fc fusion protein displayed good tumor inhibition activity in a mouse model in vivo. RBDV-IgG1 Fc fusion protein reduced the effects of proliferation, migration and tube formation induced by VEGF in murine endothelial cells in vitro. In vivo tumor therapy with RBDV-IgG1 Fc resulted in tumor inhibition by reducing angiogenesis. Pathological evidence also showed it could seriously damage vessels, causing the death of tumor cells. These findings suggest this chimeric protein has potential as an angiogenestic antagonist in tumor therapy.
4: Bone, 2010 Sep 2, 399(4)
Hypoxia increases Annexin A2 expression in osteoblastic cells via VEGF and ERK.
[Abstract]Vascular endothelial growth factor (VEGF) stimulated angiogenesis is critical for endochondral ossification that occurs during bone development, and bone repair. Under these circumstances, VEGF production appears to be driven by low oxygen tension, under the control of the hypoxia inducible factor-family of transcription factors (HIF-). Annexin 2 (AnxA2) a calcium dependant phospholipid binding protein has been implicated in VEGF-mediated retinal neovascularization and is upregulated by VEGF in choroid retinal endothelial cells. AnxA2 is also expressed in cells of the osteoblast lineage and chondrocytes and may play a role in matrix mineralization. In this paper we examined the effects of hypoxia (1% O(2)) and VEGF on the expression of AnxA2 in osteoblastic MC3T3-E1 cells. Hypoxia, desferrioxamine (hypoxia mimetic) and recombinant VEGF all increased AnxA2 mRNA and protein levels in osteoblastic cells. The hypoxia-induced increase in AnxA2 was inhibited by a blocking antibody to VEGF-R1, however, VEGF(120), a VEGF-R1 agonist demonstrated no influence upon Anxa2 expression. This suggests that VEGF induction of Annexin A2 is not mediated via VEGF-R1 agonism alone, but by VEGF-R1 and Neuropilin-1 or Neuropilin-2 heterodimers. Additionally we demonstrated that VEGF-stimulated changes in AnxA2 expression via a pathway involving Src and MEK kinase. These data demonstrate that AnxA2 expression in osteoblasts is under the control of VEGF, which may have implications for both angiogenesis and bone mineralization under low oxygen conditions.
5: Developmental biology, 2010 Sep 2, 399(4)
VEGF Signaling Has Distinct Spatiotemporal Roles During Heart Valve Development.
[Abstract]Heart valve malformations are one of the most common types of birth defects, illustrating the complex nature of valve development. Vascular endothelial growth factor (VEGF) signaling is one pathway implicated in valve formation, however its specific spatial and temporal roles remain poorly defined. To decipher these contributions, we use two inducible dominant negative approaches in mice to disrupt VEGF signaling at different stages of embryogenesis. At an early step in valve development, VEGF signals are required for the full transformation of endocardial cells to mesenchymal cells (EMT) at the outflow tract (OFT) but not atrioventricular canal (AVC) endocardial cushions. This role likely involves signaling mediated by VEGF receptor 1 (VEGFR1), which is highly expressed in early cushion endocardium before becoming downregulated after EMT. In contrast, VEGFR2 does not exhibit robust cushion endocardium expression until after EMT is complete. At this point, VEGF signaling acts through VEGFR2 to direct the morphogenesis of the AVC cushions into mature, elongated valve leaflets. This latter role of VEGF requires the VEGF-modulating microRNA, miR-126. Thus, VEGF roles in the developing valves are dynamic, transitioning from a differentiation role directed by VEGFR1 in the OFT to a morphogenetic role through VEGFR2 primarily in the AVC-derived valves.
6: Journal of cell science, 2010 Sep 15, 123(Pt 18)
ERK5 is required for VEGF-mediated survival and tubular morphogenesis of primary human microvascular endothelial cells.
[Abstract]Extracellular signal-regulated kinase 5 (ERK5) is activated in response to environmental stress and growth factors. Gene ablation of Erk5 in mice is embryonically lethal as a result of disruption of cardiovascular development and vascular integrity. We investigated vascular endothelial growth factor (VEGF)-mediated ERK5 activation in primary human dermal microvascular endothelial cells (HDMECs) undergoing proliferation on a gelatin matrix, and tubular morphogenesis within a collagen gel matrix. VEGF induced sustained ERK5 activation on both matrices. However, manipulation of ERK5 activity by siRNA-mediated gene silencing disrupted tubular morphogenesis without impacting proliferation. Overexpression of constitutively active MEK5 and ERK5 stimulated tubular morphogenesis in the absence of VEGF. Analysis of intracellular signalling revealed that ERK5 regulated AKT phosphorylation. On a collagen gel, ERK5 regulated VEGF-mediated phosphorylation of the pro-apoptotic protein BAD and increased expression of the anti-apoptotic protein BCL2, resulting in decreased caspase-3 activity and apoptosis suppression. Our findings suggest that ERK5 is required for AKT phosphorylation and cell survival and is crucial for endothelial cell differentiation in response to VEGF.
7: British journal of pharmacology, 2010 Sep, 161(1)
Intersegmental vessel formation in zebrafish: requirement for VEGF but not BMP signalling revealed by selective and non-selective BMP antagonists.
[Abstract]BACKGROUND AND PURPOSE Bone morphogenetic proteins (BMPs) were first identified through their role in inducing bone and cartilage formation, but many other important functions have since been ascribed to BMPs, including dorsoventral patterning, angiogenesis and tissue homeostasis. Using dorsomorphin and LDN193189, selective small molecule inhibitors of BMP signalling, we investigated the role of BMP signalling in early vascular patterning in zebrafish. EXPERIMENTAL APPROACH The effects of dorsomorphin and LDN193189 on vascular endothelial growth factor-a (VEGF) and BMP signalling in developing zebrafish and in human pulmonary artery endothelial cells were determined using confocal microscopy, Western blotting and quantitative PCR. KEY RESULTS We showed that dorsomorphin, similar to the VEGF inhibitor SU5416, strongly inhibits intersegmental vessel formation in zebrafish and that this is due to inhibition of VEGF activation of VEGF receptor 2 (VEGFR2), leading to reduced VEGF-induced phospho-ERK (extracellular regulated kinase) 1/2 and VEGF target gene transcription. These effects occurred at concentrations of dorsomorphin that block BMP signalling. We also showed that LDN193189, an analogue of dorsomorphin, more potently blocks BMP signalling but has no effect on VEGF signalling in zebrafish and does not disrupt early vascular patterning. CONCLUSIONS AND IMPLICATIONS Dorsomorphin inhibits both BMP and VEGF signalling, whereas LDN193189 is a more selective BMP antagonist. Results obtained in cardiovascular studies using dorsomorphin need to be interpreted with caution, and use of LDN193189 would be preferable due to its selectivity. Our data also suggest that BMP signalling is dispensable for early patterning of intersegmental vessels in zebrafish.
8: Journal of biomedical materials research. Part A, 2010 Sep 15, 94(4)
Bladder tissue engineering: Tissue regeneration and neovascularization of HA-VEGF-incorporated bladder acellular constructs in mouse and porcine animal models.
[Abstract]Successful tissue engineering requires appropriate recellularization and vascularization. Herein, we assessed the regenerative and angiogenic effects of porcine bladder acellular matrix (ACM) incorporated with hyaluronic acid (HA) and vascular endothelial growth factor (VEGF) in mouse and porcine models. Prepared HA-ACMs were rehydrated in different concentrations of VEGF (1, 2, 3, 10, and 50 ng/g ACM). Grafts were implanted in mice peritoneum in situ for 1 week. Angiogenesis was quantified with CD31 and Factor VIII immunostaining using Simple PCI. Selected optimal VEGF concentration that induced maximum vascularization was then used in porcine bladder augmentation model. Implants were left in for 4 and 10 weeks. Three groups of six pigs each were implanted with ACM alone, HA-ACM, and HA-VEGF-ACM. Histological, immunohistochemical (Uroplakin III, alpha-SMA, Factor VIII), and immunofluorescence (CD31) analysis were performed to assess graft regenerative capacity and angiogenesis. In mouse model, statistically significant increase in microvascular density was demonstrated in the 2 ng/g ACM group. When this concentration was used in porcine model, recellularization increased significantly from weeks 4 to 10 in HA-VEGF-ACM, with progressive decrease in fibrosis. Significantly increased vascularization, coupled with increased urothelium and smooth muscle cell (SMC) regeneration, was observed in HA-VEGF grafts at week 10 in the center and periphery, compared with week 4. HA-VEGF grafts displayed highest in vivo epithelialization, neovascularization, and SMCs regeneration. A total of 2 ng/g tissue VEGF when incorporated with HA proved effective in stimulating robust graft recellularization and vascularization, coordinated with increased urothelial bladder development and SMC augmentation into bundles by week 10. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2010.
9: The Journal of biological chemistry, 2010 Aug 3, 325(1-2)
Fluid flow-induced soluble vascular endothelial growth factor (VEGF) isoforms regulate actin adaptation in osteoblasts.
[Abstract]While load-induced mechanical signals play a key role in bone formation and maintenance of bone mass and structure, the cellular mechanisms involved in the translation of these signals are still not well understood. Recent identification of a novel flow-induced mechanosignaling pathway involving vascular endothelial growth factor (VEGF) in osteoblasts and the known VEGF regulation of actin reorganization in various cell types has led us to hypothesize that fluid shear stress induced Vegf upregulation underlies the actin cytoskeleton adaptation observed in osteoblasts during mechanotransduction. Our results show that MC3T3-E1 cells secret significant VEGF in response to 5h pulsatile fluid shear stress (PFSS - 5 dyne/cm2 at 1 Hz) while expression of VEGF receptors (VEGFR-1, VEGFR-2 or NRP1) is unaffected. These receptors, in particular VEGFR-2, participate in PFSS-induced VEGF release. Exposure to flow-conditioned media or exogenous VEGF significantly induces stress fiber formation in osteoblasts that is comparable to PFSS-induced stress fiber formation, whereas VEGF knock-down abrogates this response to PFSS, thereby providing evidence that flow-induced VEGF release plays a role in actin polymerization. Using neutralizing antibodies against the receptors and VEGF isoforms, we found that soluble VEGFs, in particular VEGF164, play a crucial role in transient stress fiber formation during osteoblast mechanotransduction, most likely through VEGFR-2 and NRP1. Based on these data we conclude that flow-induced VEGF release from osteoblasts regulates osteoblast actin adaptation during mechanotransduction and that VEGF paracrine signaling may provide a potent crosstalk among bone cells and endothelial cells that is essential for fracture healing, bone remodeling and osteogenesis.
10: Biochemical and biophysical research communications, 2010 Sep 3, 399(4)
New molecular mechanisms of the unexpectedly complex role of VEGF in ulcerative colitis.
[Abstract]The effects of VEGF on endothelial cells are mediated by different intracellular signaling cascades (e.g., Erk1/2, Akt, Src). VEGF plays a recently recognized role in ulcerative colitis (UC) pathogenesis, mostly by increasing vascular permeability and promoting the infiltration of inflammatory cells. We hypothesized that the excessive activation of signal transduction pathways, which is responsible for VEGF/VEGFR-2-mediated endothelial permeability (Src, Akt), is a new element in the pathogenesis of chronic UC. We demonstrated increased expression of pro-angiogenic growth factor VEGF and its receptor VEGFR-2 in colonic tissue during acute 6% iodoacetamide-induced UC in rats and chronic spontaneously developed UC in IL-10 knockout mice (IL-10 KO). Development of acute 6% iodoacetamide-induced UC in rats was accompanied by activation of Erk1/2 and Src kinase, while expression of total proteins Erk1/2 and Src was unchanged. During chronic colitis phosphorylation (i.e., activation) of Erk1/2 was significantly decreased in IL-10 KO mice vs. wild-type mice. Levels of total Erk1/2 proteins were unchanged, but the expression of total Src protein as well as its phosphorylated form was significantly increased in IL-10 KO vs. wild-type mice. There were no changes in total Akt proteins, while levels of activated Akt (pAkt) were slightly increased in IL-10 KO vs. wild-type mice. We conclude that VEGF/VEGFR-2-associated signal transduction pathways, that mediate increased vascular permeability (Src, Akt), might play a central role in perpetuation of chronic experimental UC.
11: Biomaterials, 2010 Nov, 31(31)
FGF-2 and VEGF functionalization of starPEG-heparin hydrogels to modulate biomolecular and physical cues of angiogenesis.
[Abstract]Tissue engineering therapies require biomaterials capable of encouraging an angiogenic response. To dissect the influence of different pro-angiogenic stimuli a set of starPEG-heparin hydrogels with varied physicochemical properties was used as a highly efficient reservoir and tunable delivery system for basic fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF). The engineered gel materials could be precisely tailored by decoupling the biomolecular functionalization from the variation of the viscoelastic matrix characteristics. Culture experiments with human umbilical vein endothelial cells (HUVECs) revealed the interplay of growth factor presentation, adhesive characteristics and elasticity of the gel matrices in triggering differential cellular behavior which allowed identifying effective pro-angiogenic conditions.
12: Translational stroke research, 2010 Sep 1, 1(3)
VEGF Induces More Severe Cerebrovascular Dysplasia in Endoglin than in Alk1 Mice.
[Abstract]Brain arteriovenous malformations (BAVMs) are an important cause of intracranial hemorrhage (ICH) in young adults. A small percent of BAVMs is due to hereditary hemorrhagic telangiectasia 1 and 2 (HHT1 and 2), which are caused by mutations in two genes involved in TGF-beta signaling: endoglin (ENG) and activin-like kinase 1 (ALK1). The BAVM phenotype is an incomplete penetrant in HHT patients, and the mechanism is unknown. We tested the hypothesis that a "response-to-injury" triggers abnormal vascular (dysplasia) development, using Eng and Alk1 haploinsufficient mice. Adeno-associated virus (AAV) expressing vascular endothelial growth factor (VEGF) was used to mimic the injury conditions. VEGF overexpression caused a similar degree of angiogenesis in the brain of all groups, except that the cortex of Alk1(+/-) mice had a 33% higher capillary density than other groups. There were different levels of cerebrovascular dysplasia in haploinsufficient mice (Eng(+/)>Alk1(+/-)), which simulates the relative penetrance of BAVM in HHT patients (HHT1>HHT2). Few dysplastic capillaries were observed in AAV-LacZ-injected mice. Our data indicate that both angiogenic stimulation and genetic alteration are necessary for the development of dysplasia, suggesting that anti-angiogenic therapies might be adapted to slow the progression of the disease and decrease the risk of spontaneous ICH.
13: The American journal of pathology, 2010 Sep, 177(3)
Endothelial {alpha}3{beta}1-Integrin Represses Pathological Angiogenesis and Sustains Endothelial-VEGF.
[Abstract]Integrin alpha3beta1 is a major receptor for laminin. The expression levels of laminins-8 and -10 in the basement membrane surrounding blood vessels are known to change during tumor angiogenesis. Although some studies have suggested that certain ligands of alpha3beta1 can affect angiogenesis either positively or negatively, either a direct in vivo role for alpha3beta1 in this process or its mechanism of action in endothelial cells during angiogenesis is still unknown. Because the global genetic ablation of alpha3-integrin results in an early lethal phenotype, we have generated conditional-knockout mice where alpha3 is deleted specifically in endothelial cells (ec-alpha3-/-). Here we show that ec-alpha3-/- mice are viable, fertile, and display enhanced tumor growth, elevated tumor angiogenesis, augmented hypoxia-induced retinal angiogenesis, and increased vascular endothelial growth factor (VEGF)-mediated neovascularization ex vivo and in vivo. Furthermore, our data provide a novel method by which an integrin may regulate angiogenesis. We show that alpha3beta1 is a positive regulator of endothelial-VEGF and that, surprisingly, the VEGF produced by endothelial cells can actually repress VEGF-receptor 2 (Flk-1) expression. These data, therefore, identify directly that endothelial alpha3beta1 negatively regulates pathological angiogenesis and implicate an unexpected role for low levels of endothelial-VEGF as an activator of neovascularization.
14: Gut, 2010 Aug, 59(8)
Molecular imaging of VEGF in gastrointestinal cancer in vivo using confocal laser endomicroscopy.
[Abstract]Background Vascular endothelial growth factor (VEGF) is a therapeutic target in gastrointestinal cancer (GiC). However, its in vivo visualisation could not be achieved to date with endoscopic techniques. Confocal laser endomicroscopy (CLE) is a novel imaging technique for gastrointestinal endoscopy providing in vivo microscopy at subcellular resolution. The aim of the study was to evaluate CLE for in vivo molecular imaging of VEGF in GiC. Methods Molecular imaging of tumours in APCmin mice, in xenograft models and in surgical specimens of patients with colorectal cancer (CRC) was achieved after application of labelled antibodies. The tumour sites were scanned with the probe for the strongest specific fluorescent signal. From all tumour sites examined with CLE in vivo, targeted specimens were obtained for histology, immunohistochemistry (IHC) and fluorescence microscopy. Results A VEGF-specific signal was visualised in vivo in 13/15 APCmin mice and in 9/10 xenograft tumours. CLE enabled the cytoplasmatic distribution of VEGF to be displayed due to its subcellular resolution. In human tissue, a VEGF-specific signal was observed in 12/13 malignant specimens and in 10/11 samples from healthy mucosa from the patients (p<0.03). CLE findings correlated well with ex vivo microscopy. Conclusion In vivo molecular imaging with specific targeting of VEGF is possible in murine tumours, human xenografts and tissue specimens using CLE. CLE with similar probes can be performed in human colonoscopy. Therefore-from a technical point of view-in vivo molecular imaging is transferable to stratification of patients with CRC during endoscopy even today. CLE could contribute to the identification of lesions at risk and potentially predict response to targeted treatment.
15: Journal of ethnopharmacology, 2010 Sep 15, 131(2)
Kangen-karyu improves memory deficit caused by aging through normalization of neuro-plasticity-related signaling system and VEGF system in the brain.
[Abstract]AIM OF THE STUDY: Kangen-karyu (KK) is a traditional Chinese prescription consisting of six different herbs. This study was conducted to investigate the anti-dementia effect of KK on aging-induced cognitive deficits and the underlying mechanism using senescence-accelerated mice prone (SAMP8). MATERIALS AND METHODS: Twenty-week old SAMP8 (older SAMP8) were used as an animal model of aging and age-matched senescence-resistant inbred strain (SAMR1) and 8-week-old SAMP8 (young SAMP8) were as controls. Older SAMP8 received daily administration of KK (100mg/kg, p.o.) or water vehicle for 22 days. RESULTS: Compared to the controls, older SAMP8 exhibited cognitive deficits in the object recognition and object location tests; however, KK improved the deficits caused by aging. Moreover, the older SAMP8 treated with vehicle exhibited reduced anxiety-like behavior in the elevated plus-maze test compared to SAMR1, but KK had no effect on emotional disorder of older SAMP8. The levels of biochemical factors related to neuro-plasticity and learning and memory; i.e., phosphorylated forms of N-methyl-d-aspartate receptor 1, Ca(2+)/calmodulin-dependent protein kinase II, and cyclic AMP-responsive element-binding protein, and brain-derived neurotrophic factor, were significantly decreased in older SAMP8 compared to those in the control animals. KK normalized the levels of these factors. Moreover, the mRNA and protein levels of vascular endothelial growth factor (VEGF) and its receptor type 2 in the cerebral cortices of older SAMP8 were down-regulated by aging, but these levels were reversed by KK. CONCLUSIONS: These findings suggest that normalization of neuro-plasticity-related neuronal signaling and VEGF systems in the brain may be of the mechanisms underlying the ameliorative effects of KK on the cognitive deficits in older SAMP8.
16: Experimental & molecular medicine, 2010 Jul 15, 11(8)
TGF-b1 induces mouse dendritic cells to express VEGF and its receptor (Flt-1) under hypoxic conditions.
[Abstract]Angiogenesis is a multi-step process that involves the activation, proliferation, and migration of endothelial cells. We have recently shown that TGF-b1 can induce mouse macrophages to produce VEGF, a potent angiogenic factor. In the present study, we explored whether TGF-b1 has a similar effect on mouse dendritic cells. First, we show that under hypoxic conditions, TGF-b1 induced the expression of VEGF transcripts in bone marrow-derived dendritic cells. Overexpression of Smad3/4 further augmented TGF-b1-induced VEGF transcription, while overexpression of DN-Smad3 decreased VEGF transcription in DC2.4 cells, a mouse dendritic cell line. We also show that TGF-b1 and Smads are involved in the induction of VEGF protein secretion. Interestingly, under the same conditions, the expression of VEGF receptor 1 (Flt-1) was also elevated at both the transcriptional and protein levels. Additionally, we found that the TGF-b1-induced VEGF secretion in activated DC2.4 cells has wound-healing properties. Finally, Smad7 and Smurf1 negatively regulated the TGF-b1-induced and Smad3/4-mediated VEGF expression. Taken together, these results indicate that TGF-b1 can enhance the expression of VEGF and Flt-1 through the typical Smad pathway in mouse dendritic cells.
17: Medical oncology (Northwood, London, England), 2010 Jul 13, 145(2)
Targeting integrin-linked kinase increases apoptosis and decreases invasion of myeloma cell lines and inhibits IL-6 and VEGF secretion from BMSCs.
[Abstract]In this study, we investigated ILK expression in myeloma cell lines U266 and H929 kept in suspension and cocultured with BMSCs, and studied the pharmacologic inhibitors of ILK on the apoptosis, invasive potential of myeloma cell lines, and production of pro-angiogenic factors of bone marrow stromal cells. We found that ILK protein was expressed in U266 and H929 cells and kinase activity was elevated when cocultured with BMSCs. ILK inhibitor QLT0267 reduced ILK kinase activity and increased apoptosis both in myeloma cell lines kept in suspension and cocultured with BMSCs. ILK inhibition by ILK inhibitor decreased the in vitro invasive capability of myeloma cell lines. In addition, QLT0267 significantly decreased VEGF and IL-6 secretion in BMSCs in a dose-dependent fashion. The results indicated that inhibition of ILK may provide a potential target for myeloma therapy.
18: Journal of the neurological sciences, 2010 Sep 15, 296(1-2)
VEGF/VEGFR-2 changes in frontal cortex, choroid plexus, and CSF after chronic obstructive hydrocephalus.
[Abstract]Chronic hydrocephalus (CH) is often associated with decreased cerebral blood flow (CBF) and oxygen levels. While the exact pathophysiology is not clear, vascular endothelial growth factor (VEGF) and its receptor-2 (VEGFR-2) may be involved. Because the choroid plexus (CP) is involved in cerebrospinal fluid (CSF) production and secretes numerous growth factors including VEGF, it is important to understand VEGF/VEGFR-2 levels in the CP-CSF circulatory system. Our results showed significant decreases in CBF and VEGFR-2 levels in frontal cortex (FC) in CH compared with SC; there were no significant changes in VEGF levels. CBF change in FC was positively correlated with VEGFR-2 levels (P=0.024). Immunohistochemistry (IHC) showed robust expression of VEGF/VEGFR-2 in CP. After CH induction, ventricular CSF volume and VEGF levels significantly increased. These results suggest that the decreased VEGFR-2 levels in FC may be contributed to decreased CBF and increased ventricular CSF-VEGF levels possibly reflected a hypoxic response and/or accumulation of VEGF from CP secretion after blockage of CSF outlet. Further investigation into CSF-VEGF levels in different sites may provide a better understanding of VEGF/VEGFR-2 modulation in the normal and hydrocephalic brain, and may represent a feasible approach to potential therapeutic options for hydrocephalus.
19: Biotechnology and applied biochemistry, 2010 Jul 8, 6(7)
A novel virally-inactivated human platelet lysate preparation rich in TGF-beta, EGF and IGF, depleted of PDGF and VEGF.
[Abstract]There is emerging interest in the use of standardized virally-inactivated human platelet lysate preparations rich in growth factors (GF) for cell cultures, cell therapy and clinical applications. Here we report a simple process to prepare a virally-inactivated platelet lysate preparation rich in TGF-beta1/EGF/IGF and depleted of PDGF and VEGF. Apheresis platelet concentrates were treated by the solvent-detergent (S/D) viral inactivation procedure, then subjected to one oil extraction followed by adsorption with activated charcoal, and finally sterile-filtered. The resulting preparation contained a mean of 368.4, 2.4, and 54.7 ng/mL of TGF-beta1, EGF, and IGF, respectively. PDGF-AB and VEGF were essentially completely removed by the charcoal treatment. Mean albumin, immunoglobulins G, M and A, and fibrinogen content was about 40.0, 8.5, 0.87, 1.66 and 2.65 mg/mL, respectively, cholesterol and triglyceride 15 and 20.7 mg/mL, respectively, and TnBP and Triton X-45, 8.7 and 8.8 ppm, respectively. Supplementing MEM medium with 1-10% of this S/D-treated platelet lysate promoted the proliferation of MG63 and SIRC cell lines as well as, or better than, 10% FBS, as based on the MTS assay. The process used to prepare such S/D-treated platelet lysate is easily scalable for industrial production. Our results open the possibility to evaluate the interest of this new preparation for stem cell expansion and/or bone tissue engineering and regeneration.
20: Annals of nuclear medicine, 2010 Jul 6, 6(7)
Assessment of VEGF-D expression measured by immunohistochemical staining and F-18 FDG uptake on PET as biological prognostic factors for recurrence in patients with surgically resected lung adenocarcinoma.
[Abstract]OBJECTIVE: To assess whether the combined evaluation of vascular endothelial growth factor D (VEGF-D) expression and fluorodeoxyglucose (FDG) uptake correlates with lymph node metastasis and post-operative recurrence in patients with lung adenocarcinoma. METHODS: Forty-six patients with lung adenocarcinomas, who had undergone both preoperative FDG PET imaging and thoracotomy, were enrolled in this study. The surgically resected tumor specimens were used to assess the protein levels of VEGF-D as measured by immunohistochemical assay. RESULTS: The patients were divided into the following four groups: those who were VEGF-D negative and had low FDG uptake (group I, 3 patients), VEGF-D positive and had low FDG uptake (group II, 20 patients), VEGF-D negative and had high FDG uptake (group III, 13 patients), and VEGF-D positive and had high FDG uptake (group IV, 10 patients). Lymph node metastases were seen only in group III. The 5-year disease-free survival rates were 66.7% in group I, 83.9% in group II, 8.3% in group III, and 64.0% in group IV (p < 0.0001). Thus, patients in group III exhibited the most unfavorable prognoses for recurrence. In multivariate analysis, the combined evaluation of VEGF-D expression and FDG uptake was an independent parameter for post-operative recurrence (p = 0.018). CONCLUSION: A combination of low VEGF-D expression and high FDG uptake may be a biological indicator of lymph node metastasis and post-operative recurrence in patients with lung adenocarcinoma.
21: Biotechnology letters, 2010 Jul 6, 6(7)
A fusion fragment from Flt-1 and KDR, acted as VEGF decoy receptor and exhibited anti-tumor function.
[Abstract]Angiogenesis is important in tumor development. Vascular endothelial growth factor (VEGF) is involved in this process. In this report, we constructed a recombinant protein (called FK) by fusing the second immunoglobulin-like (Ig-like) domain of a human fms-like tyrosine kinase (Flt-1) with the third Ig-like domain of human kinase insert domain-containing receptor (KDR). FK bound to VEGF(165) in a dose-dependent manner with a disocciation constant (Kd) of 2.7 pM. In addition, FK specifically inhibited the proliferation of human microvascular endothelial cell (HMEC) and human umbilical vein endothelial Cell (HUVEC) stimulated by VEGF(165). Subsequent studies also demonstrate that FK efficaciously suppresses growth of a variety of tumors, which could make FK a potential drug candidate in anti-tumor therapy.
22: European journal of cell biology, 2010 Jul 3, 6(7)
EDF-1 contributes to the regulation of nitric oxide release in VEGF-treated human endothelial cells.
[Abstract]Vascular endothelial growth factor (VEGF) induces nitric oxide (NO) release by triggering multiple intracellular signals, among others the calcium/calmodulin pathway and the activation of Akt, events which induce endothelial NO synthase (eNOS) activity. Because Endothelial Differentiation-related Factor (EDF)-1 is a calmodulin binding protein and plays a role in modulating endothelial functions, we evaluated whether EDF-1 is implicated in the regulation of eNOS activity in VEGF-treated human endothelial cells. While VEGF does not modulate the total amounts of EDF-1, it promotes the dissociation of calmodulin from EDF-1 which correlates with the increase of calmodulin bound to eNOS and the induction of NO release. To better characterize the contribution of EDF-1 to the regulation of VEGF-induced NO release, we stably silenced EDF-1 in endothelial cells. We here show that endothelial cells silencing EDF-1 produce more NO than controls and do not increase NO release in response to VEGF. The insensitivity to VEGF results from the incapability of cells silencing EDF-1 to phosphorylate eNOS Ser(1177), even though Akt is activated. Interestingly, okadaic acid, a pharmacologic inhibitor of the serine/threonine phosphatase PP2A, which preferentially dephosphorylates eNOS Ser(1177), restores NO release and eNOS Ser(1177) phosphorylation in cells silencing EDF-1. Our results suggest EDF-1 as a novel contributor to the complex regulation of eNOS activity in human endothelial cells.
23: Oncogene, 2010 Jul 5, 6(7)
RKTG inhibits angiogenesis by suppressing MAPK-mediated autocrine VEGF signaling and is downregulated in clear-cell renal cell carcinoma.
[Abstract]Vascular endothelial growth factors (VEGFs) are crucial regulators of angiogenesis and vasculogenesis. The autocrine VEGF signaling is required for maintaining the homeostasis of vasculature. Dysregulation of angiogenesis is implicated in the development of many human cancers, especially in clear-cell renal cell carcinoma (ccRCC), a highly vascularized tumor. Meanwhile, antiangiogenesis has become a mainstay in the treatment of human cancers. In this study, we analyzed the functional roles of RKTG (Raf Kinase Trapping to Golgi), a negative regulator of mitogen-activated protein kinase (Raf/MEK/ERK) signaling, by sequestration of Raf kinase to the Golgi apparatus, in angiogenesis and ccRCC. Through a series of in vitro and in vivo experiments, we found that RKTG has a negative effect on cell proliferation, migration, sprouting and angiogenesis of endothelial cells. RKTG, by suppressing mitogen-activated protein kinase signaling, negatively regulates the transactivation activity of hypoxia-inducible factor 1alpha (HIF-1alpha) by inhibiting formation of HIF-1alpha/p300 complex and suppressing VEGF transcription, thereby reducing hypoxia-induced VEGF production. The expression level of RKTG is significantly downregulated in clinical ccRCC tumor samples, with an inverse correlation with VEGF expression level. These results highlight the functional roles of RKTG and its regulated Raf/ERK/MEK signaling cascade in angiogenesis and autocrine VEGF signaling. In addition, this study indicates that RKTG is likely implicated in the development of ccRCC through its regulation on angiogenesis.Oncogene advance online publication, 5 July 2010; doi:10.1038/onc.2010.270.
24: Cancer biology & therapy, 2010 Sep 30, 10(5)
Low density lipoprotein receptor mediates anti-VEGF effect of lymphocyte T-derived microparticles in lewis lung carcinoma cells.
[Abstract]Nonstop proliferation and vigorous neovascularization are two prominent characteristics of cancer. Antiangiogenic therapy has emerged as an important modality in treatment of solid tumors. Our previous work demonstrated that microparticles derived from apoptotic T-lymphocytes (LMPs) not only reduced the viabilities of high-proliferating cells, but also exhibited potent antiangiogenic effects through inhibition of the vascular endothelial growth factor (VEGF)/VEGF receptor 2 signalling pathway. In the present study, we extended these studies to explore the anticancer potential of LMPs using a murine model of Lewis lung carcinoma (LLC). Results show that intratumoral injection of LMPs (2.5 mg/kg) decreased tumor size by more than 50% relative to control. Tumor microvessel density and VEGF-A levels were also markedly reduced upon LMPs treatment. To elucidate the underlying mechanisms of LMPs-mediated antitumor activity, LLC cells were utilized in in vitro experiments. LMPs suppressed VEGF-A protein levels in LLC cells and led to inhibition of LLC cell viability and proliferation. In addition, knockdown of the low-density lipoprotein receptor (LDLR) expression reduced the uptake of LMPs into LLC cells and attenuated the inhibitory effects of LMPs on cell growth and VEGF-A expression. Our findings demonstrate that LMPs exert antiangiogenic and proapoptotic effects that lead to inhibition of lung carcinoma by reducing VEGF-A levels and LDLR mediates the anti-VEGF effect of LMPs through translocating LMPs into LLC cells. These results suggest that LMPs are promising antiangiogenic therapeutic agent and represent a new therapeutic strategy for treating lung carcinomas.
25: The British journal of ophthalmology, 2010 Jul 3, 6(7)
Efficacy and safety of anti-vascular endothelial growth factor (VEGF) therapy with intravitreal ranibizumab (Lucentis) for naive retinal vein occlusion: 1-year follow-up.
[Abstract]Purpose To evaluate the efficacy and safety of intravitreal ranibizumab (Lucentis) in patients with treatment-naive retinal vein occlusion. Design Prospective, consecutive, non-comparative, interventional case series. Participants Seventeen eyes of 17 consecutive patients with naive retinal vein occlusion. Methods Consecutive patients were recruited and received, on demand, intravitreal 0.5 mg of ranibizumab; nine had central retinal vein occlusion (CRVO) and eight had branch retinal vein occlusion (BRVO). Pre- and postoperative clinical evaluation included measurement of best corrected visual acuity (BCVA) for distance, and near vision (MNREAD time, reading fluency), contrast sensitivity, colour fundus photography, fluorescein angiography and optical coherence tomography (OCT). All subjects were followed for a minimum of 12 months. Main outcome measures Change in BCVA, contrast sensitivity, angiographic leakage, OCT central macular thickness (CMT), number of treatments. Results Patients with CRVO had mean pre-treatment BCVA of 20/240 (1.08+/-0.25 logarithm of the minimum angle of resolution (logMAR)) and final BCVA of 20/46 (0.36+/-0.16 logMAR), with significant improvement at 1 year of follow-up (p<0.0001). At 12 months mean BCVA improved to 36.7 letters, with a gain of 6.4 lines, and OCT showed that the mean CMT was 271 mum, with a mean reduction of 360 mum (p<0.0001) from baseline (mean 631 mum). Patients with BRVO had mean pre-treatment BCVA of 20/126 (0.80+/-0.29 logMAR) and final BCVA of 20/50 (0.41+/-0.23 logMAR) (p<0.0001). The mean OCT CMT was 278 mum, with a mean reduction of 275 mum (p<0.0001) from baseline (mean 553 mum). Contrast sensitivity, MNREAD time and reading fluency improved significantly in the treated eyes. No ocular or systemic side effects were observed. Eyes with CRVO received an average of 3.0 injections (range 2-4) and those with BRVO 3.6 (range 3-4). Conclusions Intravitreal ranibizumab for the management of naive CRVO or BRVO can favourably modify the course of the occlusion, indicating that short- and long-term blockade of vascular endothelial growth factor (VEGF)-A may restore the integrity of the inner blood-retinal barrier, reduce CMT and significantly improve visual function, with a good safety profile. Further prospective long-term studies are warranted to confirm the efficacy, safety and optimal treatment regimen for intravitreal ranibizumab.
26: Biochemical pharmacology, 2010 Jul 2, 6(7)
The PKCbeta/HuR/VEGF pathway in diabetic retinopathy.
[Abstract]We investigated whether the diabetes-related PKCbeta activation affects VEGF expression through the mRNA-stabilizing human Embryonic Lethal Abnormal Vision (ELAV) protein, HuR, in the retina of streptozotocin (STZ)-induced diabetic rats. Diabetes was induced in rats by STZ injection. Retinal tissues were processed to detect PKCbetaI, PKCbetaII, VEGF and HuR contents, as well as HuR phosphorylation. Immunoprecipitation coupled to RT-PCR was employed to evaluate HuR binding to VEGF mRNA in RiboNucleoProteic (RNP) complexes. Statistical analysis was performed by ANOVA followed by an appropriate post hoc comparison test. Following experimental diabetes PKCbetaI and PKCbetaII levels were increased compared to sham; there was also a PKC-mediated phosphorylation/activation of HuR. These effects were blunted by the in vivo co-administration of a selective PKCbeta inhibitor. A specific binding between the HuR protein and the VEGF mRNA was also detected. The PKCbeta/HuR activation was accompanied by enhanced VEGF protein expression that was, again, blunted by the PKCbeta inhibitor. These findings first demonstrate the activation, in the retina, of the PKCbeta/HuR/VEGF pathway following experimental diabetes and disclose a new potential pharmacological target to counteract pathologies implicating VEGF deregulation, such as diabetic retinopathy.
27: International journal of developmental neuroscience : the official journal of the International Society for Developmental Neuroscience, 2010 Jul 2, 6(7)
Role of vasodilator stimulated phosphoprotein in VEGF induced blood-brain barrier permeability in endothelial cell monolayers.
[Abstract]The blood-brain barrier (BBB) plays an important role in the pathophysiology of central nervous system (CNS) disorders such as stroke and hypoxic-ischemic brain injury. Vascular endothelial growth factor (VEGF) is involved in angiogenesis and vasogenic edema during stroke and hypoxia. However, the role of VEGF in BBB permeability after hypoxia has not been fully elucidated. We therefore investigated VEGF effects in an in vitro BBB model using rbcec4 endothelial cell line with the stimulation of VEGF or hypoxia. In this study, BBB permeability was studied using (14)C-sucrose detection. The expression of BBB tight junction protein ZO-1, and the expression and phosphorylation of vasodilator stimulated phosphoprotein (VASP), VEGF and VEGF receptor 2 (VEGFR2) were determined using fluorescent immunocytochemistry and western blot analyses. We found that hypoxia upregulated VEGF expression, and VEGF increased BBB permeability. Hypoxia also increased VASP phosphorylation, which is mediated, in part, through VEGFR2. We also found that VASP at tight junctions was co-localized with ZO-1 in cell-cell contacts. Our findings show that VASP phosphorylation is affected by hypoxia and VEGFR2 inhibition suggesting a role for VASP in BBB permeability.
28: Annals of oncology : official journal of the European Society for Medical Oncology / ESMO, 2010 Jul 1, 6(7)
Temsirolimus in VEGF-refractory metastatic renal cell carcinoma.
[Abstract]BACKGROUND: Temsirolimus is an i.v. administered inhibitor of mammalian target of rapamycin with activity in the first-line setting in poor-prognosis patients with metastatic renal cell carcinoma (RCC). The efficacy of this agent after failure of prior inhibitors of vascular endothelial growth factor (VEGF) is unknown. METHODS: A retrospective review of patients with metastatic RCC treated at the Cleveland Clinic Taussig Cancer Institute and three regional cancer centers in Ontario, Canada, through the Torisel (temsirolimus) Compassionate Use Program was conducted. Demographic, toxicity and response data were collected. RESULTS: A total of 87 patients with metastatic RCC were identified who had previously been treated with inhibitors of VEGF subsequently treated with temsirolimus. The majority of patients had either intermediate or poor-prognosis disease at baseline. Expected toxic effects including hyperglycemia and noninfectious pneumonitis were observed. The RECIST-defined objective response rate was 5% and the stable disease rate was 65%. The median time to progression (TTP) was 3.9 months (95% confidence interval 2.8-4.8 months), and median overall survival was 11.2 months. CONCLUSIONS: In a cohort of pre-treated intermediate to poor-prognosis patients with metastatic RCC, weekly i.v. temsirolimus is associated with predictable, but manageable toxicity, and a TTP approaching 4 months.
29: Chinese journal of cancer, 2010 Jul, 29(7)
Low dosage 5-fluorouracil increases the transfection efficiency of Ad/VEGF-A in mouse lung carcinoma cell line LA795 and inhibits tumor growth.
[Abstract]Background and Objective: Adenovirus vectors were widely used in gene therapy for tumors. We used adenovirus vector to transfer small interfering RNA (siRNA) against vascular epithelium growth factor A (VEGF-A) molecules to mouse lung adenoma LA795 cells and used low dose of chemotherapeutic drugs to further elevate the infection efficiency of adenovirus vector in and therapeutic effect of RNAi on tumor cells. Methods: LA795 cells were infected by Ad/EGFP and treated with different dosages of gemcitabin, epirubicin, cisplatin, or 5-fluorouracil (5-FU). Cells were observed under fluorescence microscope continuously using green fluorescent protein (GFP) as the reporter gene. The percentage of GFP-positive cells and fluorescent intensity were tested by flow cytometry to determine optimum concentrations of drugs. Ad/siVEGF-A containing VEGF-A siRNA was constructed. Real-time PCR and ELISA were applied to measure the expression level of VEGF-A after LA795 cells were infected by Ad/siVEGF-A and treated with 5-FU. The combination of Ad/siVEGF-A and 5-FU was also applied in treating subcutaneous tumor in mice. Results: Low dose of 5-FU elevated the Ad/EGFP infection in LA795 cells significantly, and also enhanced the effect of Ad/siVEGF-A in down-regulating VEGF-A mRNA and protein levels in tumor cells. When used in tumor in vivo, the combination strategy repressed tumor growth effectively. Conclusion: Low dose of 5-FU can enhance the capability of adenovirus infecting tumor cells and promote the efficiency of gene therapy by adenovirus.
30: Journal of surgical oncology, 2010 Sep 15, 102(4)
VEGF and cortactin expression are independent predictors of tumor recurrence following curative resection of gastric cancer.
[Abstract]INTRODUCTION: To investigate the clinicopathological role of expression of vascular endothelial growth factor (VEGF) and cortactin, as well as whether their expression are independent predictors of tumor recurrence following curative resection of gastric cancer. METHODS: One hundred twenty-eight patients with gastric cancer were included in this study. Formalin-fixed paraffin-embedded specimens were stained for VEGF and cortactin, and the correlation between the staining, clinicopathological parameters and prognostic power were analyzed. RESULTS: Of the 128 patients studied, 58 (45.3%) and 71 (55.5%) cases were strongly positive for VEGF and cortactin, respectively. VEGF expression correlated with Lauren classification (P < 0.001), pathological tumor stage (P < 0.001), and pathological tumor node metastasis (TNM) stage (P = 0.003). Cortactin expression correlated with pathological lymph node stage (P = 0.018), pathological TNM stage (P < 0.001), and degree of differentiation (P < 0.001). There were statistically significant associations between tumor recurrence and VEGF expression (P = 0.023), and cortactin expression (P < 0.001). In multivariate analysis, pathological TNM stage, VEGF expression, and cortactin expression were independent prognostic influence on disease-free survival (P < 0.001, 0.022, and 0.034, respectively). CONCLUSIONS: VEGF and cortactin may be a good biomarker to be applied in clinic to predict the prognosis of patients with curatively resected gastric cancer. J. Surg. Oncol. 2010;102:325-330. (c) 2010 Wiley-Liss, Inc.
31: Diabetes, 2010 Jul, 59(7)
Response to Comment on: Biscetti et al. (2010) High-Mobility Group Box-1 Protein Promotes Angiogenesis After Peripheral Ischemia in Diabetic Mice Through a VEGF-Dependent Mechanism. Diabetes;59:1496-1505.
[Abstract]PURPOSE OF REVIEW: This review highlights the current body of knowledge regarding the role of the vascular endothelial growth factor (VEGF) and its receptor (VEGFR) in angiosarcoma, epithelioid hemangioendothelioma (EHE), and hemangiopericytoma/solitary fibrous tumor (HPC/SFT). Therapeutic agents that target this pathway are reviewed. RECENT FINDINGS: Several phase II trials in advanced soft tissue sarcoma patients have investigated the efficacy of bevacizumab, an anti-VEGF antibody, as well as sunitinib, sorafenib, and pazopanib, VEGFR tyrosine kinase inhibitors (TKIs). Although response rates and progression-free survival periods were generally low, several angiosarcoma, EHE, and HPC/SFT patients demonstrated response or durable disease stabilization on these therapies. Biological mechanisms underlying the activity of these agents in angiosarcoma, EHE, and HPC/SFT are poorly understood. Some angiosarcoma tumors, however, harbor specific activating mutations in VEGFR2, which may be effectively targeted by VEGFR TKIs. SUMMARY: Inhibition of the VEGF/VEGFR pathway may be a rational and effective therapy for certain patients with angiosarcoma, EHE, and HPC/SFT, but more studies are needed to confirm these findings and to identify which patients will benefit from these agents.
32: Diabetes, 2010 Jul, 59(7)
Comment on: Biscetti et al. (2010) High-Mobility Group Box-1 Protein Promotes Angiogenesis After Peripheral Ischemia in Diabetic Mice Through a VEGF-Dependent Mechanism. Diabetes;59:1496-1505.
[Abstract]PURPOSE OF REVIEW: This review highlights the current body of knowledge regarding the role of the vascular endothelial growth factor (VEGF) and its receptor (VEGFR) in angiosarcoma, epithelioid hemangioendothelioma (EHE), and hemangiopericytoma/solitary fibrous tumor (HPC/SFT). Therapeutic agents that target this pathway are reviewed. RECENT FINDINGS: Several phase II trials in advanced soft tissue sarcoma patients have investigated the efficacy of bevacizumab, an anti-VEGF antibody, as well as sunitinib, sorafenib, and pazopanib, VEGFR tyrosine kinase inhibitors (TKIs). Although response rates and progression-free survival periods were generally low, several angiosarcoma, EHE, and HPC/SFT patients demonstrated response or durable disease stabilization on these therapies. Biological mechanisms underlying the activity of these agents in angiosarcoma, EHE, and HPC/SFT are poorly understood. Some angiosarcoma tumors, however, harbor specific activating mutations in VEGFR2, which may be effectively targeted by VEGFR TKIs. SUMMARY: Inhibition of the VEGF/VEGFR pathway may be a rational and effective therapy for certain patients with angiosarcoma, EHE, and HPC/SFT, but more studies are needed to confirm these findings and to identify which patients will benefit from these agents.
33: Bone, 2010 Aug, 47(2)
Mechanical stimulation of the pro-angiogenic capacity of human fracture haematoma: Involvement of VEGF mechano-regulation.
[Abstract]Compromised angiogenesis appears to be a major limitation in various suboptimal bone healing situations. Appropriate mechanical stimuli support blood vessel formation in vivo and improve healing outcomes. However, the mechanisms responsible for this association are unclear. To address this question, the paracrine angiogenic potential of early human fracture haematoma and its responsiveness to mechanical loading, as well as angiogenic growth factors involved, were investigated in vitro. Human haematomas were collected from healthy patients undergoing surgery within 72h after bone fracture. The haematomas were embedded in a fibrin matrix, and cultured in a bioreactor resembling the in vivo conditions of the early phase of bone healing (20% compression, 1Hz) over 3days. Conditioned medium (CM) from the bioreactor was then analyzed. The matrices were also incubated in fresh medium for a further 24h to evaluate the persistence of the effects. Growth factor (GF) concentrations were measured in the CM by ELISAs. In vitro tube formation assays were conducted on Matrigel with the HMEC-1 cell line, with or without inhibition of vascular endothelial growth factor receptor 2 (VEGFR2). Cell numbers were quantified using an MTS test. In vitro endothelial tube formation was enhanced by CM from haematomas, compared to fibrin controls. The angiogenesis regulators, vascular endothelial growth factor (VEGF) and transforming growth factor beta1 (TGF-beta1), were released into the haematoma CM, but not angiopoietins 1 or 2 (Ang1, 2), basic fibroblast growth factor (bFGF) or platelet-derived growth factor (PDGF). Mechanical stimulation of haematomas, but not fibrin controls, further increased the induction of tube formation by their CM. The mechanically stimulated haematoma matrices retained their elevated pro-angiogenic capacity for 24h. The pro-angiogenic effect was cancelled by inhibition of VEGFR2 signalling. VEGF concentrations in CM tended to be elevated by mechanical stimulation; this was significant in haematomas from younger, but not from older patients. Other GFs were not mechanically regulated. In conclusion, the paracrine pro-angiogenic capacity of early human haematomas is enhanced by mechanical stimulation. This effect lasts even after removing the mechanical stimulus and appears to be VEGFR2-dependent.
34: Journal of cellular biochemistry, 2010 Aug 1, 110(5)
VEGF receptor binding peptide-linked high mobility box group-1 box A as a targeting gene carrier for hypoxic endothelial cells.
[Abstract]High mobility group box-1 (HMGB-1) is a nuclear protein that can bind to and condense plasmid DNA. In this study, we developed a recombinant VEGF receptor binding peptide (VRBP) linked to HMGB-1 box A (VRBP-HMGB1A) as a targeting gene carrier to hypoxic endothelial cells. Hypoxic endothelial cells in ischemic tissues of solid tumors are important targets for gene therapy. A recombinant VRBP-HMGB1A expression vector, pET21a-VRBP-HMGB1A was constructed. VRBP-HMGB1A was over-expressed in BL21 strain and purified by nickel-chelate affinity chromatography. Complex formation between VRBP-HMGB1A and pCMV-Luc was confirmed by gel retardation assay. pCMV-Luc was retarded completely at a 2/1 weight ratio (peptide/plasmid). For transfection assays, calf pulmonary artery endothelial (CPAE) cells were incubated under hypoxia for 24 h, prior to transfection to induce the VEGF receptors on the cells. VRBP-HMGB1A/pCMV-Luc complexes were transfected to hypoxic CPAE cells. The highest transfection efficiency was at a 30/1 weight ratio (peptide/plasmid). In addition, VRBP-HMGB1A had higher efficiency than poly-L-lysine (PLL) specifically in hypoxic CPAE cells, However, VRBP-HMGB1A had lower efficiency than PLL in 293, H9C2, and normoxic CPAE cells. In MTT assay, VRBP-HMGB1A was less toxic than PLL to cells. In conclusion, VRBP-HMGB1A is a potential gene carrier for targeting hypoxic endothelial cells and thus, may be useful for cancer gene therapy. J. Cell. Biochem. 110: 1094-1100, 2010. Published 2010 Wiley-Liss, Inc.
35: Journal of cellular biochemistry, 2010 Jul 1, 110(4)
Lyn kinase and ZAP70 are substrates of PTPROt in B-cells: Lyn inactivation by PTPROt sensitizes leukemia cells to VEGF-R inhibitor pazopanib.
[Abstract]We have recently shown that the gene encoding the truncated form of protein tyrosine phosphatase receptor-type O (PTPROt) expressed predominantly in hematopoietic cells is epigenetically silenced in human primary chronic lymphocytic leukemia (B-CLL). To determine whether increased phosphorylation of the PTPROt substrates following PTPROt suppression alters signal transduction pathway(s) that impart a growth advantage to the leukemic lymphocytes, it is critical to discern the key substrates of PTPROt. Here, we used substrate-trapping assay to identify two novel substrates of PTPROt, the tyrosine kinases Lyn and ZAP70. Both Lyn and ZAP70 were dephosphorylated by wild-type PTPROt, but not by its catalytic site (CS) mutant. A critical phosphorylation site in Lyn, Y397, essential for its activity was dephosphorylated by PTPROt. Consequently, the activity of Lyn kinase was compromised when co-expressed with PTPROt-WT compared to vector control or upon co-expression with PTPROt-CS. Ectopic expression of PTPROt in Raji cells reduced phosphorylation of Lyn in the absence of any change in its protein levels. These results have revealed the physiological importance of PTPROt in regulating B-cell receptor signaling at Lyn kinase. Further, ectopic expression of PTPROt also sensitized the cells to the VEGF-R inhibitor Pazopanib. J. Cell. Biochem. 110: 846-856, 2010. (c) 2010 Wiley-Liss, Inc.
36: The British journal of ophthalmology, 2010 Sep, 94(9)
Intravitreal anti-VEGF treatment in eyes with combined choroidal neovascularisation and vitreomacular traction syndrome.
[Abstract]Purpose To report the effect of intravitreal anti-vascular endothelial growth factor injections (IVI) on visual acuity in eyes with choroidal neovascularisation (CNVM) and co-existent vitreomacular traction (VMT) or when VMT has developed during the course of treatment. Methods Retrospective interventional case series of seven eyes in seven patients. VMT was monitored with serial optical coherence tomography scans. Results The mean age at presentation was 74 years (range 64-95 years). All patients presented with blurring of central vision, rather than distortion. The aetiology of CNVM was wet age-related macular degeneration in five eyes (72%), angioid streaks in one eye (14%) and pathological myopia in one eye (14%). Ranibizumab was used in four eyes (57%) and bevacizumab in three (43%) for the active CNVM component. The mean follow-up was 11 months (range 2-28 months). None of the eyes in this series required surgery for the VMT component, nor were there any cases of spontaneous resolution of VMT. Visual acuity was stabilised or improved in five of the seven eyes (71%) with IVI. Visual acuity results across the whole group were gain of three or more lines of Snellen visual acuity in two eyes (28%), gain of up to three lines in three eyes (42%), no change in visual acuity in one eye (14%) and loss of up to three lines in one eye (14%). There were no eyes losing more than three lines of Snellen visual acuity. In four eyes with pre-existing VMT, visual acuity improved in three with IVI. In three eyes that developed VMT after IVI, visual acuity improved in two with IVI. Delay from diagnosis of CNVM to treatment with IVI contributed to a poor response. Conclusions Most eyes improved visual acuity with IVI for combined CNVM and VMT. Despite the often dramatic features of VMT on optical coherence tomography, treatment of co-existing CNVM should be prompt. Vitreoretinal surgery was not required in this series, but is held in reserve if there is still potential for gain in vision following CNVM resolution.
37: Clinical cancer research : an official journal of the American Association for Cancer Research, 2010 Aug 1, 16(15)
Effects of Anti-VEGF Treatment Duration on Tumor Growth, Tumor Regrowth, and Treatment Efficacy.
[Abstract]PURPOSE: Inhibition of the vascular endothelial growth factor (VEGF) axis is the basis of all currently approved antiangiogenic therapies. In preclinical models, anti-VEGF blocking antibodies have shown broad efficacy that is dependent on both tumor context and treatment duration. We aimed to characterize this activity and to evaluate the effects of discontinuation of treatment on the dynamics of tumor regrowth. EXPERIMENTAL DESIGN: We evaluated the effects of anti-VEGF treatment on tumor growth and survival in 30 xenograft models and in genetic mouse models of cancer. Histologic analysis was used to evaluate the effects of treatment on tumor vasculature. We used a variety of treatment regimens to allow analysis of the effects of treatment duration and cessation on growth rate, survival, and vascular density. RESULTS: Preclinical tumor models were characterized for their varied dependence on VEGF, thereby defining models for testing other agents that may complement or augment anti-VEGF therapy. We also found that longer exposure to anti-VEGF monoclonal antibodies delayed tumor growth and extended survival in established tumors from both cell transplants and genetic tumor models and prevented regrowth of a subset of residual tumors following cytoablative therapy. Discontinuation of anti-VEGF in established tumors resulted in regrowth at a rate slower than that in control-treated animals, with no evidence of accelerated tumor growth or rebound. However, more rapid regrowth was observed following discontinuation of certain chemotherapies. Concurrent administration of anti-VEGF seemed to normalize these accelerated growth rates. CONCLUSIONS: In diverse preclinical models, continuous VEGF suppression provides maximal benefit as a single agent, combined with chemotherapy, or as maintenance therapy once chemotherapy has been stopped. Clin Cancer Res; 16(15); 3887-900. (c)2010 AACR.
38: Neurochemical research, 2010 Sep, 35(9)
Characterization of neural stem/progenitor cells expressing VEGF and its receptors in the subventricular zone of newborn piglet brain.
[Abstract]Neural stem/progenitor cell (NSP) biology and neurogenesis in adult central nervous system (CNS) are important both towards potential future therapeutic applications for CNS repair, and for the fundamental function of the CNS. In the present study, we report the characterization of NSP population from subventricular zone (SVZ) of neonatal piglet brain using in vivo and in vitro systems. We show that the nestin and vimentin-positive neural progenitor cells are present in the SVZ of the lateral ventricles of neonatal piglet brain. In vitro, piglet NSPs proliferated as neurospheres, expressed the typical protein of neural progenitors, nestin and a range of well-established neurodevelopmental markers. Upon dissociation and subculture, piglet NSPs differentiated into neurons and glial cells. Clonal analysis demonstrates that piglet NSPs are multipotent and retain the capacity to generate both glia and neurons. These cells expressed VEGF, VEGFR1, VEGFR2 and Neuropilin-1 and -2 mRNAs. Real time PCR revealed that SVZ NSPs from newborn piglet expressed total VEGF and all VEGF splice variants. These findings show that piglet NSPs may be helpful to more effectively design growth factor based strategies to enhance endogenous precursor cells for cell transplantation studies potentially leading to the application of this strategy in the nervous system disease and injury.
39: Diabetes, 2010 Sep, 59(9)
Ablation of 4E-BP1/2 Prevents Hyperglycemia-Mediated Induction of VEGF Expression in the Rodent Retina and in Muller Cells in Culture.
[Abstract]OBJECTIVE Vascular endothelial growth factor (VEGF) contributes to diabetic retinopathy, but control of its expression is not well understood. Here, we tested the hypothesis that hyperglycemia mediates induction of VEGF expression in a eukaryotic initiation factor 4E (eIF4E) binding protein (4E-BP) 1 and 2 dependent manner. RESEARCH DESIGN AND METHODS The retina was harvested from control and type 1 diabetic rats and mice and analyzed for VEGF mRNA and protein expression as well as biomarkers of translational control mechanisms. Similar analyses were performed in M¨¹ller cell cultures exposed to hyperglycemic conditions. The effect of 4E-BP1 and 4E-BP2 gene deletion on VEGF expression was examined in mice and in mouse embryo fibroblasts (MEFs). RESULTS Whereas VEGF mRNA in the retina remained constant, VEGF expression was increased as early as 2 weeks after the onset of diabetes. Increases in expression of 4E-BP1 protein mirrored those of VEGF and expression of 4E-BP1 mRNA was unchanged. Similar results were observed after 10 h of exposure of cells in culture to hyperglycemic conditions. Importantly, the diabetes-induced increase in VEGF expression was not observed in mice deficient in 4E-BP1 and 4E-BP2, nor in MEFs lacking the two proteins. CONCLUSIONS Hyperglycemia induces VEGF expression through cap-independent mRNA translation mediated by increased expression of 4E-BP1. Because the VEGF mRNA contains two internal ribosome entry sites, the increased expression is likely a consequence of ribosome loading at these sites. These findings provide new insights into potential targets for treatment of diabetic retinopathy.
40: American journal of physiology. Heart and circulatory physiology, 2010 Sep, 299(3)
Contraction-induced secretion of VEGF from skeletal muscle cells is mediated by adenosine.
[Abstract]The role of adenosine and contraction for secretion of vascular endothelial growth factor (VEGF) in skeletal muscle was investigated in human subjects and rat primary skeletal muscle cells. Microdialysis probes were inserted in the thigh muscle of seven male subjects, and dialysate was collected at rest, during infusion of adenosine, and during knee extensor exercise. The dialysate was analyzed for content of VEGF protein and adenosine. The mechanism of VEGF secretion from muscle cells in culture was examined in resting and electrostimulated cells and in response to the adenosine analog NECA and the adenosine A(2A) receptor specific analog CGS-21680. Adenosine receptors A(1), A(2A), and A(2B) were blocked with DPCPX, ZM-241385, and enprofylline, respectively. cAMP-dependent protein kinase A (PKA) and mitogen-activated protein kinase (MAPK) were inhibited by H-89 and PD-98509, respectively. The human experiment showed that adenosine infusion enhanced (P < 0.05) the interstitial concentration of VEGF protein approximately fourfold above baseline. Exercise increased (P < 0.05) the interstitial VEGF concentration approximately sixfold above rest in parallel with an approximately threefold increase in adenosine concentration. In accordance, in cultured muscle cells, NECA and contraction caused secretion of VEGF (P < 0.05). The contraction-induced secretion of VEGF was abolished by the A(2B) antagonist enprofylline and by inhibition of PKA or MAPK. The results demonstrate that adenosine causes secretion of VEGF from human skeletal muscle cells and that the contraction-induced secretion of VEGF protein is partially mediated via adenosine acting on A(2B) adenosine receptors. Moreover, the contraction-induced secretion of VEGF protein from muscle is dependent on both PKA and MAPK activation, but only the MAPK pathway appears to be adenosine dependent, revealing involvement of additional pathways in VEGF secretion.
41: Biomaterials, 2010 Sep, 31(25)
Functionalization of matrices by cyclically stretched osteoblasts through matrix targeting of VEGF.
[Abstract]Vascular endothelial growth factor A (VEGF) plays a central role in load-induced bone gain. We previously showed that increasing cyclic stretch frequency from 0.05 to 5 Hz induce parallel increased in entrapment of VEGF (mVEGF) into osteoblast secreted extracellular matrix. We ask in this study if mVEGF could be protective against apoptotic signals and biologically active in vitro on endothelial cell migration as well as in vivo on angiogenesis. We established that mechanically-induced VEGF entrapment using stretched silicone membrane was saturable after 3 exposures at high frequency stretches (5Hz). We found that mVEGF stimulates microvascular cells migration and enhanced angiogenesis more importantly than VEGF 165 controls suggesting the absence of potent anti-angiogenic factors in our functionalized matrices. Indeed we found that the anti-angiogenic factors, tissue inhibitor of metalloproteinase (TIMP2) and pigment epithelium-derived factor (PEDF) were specifically downregulated for 5 Hz stretch and that the release of these potent factors was increased for low frequency of stretch (0.05 Hz). This study qualifies high frequency cyclic stretch as an interesting approach for surfaces activation of deformable biomaterials.
42: Circulation research, 2010 Jun 10, 93(4)
VEGF Blockade Inhibits Lymphocyte Recruitment and Ameliorates Immune-Mediated Vascular Remodeling.
[Abstract]Rationale: There are conflicting data on the effects of vascular endothelial growth factor (VEGF) in vascular remodeling. Furthermore, there are species-specific differences in leukocyte and vascular cell biology and little is known about the role of VEGF in remodeling of human arteries. Objective: We sought to address the role of VEGF blockade on remodeling of human arteries in vivo. Methods and Results: We used an anti-VEGF antibody, bevacizumab, to study the effect of VEGF blockade on remodeling of human coronary artery transplants in severe combined immunodeficient mice. Bevacizumab ameliorated peripheral blood mononuclear cell-induced but not interferon-gamma-induced neointimal formation. This inhibitory effect was associated with a reduction in graft T-cell accumulation without affecting T-cell activation. VEGF enhanced T-cell capture by activated endothelium under flow conditions. The VEGF effect could be recapitulated when a combination of recombinant intercellular adhesion molecule 1 and VCAM-1 rather than endothelial cells was used to capture T cells. A subpopulation of CD3+ T cells expressed receptor (VEGFR)-1 by immunostaining and FACS analysis. VEGFR-1 mRNA was also detectable in purified CD4+ T cells and Jurkat and HSB-2 T-cell lines. Stimulation of HSB-2 and T cells with VEGF triggered downstream ERK phosphorylation, demonstrating the functionality of VEGFR-1 in human T cells. Conclusions: VEGF contributes to vascular remodeling in human arteries through a direct effect on human T cells that enhances their recruitment to the vessel. These findings raise the possibility of novel therapeutic approaches to vascular remodeling based on inhibition of VEGF signaling.
43: Biochemical and biophysical research communications, 2010 Jul 9, 397(4)
Albendazole inhibits endothelial cell migration, tube formation, vasopermeability, VEGF receptor-2 expression and suppresses retinal neovascularization in ROP model of angiogenesis.
[Abstract]The angiogenic process begins with the cell proliferation and migration into the primary vascular network, and leads to vascularization of previously avascular tissues and organs as well to growth and remodeling of the initially homogeneous capillary plexus to form a new microcirculation. Additionally, an increase in microvascular permeability is a crucial step in angiogenesis. Vascular endothelial growth factor (VEGF) plays a central role in angiogenesis. We have previously reported that albendazole suppresses VEGF levels and inhibits malignant ascites formation, suggesting a possible effect on angiogenesis. This study was therefore designed to investigate the antiangiogenic effect of albendazole in non-cancerous models of angiogenesis. In vitro, treatment of human umbilical vein endothelial cells (HUVECs) with albendazole led to inhibition of tube formation, migration, permeability and down-regulation of the VEGF type 2 receptor (VEGFR-2). In vivo albendazole profoundly inhibited hyperoxia-induced retinal angiogenesis in mice. These results provide new insights into the antiangiogenic effects of albendazole.
44: Der Ophthalmologe : Zeitschrift der Deutschen Ophthalmologischen Gesellschaft, 2010 Jun 11, 93(4)
[Anti-VEGF therapy of exudative AMD : Prognostic factors for therapy success.]
[Abstract]BACKGROUND: The aim of the study was to evaluate possible prognostic and predictive factors (morphologic and functional) of the individual visual gain/decline after anti-VEGF therapy of exudative AMD. PATIENTS/METHODS: Best corrected visual acuity (VA), microperimetric sensitivity (RS), retinal thickness (RT) and autofluorescence pattern (AF) were documented in128 patients with exudative AMD. RESULTS: Eyes with classic choroidal neovascularization (CNV) had the best visual gain but still remained at a lower level. Eyes which initially had the lowest VA had the largest gain and those with good initial VA could maintain this level. There was no correlation between RT and visual outcome. Eyes with initially normal AF had a significantly greater visual gain. CONCLUSIONS: The type of CNV, initial VA, RS and the initial RT were only of limited usefulness, while the initial foveal AF was most important predictive factor. This may indicate that preexisting changes and irreversible damage in the outer retina and/or retinal pigment epithelium are responsible for the resulting VA after therapy.
45: Diabetes, 2010 Sep, 59(9)
Muller cell-derived VEGF is essential for diabetes-induced retinal inflammation and vascular leakage.
[Abstract]OBJECTIVE Vascular endothelial growth factor (VEGF-A or VEGF) is a major pathogenic factor and therapeutic target for diabetic retinopathy (DR). Since VEGF has been proposed as a survival factor for retinal neurons, defining the cellular origin of pathogenic VEGF is necessary for the effectiveness and safety of long-term anti-VEGF therapies for DR. To determine the significance of M¨¹ller cell-derived VEGF in DR, we disrupted VEGF in M¨¹ller cells with an inducible Cre/lox system and examined diabetes-induced retinal inflammation and vascular leakage in these conditional VEGF knockout (KO) mice. RESEARCH DESIGN AND METHODS Leukostasis was determined by counting the number of fluorescently labeled leukocytes inside retinal vasculature. Expression of biomarkers for retinal inflammation was assessed by immunoblotting of TNF-alpha, ICAM-1, and NF-kappaB. Vascular leakage was measured by immunoblotting of retinal albumin and fluorescent microscopic analysis of extravascular albumin. Diabetes-induced vascular alterations were examined by immunoblotting and immunohistochemistry for tight junctions, and by trypsin digestion assays for acellular capillaries. Retinal integrity was analyzed with morphologic and morphometric analyses. RESULTS Diabetic conditional VEGF KO mice exhibited significantly reduced leukostasis, expression of inflammatory biomarkers, depletion of tight junction proteins, numbers of acellular capillaries, and vascular leakage compared to diabetic control mice. CONCLUSIONS M¨¹ller cell-derived VEGF plays an essential and causative role in retinal inflammation, vascular lesions, and vascular leakage in DR. Therefore, M¨¹ller cells are a primary cellular target for proinflammatory signals that mediates retinal inflammation and vascular leakage in DR.
46: Molecular and cellular biochemistry, 2010 Jun 4, 58(11)
Intermittent high glucose enhances cell proliferation and VEGF expression in retinal endothelial cells: the role of mitochondrial reactive oxygen species.
[Abstract]Proliferation of human retinal endothelial cells (HRECs) is an important event in the development of diabetic retinopathy. Glucose fluctuations are strong predictor of diabetic vascular complications. In this study we have investigated the effect of intermittent high glucose on proliferation and expression of vascular endothelial growth factor (VEGF) in HRECs. The possible involvement of mitochondrial reactive oxygen species (ROS) was assessed. HRECs were incubated for 72 h in media containing different glucose concentrations: 5, 25, 5 mmol/l alternating with 25 mmol/l glucose, with or without Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP) and thenoyltri-fluoroacetone (TTFA). The cell proliferation, VEGF expression, mitochondrial ROS, nitrotyrosine and 8-hydroxydeoxyguanosine (8-OHdG) were measured. In cultured HRECs, treatment with constant or intermittent high glucose significantly increased [(3)H]thymidine incorporation in a time-dependent manner. Treatment with constant high glucose for 48 h resulted in significant increases in [(3)H]thymidine incorporation, mRNA and protein levels of VEGF compared with HRECs treated with the normal glucose, which were markedly enhanced in cells exposed to intermittent high glucose. The levels of mitochondrial ROS, nitrotyrosine and 8-OhdG were significantly elevated under both intermittent and constant high glucose conditions, the effect being greater under intermittent high glucose. In addition, the antioxidants MnTBAP or TTFA can effectively prevent cell proliferation and overexpression of VEGF, as well as overproduction of mitochondrial ROS, nitrotyrosine and 8-OhdG in HRECs induced by constant or intermittent high glucose. Intermittent high glucose enhances cell proliferation and overexpression of VEGF through reactive oxygen species (ROS) overproduction at the mitochondrial transport chain level in HRECs, indicating that glycemic variability have important pathological effects on the development of diabetic retinopathy dependent of mitochondrial ROS.
47: Chemical research in toxicology, 2010 Jun 3, 115(22)
Application of Genomics for Identification of Systemic Toxicity Triggers Associated With VEGF-R Inhibitors.
[Abstract]The key to the discovery of new pharmaceuticals is to develop molecules that interact with the intended target and minimize interaction with unintended molecular targets, therefore minimizing toxicity. This is aided by the use of various in vitro selectivity assays that are used to select agents most potent for the desired target. Typically, molecules from similar chemical series, with similar in vitro potencies, are expected to yield comparable in vivo pharmacological and toxicological profiles, predictive of target effects. However, in this study, we investigated the in vivo effects of two analogue compounds that similarly inhibit several receptor tyrosine kinases such as vascular endothelial growth factor receptor 1 (VEGFR/Flt1), vascular endothelial growth factor 2 (VEGFR2/kinase domain receptor/Flk-1), vascular endothelial growth factor receptor 3 (VEGFR3/Flt4), platelet-derived growth factor receptor (PDGFR), and Kit receptors, which bear similar chemical structures, have comparable potencies, but differ markedly in their rodent toxicity profiles. Global gene expression data were used to generate hypotheses regarding the existence of toxicity triggers that would reflect the perturbation of signaling in multiple organs such as the liver, adrenal glands, and the pancreas in response to compound treatment. We concluded that differences in pharmacokinetic properties of the two analogues, such as volume of distribution, half-life, and organ concentrations, resulted in marked differences in the chemical burden on target organs and may have contributed to the vast differences in toxicity profiles observed with the two otherwise similar molecules. We propose including select toxicokinetic parameters such as V(ss), T (1/2), and T (max) as additional criteria that could be used to rank order compounds from the same pharmacological series to possibly minimize organ toxicity. Assessment of toxicokinetics is not an atypical activity on toxicology studies, even in early screening studies; however, these data may not always be used in decision making for selecting or eliminating one compound over another. Finally, we illustrate that in vivo gene expression profiles can serve as a complementary assessor of this activity and simultaneously help provide an assessment of on or off-target biological activity.
48: Clinica chimica acta; international journal of clinical chemistry, 2010 Sep 6, 411(17-18)
Vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (NOS3) polymorphisms are associated with high relapse risk in childhood acute lymphoblastic leukemia (ALL).
[Abstract]BACKGROUND: Angiogenesis has been shown as an important process in hematological malignancies. It consists in endothelial proliferation, migration, and tube formation following pro-angiogenic factors releasing, specially the vascular endothelial growth factor (VEGF), which angiogenic effect seems to be dependent on nitric oxide (NO). We examined the association among functional polymorphisms in these two angiogenesis related genes: VEGF (-2578C>A, -1154G>A, and -634G>C) and NOS3 (-786T>C, intron 4 b>a, and Glu298Asp) with prognosis of childhood acute lymphoblastic leukemia (ALL). METHODS: The genotypes were determined and haplotypes estimated in 105 ALL patients that were divided in 2 groups: high risk (HR) and low risk of relapse (LR) patients. In addition, event-free survival curves according to genotypes were assessed. RESULTS: The group HR compared to the LR showed a higher frequency of the alleles -2578C and -634C and the haplotype CGC for VEGF (0.72 vs. 0.51, p<0.008; 0.47 vs. 0.26, p<0.008; and 42.1 vs. 14.5, p<0.006; respectively) and a lower frequency of the haplotype CbGlu (0.4 vs. 8.8,p<0.006), for NOS3. CONCLUSION: Polymorphisms of VEGF and NOS3 genes are associated with high risk of relapse, therefore may have a prognostic impact in childhood ALL.
49: European journal of cancer prevention : the official journal of the European Cancer Prevention Organisation (ECP), 2010 Sep, 19(5)
VEGF and prostatic cancer: a systematic review.
[Abstract]Elevated vascular endothelial growth factor (VEGF) blood concentration reflects its prostatic production, making this a potentially interesting tumour marker to support the decision of submitting a patient for prostatic biopsy. The objective was to review systematically the evidence on the role of VEGF blood concentration in prostate cancer detection. Published studies addressing the relation between serum or plasma VEGF levels and prostate cancer were identified by searching Pubmed, ISI Web of Knowledge, SCOPUS and LILACS up to January 2010, and reviewed following a standardized protocol. Three studies reported higher plasma VEGF (pg/ml) in patients with localized prostate cancer than in healthy controls (7.0 vs. 0.0, 9.9 vs. 2.2, and 210 vs. 26.5, P<0.01), and two showed higher serum VEGF (pg/ml) in prostate cancer patients than in patients with benign prostate hypertrophy (518.9 vs. 267.9, P<0.001; no specific values, P<0.05). In one study, serum VEGF was significantly lower in healthy controls than in patients with benign prostate hypertrophy, localized or metastatic prostate cancer. The three studies that used controls with previous suspicion of prostatic cancer but a negative biopsy reported non-statistically significant difference in VEGF serum levels (pg/ml) between controls and localized prostate cancer patients (241 vs. 206; 69.5 vs. 55; 215.2 vs. 266.4). Higher VEGF plasma levels are observed in prostatic cancer patients compared with healthy controls, but serum levels do not appear to be useful in differentiating benign from malignant prostatic disease using, as controls, individuals with high risk of prostate cancer and negative biopsy.
50: Acta ophthalmologica, 2010 Aug, 88(5)
Toxicity testing of the VEGF inhibitors bevacizumab, ranibizumab and pegaptanib in rats both with and without prior retinal ganglion cell damage.
[Abstract]Purpose: To evaluate the effects of intravitreally introduced vascular endothelial growth factor (VEGF) inhibitors in rat eyes with healthy retinal ganglion cells (RGC) and into others with N-methyl-D-aspartate (NMDA)-induced RGC damage. Methods: Bevacizumab, ranibizumab and pegaptanib were intravitreally injected each at two different concentrations. Respective vehicles of the three substances served as controls. In a different group, additionally a rat anti-VEGF antibody was injected after NMDA treatment. Retrogradely labelled RGC were counted on retinal wholemounts 1 week or 2 months after intravitreal introduction of the VEGF inhibitors. Electron microscopy (EM) was performed on normal rat eyes 2 months after introduction of the VEGF inhibitors. Results: RGC counts in healthy rat eyes were essentially unchanged from those of the control animals after the administration of both low and high concentrations of bevacizumab, ranibizumab or pegaptanib. Compared to the other two substances, however, high doses of pegaptanib and its respective vehicle significantly decreased RGC after 1 week and led to a marked increase of mitochondrial swelling in EM. In eyes with NMDA-induced RGC damage, no changes of RGC numbers were detected after rat anti-VEGF antibody or bevacizumab, ranibizumab and pegaptanib at both tested concentrations. Conclusions: Even at higher doses, bevacizumab and ranibizumab showed no toxic effects on RGC in vivo in either untreated rats or in the NMDA-induced RGC damage model. Also a rat anti-VEGF antibody showed no adverse effects after NMDA. Anti-VEGF therapy therefore appears safe even for eyes with additional excitotoxic RGC damage. Potential harm from the pegaptanib carrier solution at very high local concentrations cannot be excluded.
51: Current opinion in oncology, 2010 Jul, 22(4)
Inhibiting the VEGF-VEGFR pathway in angiosarcoma, epithelioid hemangioendothelioma, and hemangiopericytoma/solitary fibrous tumor.
[Abstract]PURPOSE OF REVIEW: This review highlights the current body of knowledge regarding the role of the vascular endothelial growth factor (VEGF) and its receptor (VEGFR) in angiosarcoma, epithelioid hemangioendothelioma (EHE), and hemangiopericytoma/solitary fibrous tumor (HPC/SFT). Therapeutic agents that target this pathway are reviewed. RECENT FINDINGS: Several phase II trials in advanced soft tissue sarcoma patients have investigated the efficacy of bevacizumab, an anti-VEGF antibody, as well as sunitinib, sorafenib, and pazopanib, VEGFR tyrosine kinase inhibitors (TKIs). Although response rates and progression-free survival periods were generally low, several angiosarcoma, EHE, and HPC/SFT patients demonstrated response or durable disease stabilization on these therapies. Biological mechanisms underlying the activity of these agents in angiosarcoma, EHE, and HPC/SFT are poorly understood. Some angiosarcoma tumors, however, harbor specific activating mutations in VEGFR2, which may be effectively targeted by VEGFR TKIs. SUMMARY: Inhibition of the VEGF/VEGFR pathway may be a rational and effective therapy for certain patients with angiosarcoma, EHE, and HPC/SFT, but more studies are needed to confirm these findings and to identify which patients will benefit from these agents.
52: Endocrine pathology, 2010 Sep, 21(3)
VEGF and CD31 Association in Pituitary Adenomas.
[Abstract]Pituitary tumors are usually less vascularized than the normal pituitary, and the role of angiogenesis in these adenomas is contentious. Appraisal of microvascular density and expression of the potent angiogenic vascular endothelial growth factor (VEGF) by immunohistochemistry has yielded controversial results, as a broad spectrum of immunostaining can be found. We determined the protein expression of VEGF and CD31, an endothelial marker, in a series of 56 surgically removed pituitary adenomas using Western blot assay. Prolactinomas had higher VEGF protein expression compared to nonfunctioning or ACTH- and GH-secreting adenomas, while CD31 was similar in the different adenoma histotypes. VEGF and CD31 were not affected by sex, age, years of adenoma evolution, or proliferation rate (Ki67 and PCNA) for all adenoma types. Only in nonfunctioning adenomas CD31 concentration increased significantly with age. There was a positive correlation between CD31 and VEGF expression when all adenoma histotypes were considered, or when prolactinomas and nonfunctioning adenomas were evaluated separately. The positive association of VEGF and CD31 expression suggests the participation of angiogenesis in adenoma development, while epithelial cell proliferation in pituitary tumors is not directly related to VEGF or CD31 expression, and other factors, such as primary genetic alterations may be involved.
53: Cancer letters, 2010 Oct 28, 296(2)
mTOR inhibition by everolimus counteracts VEGF induction by sunitinib and improves anti-tumor activity against gastric cancer in vivo.
[Abstract]VEGF receptor blockage has been reported to increase serum VEGF. We hypothesized that mTOR inhibition by everolimus counteracts VEGF induction by sunitinib resulting in an improved anti-tumor activity of sunitinib. In vitro, sunitinib in combination with everolimus did not outperform the respective monotherapies. In vivo, monotherapies reduced tumor growth by 60%, whereas the combination of sunitinib and everolimus led to an almost complete tumor growth inhibition. This superior anti-tumor activity coincided with attenuation of VEGF peaks. In conclusion mTOR inhibition by everolimus counteracts VEGF induction by sunitinib and results in significant reduction of tumor burden and long-lasting tumor growth control.
54: Gynecologic oncology, 2010 Aug 1, 118(2)
The predictive value of serum VEGF in multiresistant ovarian cancer patients treated with bevacizumab.
[Abstract]OBJECTIVE: Bevacizumab, a humanized monoclonal antibody against VEGF (vascular endothelial growth factor), has shown antitumor activity, but so far no biomarkers have been identified to predict outcome. The purpose of the present study was to investigate the efficacy of bevacizumab in patients with multiresistant ovarian cancer and, furthermore, to investigate the possible predictive value of serum VEGF, VEGFR1-2 and VEGF gene polymorphisms. METHODS: Patients received single-agent bevacizumab 10 mg/kg every 3 weeks. All patients were followed with CA 125 measurements and serum VEGF/VEGFR1-2 levels prior to each cycle. Endpoints were response rate (RR), progression-free survival (PFS) and overall survival (OS). RESULTS: Thirty-eight patients were included. All patients were heavily pre-treated with a median of five prior regimens. The median number of bevacizumab treatments was 4. Overall response rate was 30% according to CA 125 (GCIG criteria). Median PFS was 5.9 months (95% CI, 3.5-9.4) and median OS was 8.6 months (95% CI, 6.6-12.8). The VEGF serum level decreased during treatment in all patients. A low pre-treatment VEGF level was predictive to response. The median value was 540 pg/ml and divided the patients into two groups with a response rate of 60% and 0%, respectively (p=0.0007). The difference translated to a significant difference in PFS (p=0.047) and OS (p=0.01). VEGF gene polymorphisms -2578, -1154, -460, +405, +936 did not reveal any association with response or survival and the same applied to serum VEGFR1-2. CONCLUSIONS: Single agent bevacizumab has activity in ovarian cancer patients. Pre-treatment serum VEGF seems to have predictive value.
55: British journal of neurosurgery, 2010 Jun, 24(3)
Release of VEGF and FGF in the extracellular space following severe subarachnoidal haemorrhage or traumatic head injury in humans.
[Abstract]Microdialysate fluid from 145 severely injured NSICU-patients, 88 with subarachnoidal haemorrage (SAH), and 57 with traumatic brain injury (TBI), was collected by microdialysis during the first 7 days following impact, and levels of the neurotrophins fibroblast growth factor-2 (FGF2) and vascular endothelial growth factor (VEGF) were analysed. The study illustrates both similarities and differences in the reaction patterns of the 2 inflammatory proteins. The highest concentrations of both FGF2 and VEGF were measured on Day 2 (mean (+/- SE) values being 47.1 +/- 15.33 and 116.9 +/- 41.85 pg/ml, respectively, in the pooled patient material). The VEGF concentration was significantly higher in TBI-patients, while the FGF2 showed a tendency to be higher in SAH-patients. This is the first report presenting in some detail the human cerebral response of FGF2 and VEGF following SAH and TBI. Apart from increasing the understanding of the post-impact inflammatory response of the human brain, the study identifies potential threshold values for these chemokines that may serve as monitoring indicators in the NSICU.
56: Molecular imaging and biology : MIB : the official publication of the Academy of Molecular Imaging, 2010 May 7, 143(3)
Association of the VEGF 936C>T Polymorphism with FDG Uptake, Clinical, Histopathological, and Metabolic Response in Patients with Adenocarcinomas of the Esophagogastric Junction.
[Abstract]PURPOSE: The MUNICON trial confirmed prospectively the usefulness of early response evaluation by 2-deoxy-2-[F-18]fluoro-D-glucose-positron emission tomography (FDG-PET) . Metabolic responders (R) showed initially a higher FDG uptake compared with nonresponders (p = 0.018). An association of the vascular endothelial growth factor (VEGF) 936C>T polymorphism and FDG uptake was reported for breast cancer. Therefore, we investigated the VEGF 936C>T polymorphism for an association with response and survival. PROCEDURES: The study was based on 110 patients included in the MUNCON trial (103 male, seven female; 75 AEG I, 35 AEG II, event-free survival (EFS) median 21.1 +/- 4.6 months). Response was significantly associated with EFS. The VEGF 936C>T polymorphism was determined by PCR and restriction fragment length polymorphism analysis. For analysis, the T-variants were combined. RESULTS: One hundred two patients were evaluable. Seventy-two patients showed the CC, 24 the CT, and six the TT genotype. Median EFS was 29.3 months for CC and 11.7 months for CT/TT (p = 0.04). No association of the genotypes (CC or CT/TT) with the SUV or response was found. Multivariate analysis revealed histopathological regression (p = 0.003) and genotype (p = 0.04) as independent prognostic factors. A combination of genotype and PET response (Gen-PET) defines three prognostic groups early in the course of treatment (p = 0.002). Cox regression analysis including clinical and histopathological response and Gen-PET reveals Gen-PET as independent prognostic factor (p = 0.003). CONCLUSION: The VEGF 936C>T polymorphism is a prognostic factor in patients undergoing neoadjuvant chemotherapy, although it is not associated with FDG uptake and response. The combination of metabolic response and VEGF 936C>T polymorphism defines three different prognostic groups. These findings need to be confirmed prospectively. This study has been registered in the European Clinical Trials Database as trial 2007-003356-11.
57: Clinical & experimental metastasis, 2010 May 6, 143(3)
Localization of osteoblast inflammatory cytokines MCP-1 and VEGF to the matrix of the trabecula of the femur, a target area for metastatic breast cancer cell colonization.
[Abstract]Bone likely provides a hospitable environment for cancer cells as suggested by their preferential localization to the skeleton. Previous work has shown that osteoblast-derived cytokines increased in the presence of metastatic breast cancer cells. Thus, we hypothesized that osteoblast-derived cytokines, in particular IL-6, MCP-1, and VEGF, would be localized to the bone metaphyses, an area to which breast cancer cells preferentially traffic. Human metastatic MDA-MB-231 breast cancer cells were inoculated into the left ventricle of the heart of athymic mice. Three to four weeks later, tumor localization within isolated femurs was examined using muCT and MRI. In addition, IL-6, MCP-1, and VEGF localization were assayed via immunohistochemistry. We found that MDA-MB-231 cells colonized trabecular bone, the area in which murine MCP-1 and VEGF were visualized in the bone matrix. In contrast, IL-6 was expressed by murine cells throughout the bone marrow. MDA-MB-231 cells produced VEGF, whose expression was not only associated with the breast cancer cells, but also increased with tumor growth. This is the first study to localize MCP-1, VEGF, and IL-6 in bone compartments via immunohistochemistry. These data suggest that metastatic cancer cells may co-opt bone cells into creating a niche facilitating cancer cell colonization.
58: Cardiovascular research, 2010 May 5, 9(9)
ERYTHROPOIETIN AND VENTRICULAR REMODELLING: A VEGF-DEPENDENT NEOVASCULARITY.
[Abstract]Bone likely provides a hospitable environment for cancer cells as suggested by their preferential localization to the skeleton. Previous work has shown that osteoblast-derived cytokines increased in the presence of metastatic breast cancer cells. Thus, we hypothesized that osteoblast-derived cytokines, in particular IL-6, MCP-1, and VEGF, would be localized to the bone metaphyses, an area to which breast cancer cells preferentially traffic. Human metastatic MDA-MB-231 breast cancer cells were inoculated into the left ventricle of the heart of athymic mice. Three to four weeks later, tumor localization within isolated femurs was examined using muCT and MRI. In addition, IL-6, MCP-1, and VEGF localization were assayed via immunohistochemistry. We found that MDA-MB-231 cells colonized trabecular bone, the area in which murine MCP-1 and VEGF were visualized in the bone matrix. In contrast, IL-6 was expressed by murine cells throughout the bone marrow. MDA-MB-231 cells produced VEGF, whose expression was not only associated with the breast cancer cells, but also increased with tumor growth. This is the first study to localize MCP-1, VEGF, and IL-6 in bone compartments via immunohistochemistry. These data suggest that metastatic cancer cells may co-opt bone cells into creating a niche facilitating cancer cell colonization.
59: Free radical biology & medicine, 2010 Aug 1, 49(3)
alphaB-crystallin is involved in oxidative stress protection determined by VEGF in skeletal myoblasts.
[Abstract]Recent studies suggest that the effects of VEGF-A, the prototype VEGF ligand, may extend to a variety of cell types other than endothelial cells. The expression of VEGF-A and its main receptors, Flt-1/VEGFR-1 and KDR/Flk-1/VEGFR-2, was indeed detected in several cell types, including cardiac myocytes and regenerating myotubes. In addition to its proangiogenic activity, evidence indicates that VEGF-A can sustain skeletal muscle regeneration by enhancing the survival and migration of myogenic cells and by promoting the growth of myogenic fibers. In this study, our aim was to investigate whether VEGF could protect skeletal muscle satellite cells from apoptotic cell death triggered by reactive oxygen species and to identify the main molecular mechanisms. C2C12 mouse myoblasts, cultured in vitro in the presence of exogenous VEGF or stably transfected with a plasmid vector expressing VEGF-A, were subjected to oxidative stress and analyzed for cell growth and survival, induction of apoptosis, and molecular signaling. The results of our study demonstrated that VEGF protects C2C12 myoblasts from apoptosis induced by oxidative or hypoxic-like stress. This protection did not correlate with the modulation of the expression of VEGF receptors, but is clearly linked to the phosphorylation of the KDR/Flk-1 receptor, the activation of NF-kappaB, and/or the overexpression of the antiapoptotic protein alphaB-crystallin.
60: Journal of cellular physiology, 2010 Sep, 224(3)
Expression profiling of ETS and MMP factors in VEGF-activated endothelial cells: role of MMP-10 in VEGF-induced angiogenesis.
[Abstract]In the process of angiogenesis, working of many transcription factors at the proper time is important to activate angiogenesis-related genes such as cytokine, matrix protease and adhesion molecules. In this study, we searched for Ets transcription factors and matrix metalloproteinases (MMPs) that respond to VEGF in endothelial cells. We first analyzed the expression of 27 human Ets factors and 15 human MMPs in VEGF-treated human umbilical vein endothelial cells (HUVEC) using quantitative RT-PCR. The most abundant Ets factors in HUVEC were ETS-1, Fli-1, ERP/NET/ELK3, and ERG. MMP-1, -2, -10, -11, -14, -15, and -16 were also detected in HUVEC. We also found that ETV-1, Fli-1, ERG, MMP-1, -3, -7, -8, -9, -10, -13, and -19 expression is up-regulated more than 1.5-fold in HUVEC after 2 h of VEGF treatment. In addition, the expression of MMP-10 induced by VEGF remained twofold higher for 24 h compared to non-treated control. The elevation of MMP10 mRNA and protein levels was confirmed to be both time- and dosage-dependent. In addition, MMP-10 transcription was mediated by Ets-1 but not ERP/NET/ELK3. The inhibition of PI3K and MAPK inhibited VEGF-induced MMP-10 expression. Furthermore, transfection of MMP-10 siRNA inhibited VEGF-induced migration and tube formation in HUVEC, and it also inhibited vessel formation in matrigel plugs in vivo. In conclusion, our study demonstrated induction of MMP-10 by VEGF in HUVEC and supports an angiogenic role for MMP-10 in response to VEGF stimulation in vitro and in vivo.
61: Journal of cellular biochemistry, 2010 May 15, 110(2)
VEGF-A expression in osteoclasts is regulated by NF-kappaB induction of HIF-1alpha.
[Abstract]Large osteoclasts (10+ nuclei), predominant in rheumatoid arthritis and periodontal disease, have higher expression of proteases and activating receptors and also have increased resorptive activity when compared to small (2-5 nuclei) osteoclasts. We hypothesized that large and small osteoclasts activate different signaling pathways. A Signal Transduction Pathway Finder Array was used to compare gene expression of large and small osteoclasts in RAW 264.7-derived osteoclasts. Expression of vascular endothelial growth factor A (Vegfa) was higher in large osteoclasts and this result was confirmed by RT-PCR. RT-PCR further showed that RANKL treatment of RAW cells induced Vegfa expression in a time-dependent manner. Moreover, VEGF-A secretion in conditioned media was also increased in cultures with a higher proportion of large osteoclasts. To investigate the mechanism of Vegfa induction, specific inhibitors for the transcription factors NF-kappaB, AP-1, NFATc1, and HIF-1 were used. Dimethyl bisphenol A, the HIF-1alpha inhibitor, decreased Vegfa mRNA expression, whereas blocking NF-kappaB, AP-1, and NFATc1 had no effect. Furthermore, the NF-kappaB inhibitor gliotoxin inhibited Hif1alpha mRNA expression. In conclusion, VEGF-A gene and protein expression are elevated in large osteoclasts compared to small osteoclasts and this increase is regulated by HIF-1. In turn, Hif1alpha mRNA levels are induced by RANKL-mediated activation of NF-kappaB. These findings reveal further differences in signaling between large and small osteoclasts and thereby identify novel therapeutic targets for highly resorptive osteoclasts in inflammatory bone loss.
62: Circulation research, 2010 Jun 25, 106(12)
Intramyocardial VEGF-B167 Gene Delivery Delays the Progression Towards Congestive Failure in Dogs With Pacing-Induced Dilated Cardiomyopathy.
[Abstract]Rationale: Vascular endothelial growth factor (VEGF)-B selectively binds VEGF receptor (VEGFR)-1, a receptor that does not mediate angiogenesis, and is emerging as a major cytoprotective factor. Objective: To test the hypothesis that VEGF-B exerts non-angiogenesis-related cardioprotective effects in nonischemic dilated cardiomyopathy. Methods and Results: AAV-9-carried VEGF-B(167) cDNA (10(12) genome copies) was injected into the myocardium of chronically instrumented dogs developing tachypacing-induced dilated cardiomyopathy. After 4 weeks of pacing, green fluorescent protein-transduced dogs (AAV-control, n=8) were in overt congestive heart failure, whereas the VEGF-B-transduced (AAV-VEGF-B, n=8) were still in a well-compensated state, with physiological arterial Po(2). Left ventricular (LV) end-diastolic pressure in AAV-VEGF-B and AAV-control was, respectively, 15.0+/-1.5 versus 26.7+/-1.8 mm Hg and LV regional fractional shortening was 9.4+/-1.6% versus 3.0+/-0.6% (all P<0.05). VEGF-B prevented LV wall thinning but did not induce cardiac hypertrophy and did not affect the density of alpha-smooth muscle actin-positive microvessels, whereas it normalized TUNEL-positive cardiomyocytes and caspase-9 and -3 activation. Consistently, activated Akt, a major negative regulator of apoptosis, was superphysiological in AAV-VEGF-B, whereas the proapoptotic intracellular mediators glycogen synthase kinase (GSK)-3beta and FoxO3a (Akt targets) were activated in AAV-control, but not in AAV-VEGF-B. Cardiac VEGFR-1 expression was reduced 4-fold in all paced dogs, suggesting that exogenous VEGF-B(167) exerted a compensatory receptor stimulation. The cytoprotective effects of VEGF-B(167) were further elucidated in cultured rat neonatal cardiomyocytes exposed to 10(-8) mol/L angiotensin II: VEGF-B(167) prevented oxidative stress, loss of mitochondrial membrane potential, and, consequently, apoptosis. Conclusions: We determined a novel, angiogenesis-unrelated cardioprotective effect of VEGF-B(167) in nonischemic dilated cardiomyopathy, which limits apoptotic cell loss and delays the progression toward failure.
63: Carcinogenesis, 2010 Jul, 31(7)
PTEN regulates angiogenesis and VEGF expression through phosphatase-dependent and -independent mechanisms in HepG2 cells.
[Abstract]Hepatocellular carcinoma (HCC) is a typical hypervascular tumor, and increased levels of vascular endothelial growth factor (VEGF) are associated with progression of HCC. Tumor suppression gene PTEN (phosphatase and tensin homolog deleted on chromosome 10), an important antagonist of the phosphoinositide-3-kinase (PI3K)/adenosine triphosphate-dependent tyrosine kinase (Akt) pathway, is also commonly lost or mutated in HCC. However, the effect of PTEN on VEGF-mediated angiogenesis in HCC remains unknown. To explore this relationship, we expressed a panel of PTEN mutants in human HCC cells with low expression of PTEN (HepG2 cells). Overexpression of PTEN in HepG2 cells resulted in the downregulation of proliferation and migration of cocultured endothelial cells and decreased expression of hypoxia-inducible factor 1 (HIF-1) and VEGF. Similarly, using a nude mouse model, we demonstrated that PTEN decreased expression of HIF-1 and VEGF and suppressed HepG2-induced angiogenesis. This inhibitory effect was not observed in cells expressing a phosphatase-deficient PTEN mutant, suggesting that PTEN inhibits angiogenesis and VEGF through a phosphatase-dependent pathway. Strikingly, reintroducing the C2 domain of PTEN also resulted in a significant decrease in angiogenesis and VEGF expression, although it did not affect Akt phosphorylation or HIF-1 expression. In summary, this study suggests the novel viewpoint that PTEN suppresses angiogenesis and VEGF expression in HCC through both phosphatase-dependent and -independent mechanisms.
64: Bioorganic & medicinal chemistry letters, 2010 Jun 1, 20(11)
8-THP-DHI analogs as potent Type I dual TIE-2/VEGF-R2 receptor tyrosine kinase inhibitors.
[Abstract]A novel series of 8-(2-tetrahydropyranyl)-12,13-dihydroindazolo[5,4-a]pyrrolo[3,4-c]carbazoles (THP-DHI) was synthesized and evaluated as dual TIE-2 and VEGF-R2 receptor tyrosine kinase inhibitors. Development of the structure-activity relationships (SAR) with the support of X-ray crystallography led to identification of 7f and 7g as potent, selective dual TIE-2/VEGF-R2 inhibitors with excellent cellular potency and acceptable pharmacokinetic properties. Compounds 7f and 7g were orally active in tumor models with no observed toxicity.
65: Neuropeptides, 2010 Aug, 44(4)
Multiple neurotrophic effects of VEGF on cultured neurons.
[Abstract]A large literature demonstrates the multifunctional nature of vascular endothelial growth factor (VEGF). Though initially characterized as an endothelial cell-specific factor, recent studies reveal that VEGF has numerous effects on diverse cell types in the brain including neurons. The objective of this study is to examine the effects of VEGF in cultured cortical neurons on survival, p38 mitogen-activated protein kinase (p38 MAP kinase) activity, pro- and anti-apoptotic protein expression and on release of neurotrophic and neurotoxic factors. The results show that VEGF dose-dependently enhances the survival of neurons in culture. VEGF decreases active caspase 3 levels and increases expression of the anti-apoptotic protein Bcl-2. VEGF decreases phosphorylated p38 MAP kinase level and activity in cortical neurons. In addition to modulating survival/death pathways in cortical neurons, VEGF also regulates release of proteins that affect neuronal viability. VEGF causes a dose-dependent release of the neurotrophic protein pigment epithelial-derived factor (PEDF), while significantly decreasing release of the neurotoxic protein amyloid beta. The VEGF-mediated decrease in amyloid beta is dependent on a functional Flt-1 receptor and is inhibited by dicoumarol, a multifunctional inhibitor of stress-activated protein kinase (SAPK)/JNK and NFkappaB pathways. Taken together, these data demonstrate that the neurotrophic effects of VEGF are likely mediated directly by increasing survival and decreasing apoptotic proteins and signals as well as indirectly by modulating release of proteins that affect neuronal viability.
66: International journal of oncology, 2010 Jun, 36(6)
VEGF-induced ROS generation from NAD(P)H oxidases protects human leukemic cells from apoptosis.
[Abstract]Vascular endothelial growth factor (VEGF) and reactive oxygen species (ROS) play critical roles in vascular pathophysiology and in hematological malignancies. VEGF is supposed to utilize ROS as messenger intermediates downstream of the VEGF receptor-2. NAD(P)H oxidase (Nox) family is a major source of cellular ROS and is implicated in increased ROS production in tumor cells. We previously demonstrated that B1647 cells, a human leukemic cell line, express Nox2 and Nox4, both at mRNA and protein level. We suggest here that the VEGF-induced increase in ROS can be related to Nox2 and Nox4 activities. Nox-derived ROS are involved in early signaling events such as the autophosphorylation of VEGF receptor-2, and in the modulation of glucose uptake, a cellular activity strictly bound to VEGF-induced leukemic cell proliferation, as shown by experiments with antioxidants and Nox inhibitors and siRNA. Nox-generated ROS are required to sustain B1647 cell viability and proliferation; in fact, antioxidants such as EUK-134 or Nox inhibitors and siRNA direct cells to apoptotic cell death, suggesting that manipulation of cellular Nox2 and Nox4 could affect survival of leukemic cells.
67: Molecular bioSystems, 2010 Jul 15, 6(7)
Signal sequence as a determinant in expressing disulfide-stabilized single chain antibody variable fragments (sc-dsFv) against human VEGF.
[Abstract]Phage-displayed single chain variable fragment (scFv) libraries have been powerful tools in antibody engineering. But the scFv structures are frequently unstable due to the dissociation of the dimeric interface between the two variable domains. One solution is the sc-dsFv construct, where the single chain variable domain fragment is stabilized with an additional interface disulfide bond, leading to stable and homogeneous dimeric interface for the sc-dsFv structure. However, the phagemid system that is capable of effective expression for both sc-dsFv-pIII fusion proteins on phage surface and secreted non-fusion sc-dsFv in bacterial culture medium has not been demonstrated. In this work, a biological combinatorial approach was applied to optimize the signal sequence N-terminal to the sc-dsFv-pIII fusion protein encoded in a phagemid. The optimized sc-dsFv phage display systems were compatible with both the phage-based directed evolution procedure and the high throughput screening of the soluble sc-dsFv. The utility of the phagemid systems was demonstrated in generating anti-VEGF sc-dsFv with VEGF-binding affinity one order of magnitude higher than the corresponding scFv, due only to the interface disulfide bond in the sc-dsFv. Moreover, the protein stability of the sc-dsFv construct was unmatched by the corresponding scFv. These advantages of the sc-dsFv were gained through the interface disulfide bond of the sc-dsFv and the novel signal sequence in the phagemid.
68: American journal of physiology. Regulatory, integrative and comparative physiology, 2010 May, 298(5)
Inhibition of VEGF- and NO-dependent angiogenesis does not impair liver regeneration.
[Abstract]Angiogenesis occurs through a convergence of diverse signaling mechanisms with prominent pathways that include autocrine effects of endothelial nitric oxide (NO) synthase (eNOS)-derived NO and vascular endothelial growth factor (VEGF). However, the redundant and distinct roles of NO and VEGF in angiogenesis remain incompletely defined. Here, we use the partial hepatectomy model in mice genetically deficient in eNOS to ascertain the influence of eNOS-derived NO on the angiogenesis that accompanies liver regeneration. While sinusoidal endothelial cell (SEC) eNOS promotes angiogenesis in vitro, surprisingly the absence of eNOS did not influence the angiogenesis that occurs after partial hepatectomy in vivo. While this observation could not be attributed to induction of alternate NOS isoforms, it was associated with induction of VEGF signaling as evidenced by enhanced levels of VEGF ligand in regenerating livers from mice genetically deficient in eNOS. However, surprisingly, mice that were genetically heterozygous for deficiency in the VEGF receptor, fetal liver kinase-1, also maintained unimpaired capacity for liver regeneration. In summary, inhibition of VEGF- and NO-dependent angiogenesis does not impair liver regeneration, indicating signaling redundancies that allow liver regeneration to continue in the absence of this canonical vascular pathway.
69: Journal of controlled release : official journal of the Controlled Release Society, 2010 Aug 3, 145(3)
Topical and intravitreous administration of cationic nanoemulsions to deliver antisense oligonucleotides directed towards VEGF KDR receptors to the eye.
[Abstract]Antisense oligonucleotides (ODNs) specific for VEGFR-2-(17 MER) and inhibiting HUVEC proliferation in-vitro were screened. One efficient sequence was selected and incorporated in different types of nanoemulsions the potential toxicity of which was evaluated on HUVEC and ARPE19 cells. Our results showed that below 10microl/ml, a 2.5% mid-chain triglycerides cationic DOTAP nanoemulsion was non-toxic on HUVEC and retinal cells. This formulation was therefore chosen for further experiments. In-vitro transfection of FITC ODNs in ARPE cells using DOTAP nanoemulsions showed that nanodroplets do penetrate into the cells. Furthermore, ODNs are released from the nanoemulsion after 48h and accumulate into the cell nuclei. In both ex-vivo and in-vivo ODN stability experiments in rabbit vitreous, it was noted that the nanoemulsion protected at least partially the ODN from degradation over 72h. The kinetic results of fluorescent ODN (Hex) distribution in DOTAP nanoemulsion following intravitreal injection in the rat showed that the nanoemulsion penetrates all retinal cells. Pharmacokinetic and ocular tissue distribution of radioactive ODN following intravitreal injection in rabbits showed that the DOTAP nanoemulsion apparently enhanced the intraretinal penetration of the ODNs up to the inner nuclear layer (INL) and might yield potential therapeutic levels of ODN in the retina over 72h post injection.
70: Neuroscience letters, 2010 Jun 25, 477(3)
Vascular endothelial growth factor (VEGF) polymorphism is associated with treatment resistant depression.
[Abstract]Antidepressive medication and electroconvulsive therapy (ECT) increase hippocampal neurogenesis by promoting expression of trophic factors, including brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF). The aims were to test for an association between the VEGF 2578 C/A polymorphism and major depressive disorder (MDD) in two patient populations compared to controls, and the association between this polymorphism and response to serotonin selective reuptake inhibitors (SSRI) and to ECT. The first patient sample consisted of 119 subjects with treatment resistant major depressive disorder who were treated with ECT and the second of 98 depressive patients treated with SSRI. Treatment response was assessed by the Montgomery and Asberg Depression Rating Scale (MADRS). Patients scoring <8 in post-treatment MADRS were considered remitters. There was a trend that CC genotype of VEGF 2578C/A polymorphism was more common in ECT-treated and SSRI-treated patients than in controls (31.1%, 25.5% and 18.7% respectively; p=0.056). The VEGF 2578 C/A polymorphism was associated with treatment resistant MDD. CC genotype was more common in ECT patients than in controls (31.1% and 18.7% respectively; p=0.015). The VEGF 2578 C/A polymorphism was not associated with treatment response to SSRI or to ECT. The finding suggests an association between VEGF 2578 C/A polymorphism and treatment resistant depression which is reported for the first time. Further studies with larger samples will be required to confirm the results.
71: Molecular and cellular endocrinology, 2010 Aug 30, 325(1-2)
Direct survival role of vascular endothelial growth factor (VEGF) on rat ovarian follicular cells.
[Abstract]The aim of the present work was to analyze the direct effect of VEGF in follicular cell proliferation, apoptosis and activation of the PI3K/AKT and ERK/MEK signaling pathways in early antral follicles or granulosa cells. Antral follicles or granulosa cells were isolated from prepubertal female Sprague Dawley rats treated with DES.VEGF directly stimulates follicular cell proliferation and it also decreases apoptosis by inhibiting caspase 3 activation. In addition, VEGF increases the proliferation and inhibits the apoptosis of isolated granulosa cells in culture. VEGF activates the PI3K/AKT pathway evidenced by an increase in AKT phosphorylation levels and induces the phosphorylation of ERK1/2 in cultured antral follicles. These results demonstrate for the first time that VEGF has a proliferative and cytoprotective role in early antral follicles and in granulosa cells isolated from DES treated prepubertal rats and suggest that PI3K/AKT and ERK/MEK signaling pathways are involved in these processes.
72: Blood, 2010 Apr 22, 115(16)
alphaB-crystallin: a novel VEGF chaperone.
[Abstract]We have developed a cancer vaccine candidate (hereafter denominated CIGB-247), based on recombinant modified human vascular endothelial growth factor (VEGF) as antigen, and the adjuvant VSSP (very small sized proteoliposomes of Neisseria meningitidis outer membrane). In mice, previous work of our group had shown that vaccination with CIGB-247 extended tumor-take time, slowed tumor growth, and increased animal survival. Immunization elicited anti-human and murine VEGF-neutralizing antibodies, and spleen cells of vaccinated mice are cytotoxic in vitro to tumor cells that produce VEGF. We have now tested the immunogenicity of CIGB-247 in Wistar rats, New Zealand White rabbits and the non-human primate Chlorocebus aethiops sabaeus. Using weekly, biweekly and biweekly plus montanide immunization schemes, all three species develop antigen-specific IgG antibodies that can block the interaction of VEGF and VEGF receptor 2 in an ELISA assay. Antibody titers decline after vaccination stops, but can be boosted with new immunizations. In monkeys, DTH and direct cell cytotoxicity experiments suggest that specific T-cell responses are elicited by vaccination. Immunization with CIGB-247 had no effect on normal behavior, hematology, blood biochemistry and histology of critical organs, in the tested animals. Skin deep wound healing was not affected in vaccinated rats and monkeys.
73: European journal of cancer (Oxford, England : 1990), 2010 Apr 20, 18(4)
Prolonged tamoxifen treatment increases relapse-free survival for patients with primary breast cancer expressing high levels of VEGF.
[Abstract]Previous retrospective studies have shown that high intratumoural levels of vascular endothelial growth factor (VEGF) correlate with an inferior outcome for patients treated with adjuvant tamoxifen. Our objectives were to validate the impact of VEGF on survival after adjuvant tamoxifen and to investigate the interaction between VEGF and treatment duration. For this purpose tumour homogenates from 402 patients with operable oestrogen receptor positive breast cancer (BC), treated with tamoxifen for 2 (n=149) or 5years (n=253) as the only systemic adjuvant therapy were included. The median follow-up time for surviving patients was 9.8years (range 0.5-14.8years). Expression of VEGF was assessed by an enzyme-linked immunosorbent assay and investigated in relation to the standard BC parameters and survival. In the total population, higher VEGF was significantly correlated with shorter recurrence-free survival (RFS) (HR=1.63, 95%CI=1.11-2.39, p=0.010), breast cancer corrected survival (BCCS) (HR=1.82, 95%CI=1.13-2.93, p=0.014) and overall survival (OS) (HR=1.51, 95%CI=1.11-2.05, p=0.009). High VEGF was significantly associated with reduced RFS (HR=2.61, 95%CI=1.45-4.70, p=0.001) after two years of tamoxifen, whilst no difference was seen in patients treated for five years (HR=1.09, 95%CI=0.64-1.84, p=0.760). A statistically significant interaction was observed between high VEGF expression and improved RFS after 5-year tamoxifen (p=0.034). In concordance with previous studies, high VEGF was significantly correlated with shorter survival. We present data not reported previously revealing that patients expressing high levels of VEGF display a better outcome provided that tamoxifen is given for five years. Further studies on the impact of VEGF on a 5-year regimen are motivated.
74: Developmental cell, 2010 Apr 20, 18(4)
Tel1/ETV6 specifies blood stem cells through the agency of VEGF signaling.
[Abstract]The regulation of stem cell ontogeny is poorly understood. We show that the leukemia-associated Ets transcription factor, Tel1/ETV6, specifies the first hematopoietic stem cells (HSCs) in the dorsal aorta (DA). In contrast, Tel1/ETV6 has little effect on embryonic blood formation, further distinguishing the programming of the long- and short-term blood populations. Consistent with the notion of concordance of arterial and HSC programs, we show that Tel1/ETV6 is also required for the specification of the DA as an artery. We further show that Tel1/ETV6 acts by regulating the transcription of VegfA in both the lateral plate mesoderm and also in the somites. Exogenous VEGFA rescues Tel1/ETV6 morphants, and depletion of VEGFA or its receptor, Flk1, largely phenocopies Tel1/ETV6 depletion. Few such links between intrinsic and extrinsic programming of stem cells have been reported previously. Our data place Tel1/ETV6 at the apex of the genetic regulatory cascade leading to HSC production.
75: Cell transplantation, 2010 Apr 21, 464(7292)
INJECTABLE VEGF HYDROGELS PRODUCE NEAR COMPLETE NEUROLOGICAL AND ANATOMICAL PROTECTION FOLLOWING CEREBRAL ISCHEMIA IN RATS.
[Abstract]Vascular endothelial growth factor (VEGF) is a potent pro-angiogenic peptide and its administration has been considered as a potential neuroprotective strategy following cerebral stroke. Because VEGF has a short half-life and limited access to the brain parenchyma following systemic administration, approaches are being developed to deliver it directly to the site of infarction. In the present study, VEGF was incorporated into a sustained release hydrogel delivery system to examine its potential benefits in a rat model of cerebral ischemia. The hydrogel loaded with VEGF (1 ug) was stereotaxically injected into the striatum of adult rats 15 minutes prior to a 1-hour occlusion of the middle cerebral artery. Two days after surgery, animals were tested for motor function using the elevated bias swing test (EBST) and Bederson neurological battery. Control animals received either stroke alone, stroke plus injections of a blank gel, or a single bolus injection of VEGF (1 ug). Behavioral testing confirmed that the MCA occlusion resulted in significant deficits in the the EBST and Bederson tests. In contrast, the performance of animals receiving VEGF gels was significantly improved relative to controls, with only modest impairments observed. Cerebral infarction analyzed using 2,3,5-triphenyl-tetrazolium chloride staining confirmed that the VEGF gels significantly and potently reduced the lesion volume. No neurological or histological benefits were conferred by either blank gel or bolus VEGF injections. These data demonstrate that VEGF, delivered from a hydrogel directly to the brain can induce significant functional and structural protection from ischemic damage in a rat model of stroke. Vascular endothelial growth factor (VEGF) is a potent pro-angiogenic peptide and its administration has been considered as a potential neuroprotective strategy following cerebral stroke. Because VEGF has a short half-life and limited access to the brain parenchyma following systemic administration, approaches are being developed to deliver it directly to the site of infarction. In the present study, VEGF was incorporated into a sustained release hydrogel delivery system to examine its potential benefits in a rat model of cerebral ischemia. The hydrogel loaded with VEGF (1 ug) was stereotaxically injected into the striatum of adult rats 15 minutes prior to a 1-hour occlusion of the middle cerebral artery. Two days after surgery, animals were tested for motor function using the elevated bias swing test (EBST) and Bederson neurological battery. Control animals received either stroke alone, stroke plus injections of a blank gel, or a single bolus injection of VEGF (1 ug). Behavioral testing confirmed that the MCA occlusion resulted in significant deficits in the the EBST and Bederson tests. In contrast, the performance of animals receiving VEGF gels was significantly improved relative to controls, with only modest impairments observed. Cerebral infarction analyzed using 2,3,5-triphenyl-tetrazolium chloride staining confirmed that the VEGF gels significantly and potently reduced the lesion volume. No neurological or histological benefits were conferred by either blank gel or bolus VEGF injections. These data demonstrate that VEGF, delivered from a hydrogel directly to the brain can induce significant functional and structural protection from ischemic damage in a rat model of stroke.
76: Cancer investigation, 2010 May, 120(5)
Vascular endothelial growth factor (VEGF) serum levels are associated with survival in early stages of lung cancer patients.
[Abstract]AIM: Evaluate the serum vascular endothelial growth factor (VEGF) levels in the prognosis of lung cancer patients. METHODS: Fifty-four serum samples were analyzed for VEGF concentrations (79.3% nonsmall cell lung cancer (NSCLC) and 20.7% small cell lung cancer). RESULTS: Patients with serum VEGF-A levels higher than the mean of the patients studied (434.93 pg/mL) presented a shorter median survival time than those with lower levels (p =.04), as in patients with NSCLC tumors (p =.04) and in those with stages I-II (p <.05), and high serum VEGF-A levels. CONCLUSION: Elevated VEGF serum levels have a negative prognostic impact on survival in NSCLC and early stages of lung cancer patients.
77: Molecular pharmacology, 2010 Apr 20, 18(4)
The Farnesyltransferase Inhibitor LB42708 Suppresses VEGF-induced Angiogenesis by Inhibiting Ras-dependent MAPK and PI3K/Akt Signal Pathways.
[Abstract]Farnesyltransferase (FTase) inhibitors induce growth arrest and apoptosis in various human cancer cells by inhibiting the post-translational activation of Ras. FTase inhibitors also function to suppress the release of vascular endothelial growth factor (VEGF) from tumor cells by inhibiting Ras activation; however, the effects of FTase inhibitors on VEGF-induced angiogenesis in endothelial cells have not been studied. We have investigated the anti-angiogenic effect and molecular mechanism of LB42708, a selective non-peptidic FTase inhibitor, using in vitro and in vivo assay systems. LB42708 inhibited VEGF-induced Ras activation and subsequently suppressed angiogenesis in vitro and in vivo by blocking the MEK/ERK/p38 MAPK and phosphatidylinositol 3-kinase (PI3K)/Akt/eNOS pathways in endothelial cells, without altering FAK/Src activation. In addition, this inhibitor suppressed VEGF-induced endothelial cell cycle progression at the G(1) phase by suppressing cyclin D1 expression and Rb phosphorylation as well as upregulating the cyclin-dependent kinase inhibitors p21 and p27. Knockdown of Ras by siRNA revealed similar inhibitory effects on VEGF-induced angiogenic signal events as compared to LB42708. Moreover, the inhibitory effects of LB42708 were significantly higher than those of SCH66336, a well-known FTase inhibitor. LB42708 suppressed tumor growth and tumor angiogenesis in both xenograft tumor models of Ras-mutated HCT116 cells and its wild-type Caco-2 cells, indicating its potential application in the treatment for both Ras-mutated and wild type tumors. These data indicate that the anti-tumor effect of LB42708 can be associated with direct inhibition of VEGF-induced tumor angiogenesis by blocking Ras-dependent MAPK and PI3K/Akt signal pathways in tumor-associated endothelial cells.
78: Blood, 2010 Apr 19, 28(19)
Tissue macrophages act as cellular chaperones for vascular anastomosis downstream of VEGF-mediated endothelial tip cell induction.
[Abstract]Blood vessel networks expand in a two-step process that begins with vessel sprouting and is followed by vessel anastomosis. Vessel sprouting is induced by chemotactic gradients of the vascular endothelial growth factor VEGF, which stimulates tip cell protrusion. Yet, it is not known which factors promote the fusion of neighbouring tip cells to add new circuits to the existing vessel network. By combining the analysis of mouse mutants defective in macrophage development or VEGF signalling with live imaging in zebrafish, we now show that macrophages promote tip cell fusion downstream of VEGF-mediated tip cell induction. Macrophages therefore play a hitherto unidentified and unexpected role as vascular fusion cells. Moreover, we show that there are striking molecular similarities between the pro-angiogenic tissue macrophages essential for vascular development and those that promote the angiogenic switch in cancer, including the expression of the cell surface proteins TIE2 and NRP1. Our findings suggest that tissue macrophages are a target for anti-angiogenic therapies, but that they could equally well be exploited to stimulate tissue vascularisation in ischemic disease.
79: Free radical biology & medicine, 2010 Apr 16, 474(1)
NADPH oxidase 4 mediates reactive oxygen species induction of CD146 dimerization in VEGF signal transduction.
[Abstract]CD146 dimerization plays an important role in tumor-induced angiogenesis. Stimulation of target cells with vascular endothelial growth factor (VEGF), a major angiogenic factor produced by tumor cells, elicits a burst of reactive oxygen species (ROS) that enhances angiogenesis. However, the molecular mechanism coupling CD146 dimerization with the VEGF-related oxidant-generating apparatus has not been elucidated. Here, we show that CD146 dimerization is induced by VEGF and is significantly diminished by pretreatment with diphenyliodonium (DPI), an inhibitor of NADPH oxidase, suggesting a potential role of NADPH oxidase (NOX) in VEGF-induced CD146 dimerization. Importantly, we found that overexpression of NADPH oxidase 4 (NOX4), which is the predominant NOX expressed in endothelial cells, significantly enhances VEGF-induced ROS generation and CD146 dimerization. By contrast, these VEGF effects were dramatically attenuated after transfection with siRNA to reduce NOX4 expression. Furthermore, expression of Rac1 N17, a dominant negative mutant of Rac1, a member of the Rho family of small GTPases, suppressed VEGF-induced ROS generation and CD146 dimerization. These studies show for the first time that VEGF alteration of CD146 dimerization is mediated via a NOX4-dependent pathway and provide novel insights into the significant role of NOX in redox-regulation of the dimerization of cell adhesion molecules.
80: Experimental eye research, 2010 Apr 13, 17(4)
Genetic Deletion of COX-2 Diminishes VEGF Production in Mouse Retinal M¨¹ller Cells.
[Abstract]High Gleason grade prostate carcinomas are aggressive, poorly differentiated tumors that exhibit diminished estrogen receptor beta (ERbeta) expression. We report that a key function of ERbeta and its specific ligand 5alpha-androstane-3beta,17beta-diol (3beta-adiol) is to maintain an epithelial phenotype and repress mesenchymal characteristics in prostate carcinoma. Stimuli (TGF-beta and hypoxia) that induce an epithelial-mesenchymal transition (EMT) diminish ERbeta expression, and loss of ERbeta is sufficient to promote an EMT. The mechanism involves ERbeta-mediated destabilization of HIF-1alpha and transcriptional repression of VEGF-A. The VEGF-A receptor neuropilin-1 drives the EMT by promoting Snail1 nuclear localization. Importantly, this mechanism is manifested in high Gleason grade cancers, which exhibit significantly more HIF-1alpha and VEGF expression, and Snail1 nuclear localization compared to low Gleason grade cancers.
81: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association, 2010 Apr 15, 126(8)
Autocrine VEGF-VEGF-R loop on podocytes during glomerulonephritis in humans.
[Abstract]BACKGROUND: Vascular endothelial growth factor (VEGF) is the most important and tightly regulated angiogenic cytokine in the kidney. Its activity is critical for capillary/glomerular preservation and repair, and recent studies have also demonstrated its relevance for the preservation of podocytes. METHODS: The present study investigated a large number (n = 153) of renal biopsies from patients with glomerulonephritis (GN) and evaluated the expression and activity of the glomerular VEGF system [VEGF, VEGF-R1, VEGF-R2 and biologically active VEGF as identified by VEGF-VEGF receptor complexes (VEGF-VEGF-R)] in parallel with markers of renal function, injury and repair. RESULTS: Whereas glomerular VEGF expression was clearly elevated, VEGF-R expression levels were widely unchanged. In parallel to the overall VEGF expression, the biological activity of VEGF on its receptors was uniformly significantly enhanced. Interestingly, the expression pattern of VEGF-R1 and VEGF-R2 significantly changed during GN where a very prominent podocytic pattern appeared, which was also detected for receptor-bound VEGF. VEGF expression and activity could be linked with indicators of renal injury such as glomerular proliferation and creatinine, respectively. CONCLUSIONS: This study shows, for the first time, increased podocytic VEGF-VEGF-R binding during human GN, suggesting not only the existence of a glomerular paracrine proangiogenic, but also an autocrine role of the VEGF-VEGF-R system in diseased podocytes.
82: Investigative ophthalmology & visual science, 2010 Apr 14, 24(7)
Relative contribution of VEGF and TNF-alpha in the cynomolgus laser-induced CNV model: Comparing efficacy of bevacizumab, adalimumab and ESBA105.
[Abstract]PURPOSE. To compare the relative contribution of VEGF and TNF-alpha in the development of laser-induced choroidal neovascularization (CNV) in monkeys and to exploit the feasibility of topical use of suitable antibody fragments for prevention of experimental CNV. METHODS. In order to induce experimental CNV, small high energy laser spots were used to treat several areas of the macula in the retinas of cynomolgus monkeys according to previously published protocols. For prevention of pathology, bevacizumab/Avastin(R) (a potent VEGF inhibitor), adalimumab/Humira(R) or ESBA105 (potent TNF-alpha inhibitors) were given by intravitreal injection one week prior to as well as one and three weeks after laser treatment. ESBA105 was also applied topically in a separate group. Control animals were either treated with intravitreal or topical saline. Eyes were monitored by ophthalmic examination, color photographs, and fluorescein angiography. RESULTS. Inhibition of VEGF by bevacizumab completely blocked formation of CNV. Both TNF-alpha inhibitors also significantly reduced laser-induced CNV pathology following intravitreal administration. Most importantly, topical use of the anti-TNF-alpha single-chain antibody fragment ESBA105 also reduced formation of CNV. CONCLUSIONS. TNF-alpha contributes to laser-induced CNV formation and its inhibition can be a new therapeutic target for CNV Our data suggest TNF-alpha as another therapeutic target for the prevention and treatment of CNV and add to the emerging clinical data suggesting therapeutic value of TNF-alpha inhibitors in age-related macular degeneration (AMD). Further, our study shows that topical therapy with suitable antibody fragments has the potential of being introduced to retinal disease treatment regimens.
83: BMC cancer, 2010 Apr 13, 10(1)
TIMP-1 and VEGF-165 serum concentration during first-line therapy of ovarian cancer patients.
[Abstract]ABSTRACT: BACKGROUND: Angiogenesis appears to play an important role in ovarian cancer. Vascular endothelial growth factor (VEGF) has recently been implicated as a therapeutic target in ovarian cancer. The tissue inhibitor of metalloproteinase 1 (TIMP-1) is involved in tissue invasion and angiogenesis. The application of serum TIMP-1 and VEGF to monitor primary therapy and predict clinical outcome of patients with ovarian cancer is unclear. METHODS: Patients with epithelial ovarian cancer who presented for primary surgery were included in this study. A total of 148 serum samples from 37 patients were analyzed. Samples were prospectively collected at 4 predefined time points: 1. before radical debulking surgery, 2. after surgery and before platinum/taxane based chemotherapy, 3. during chemotherapy, 4. after chemotherapy. Serum VEGF-165 and TIMP-1 as well as CA-125 were quantified by ELISA or ECLIA and correlation with response and long-term clinical outcome was analyzed. RESULTS: Serum levels of all markers changed substantially during first-line therapy. High CA-125 (p=0.002), TIMP-1 (p=0.007) and VEGF-165 (p=0.02) after chemotherapy were associated with reduced overall survival. In addition, elevated CA-125 (p<0.001) and VEGF-165 (p=0.006) at this time point predicted poor progression-free survival. TIMP-1 and VEGF-165 were closely correlated at all time-points during therapy. CONCLUSIONS: TIMP-1 and VEGF serum levels changed significantly during first-line therapy of ovarian cancer patients and predicted prognosis. These findings support the role of angiogenesis in ovarian cancer progression and the use of antiangiogenic therapy.
84: Cancer cell, 2010 Apr 13, 17(4)
ERbeta impedes prostate cancer EMT by destabilizing HIF-1alpha and inhibiting VEGF-mediated snail nuclear localization: implications for Gleason grading.
[Abstract]High Gleason grade prostate carcinomas are aggressive, poorly differentiated tumors that exhibit diminished estrogen receptor beta (ERbeta) expression. We report that a key function of ERbeta and its specific ligand 5alpha-androstane-3beta,17beta-diol (3beta-adiol) is to maintain an epithelial phenotype and repress mesenchymal characteristics in prostate carcinoma. Stimuli (TGF-beta and hypoxia) that induce an epithelial-mesenchymal transition (EMT) diminish ERbeta expression, and loss of ERbeta is sufficient to promote an EMT. The mechanism involves ERbeta-mediated destabilization of HIF-1alpha and transcriptional repression of VEGF-A. The VEGF-A receptor neuropilin-1 drives the EMT by promoting Snail1 nuclear localization. Importantly, this mechanism is manifested in high Gleason grade cancers, which exhibit significantly more HIF-1alpha and VEGF expression, and Snail1 nuclear localization compared to low Gleason grade cancers.
85: American journal of physiology. Heart and circulatory physiology, 2010 Jun, 298(6)
VEGF and soluble VEGF receptor-1 (sFlt-1) distributions in peripheral arterial disease: an in silico model.
[Abstract]Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis, the growth of new capillaries from existing microvasculature. In peripheral arterial disease (PAD), lower extremity muscle ischemia develops downstream of atherosclerotic obstruction. A working hypothesis proposed that the maladaptive overexpression of soluble VEGF receptor 1 (sVEGFR1) in ischemic muscle tissues, and its subsequent antagonism of VEGF bioactivity, may contribute to the deficient angiogenic response in PAD, as well as the limited success of therapeutic angiogenesis strategies where exogenous VEGF genes/proteins are delivered. The objectives of this study were to develop a computational framework for simulating the systemic distributions of VEGF and sVEGFR1 (e.g., intramuscular vs. circulating, free vs. complexed) as observed in human PAD patients and to serve as a platform for the systematic optimization of diagnostic tools and therapeutic strategies. A three-compartment model was constructed, dividing the human body into the ischemic calf muscle, blood, and the rest of the body, connected through macromolecular biotransport processes. Detailed molecular interactions between VEGF, sVEGFR1, endothelial surface receptors (VEGFR1, VEGFR2, NRP1), and interstitial matrix sites were modeled. Our simulation results did not support a simultaneous decrease in plasma sVEGFR1 during PAD-associated elevations in plasma VEGF reported in literature. Furthermore, despite the overexpression in sVEGFR1, our PAD control demonstrated increased proangiogenic signaling complex formation, relative to our previous healthy control, due to sizeable upregulations in VEGFR2 and VEGF expression, thus leaving open the possibility that impaired angiogenesis in PAD may be rooted in signaling pathway disruptions downstream of ligand-receptor binding.
86: Biochemical pharmacology, 2010 Sep 1, 80(5)
Building on a foundation of VEGF and mTOR targeted agents in renal cell carcinoma.
[Abstract]Since late 2005 six new drugs have been approved by the Food and Drug Administration (FDA) for the treatment of metastatic renal cell carcinoma (RCC). However, the similarity of these agents with regard to mechanism of action makes it unclear if each agent has unique clinical utility. This flurry of drug development activity stems from the understanding of the central role that loss of von Hippel Lindau (VHL) gene function plays in the pathophysiology of clear cell RCC. The first agent to establish the therapeutic value of targeting the downstream consequences of VHL loss of function was a vascular endothelial growth factor (VEGF) directed monoclonal antibody, bevacizumab. Following the initial observations with bevacizumab, three VEGF receptor (VEGFR) tyrosine kinase inhibitors, with varied spectra beyond VEGFR, have been successfully developed clinically. Unanticipated clinical activity was observed with inhibitors of mTOR, a central component of the nutrient-sensing PI3 kinase pathway, in RCC. Subsequent work identified that mTOR also regulates the expression of hypoxia inducible factor (HIF), which is regulated by VHL outside of the setting of inactivating mutations or deletions. This appears to tie all of the six approved therapies to the direct consequences of loss of VHL function in clear cell RCC. It remains poorly understood to what extent these therapies differ from one another. Although the outcome of patients with metastatic RCC has been substantially altered with administration of the currently available therapies, the proper selection of currently available therapy, rational development of agents with novel mechanism of action and development of predictive biomarkers of response remains a challenge.
87: Journal of controlled release : official journal of the Controlled Release Society, 2010 Jul 14, 145(2)
VEGF-induced angiogenesis following localized delivery via injectable, low viscosity poly(trimethylene carbonate).
[Abstract]The purpose of this study was to examine the potential of low molecular weight poly(trimethylene carbonate) for localized vascular endothelial growth factor (VEGF) delivery. Poly(trimethylene carbonate) of various molecular weights was prepared by ring-opening polymerization initiated by 1-octanol. The resultant polymers were liquid at room temperature with low glass transition temperatures and viscosities at 37 degrees C that permitted their injection through an 18 (1/2) G 1.5' needle. Particles consisting of VEGF co-lyophilized with trehalose were mixed into the polymers and the rate of release of VEGF was assessed in vitro. With a 1% particle loading, VEGF was released from the polymer at a rate of 20ng/day over a period of 3weeks. This release behavior was independent of the molecular weight of polymer used. Increasing the VEGF content in the lyophilized particles did not increase the VEGF release rate, an effect attributed to the solubility limit of VEGF in the solution formed upon dissolution of the particles. The VEGF released retained its bioactivity at greater than 95% of that of as-lyophilized VEGF, as assessed using a human aortic endothelial cell proliferation assay. This high bioactivity was supported by in vivo release experiments, wherein VEGF containing polymer implants induced the generation of significantly greater numbers of blood vessels towards the polymer implant than controls. The blood vessels did not remain stable and were reduced in number by three weeks, due to the unsustained and low concentration of VEGF released. This formulation approach, of using a low viscosity polymer delivery vehicle, is potentially useful for localized delivery of acid-sensitive proteins, such as VEGF.
88: Biomaterials, 2010 Apr 6, 24(7)
Target specific tumor treatment by VEGF siRNA complexed with reducible polyethyleneimine-hyaluronic acid conjugate.
[Abstract]Target specific delivery of small interfering RNA (siRNA) has been regarded as one of the most important technologies for the development of siRNA therapeutics. In this work, non-toxic low molecular weight (MW) polyethyleneimine (PEI, 2000Da) was cross-linked with cystamine bisacrylamide (CBA) to prepare reducible PEI-SS in the body. Then, PEI-SS was conjugated with hyaluronic acid (HA) in the form of block-copolymer to enhance serum stability and facilitate target specific cellular uptake of siRNA by HA receptor mediated endocytosis. The cytotoxicity of (PEI-SS)-b-HA conjugate appeared to be negligible likely due to the degradation of PEI-SS to low MW PEI in the cytosol. Flow cytometric and confocal microscopic analyses confirmed the HA receptor mediated endocytosis of siRNA/(PEI-SS)-b-HA complex. The siRNA/(PEI-SS)-b-HA complex demonstrated an excellent in vitro gene silencing efficiency in the range of 50-80% reducing the mRNA expression level in the absence and presence of 50 vol% serum. Moreover, intra-tumoral injection of vascular endothelial growth factor (VEGF) siRNA/(PEI-SS)-b-HA complex resulted in dramatically inhibited tumor growth with reduced VEGF mRNA and VEGF levels in the tumors.
89: Molecular biology reports, 2010 Apr 8, 24(7)
The polymorphisms of bovine VEGF gene and their associations with growth traits in Chinese cattle.
[Abstract]PCR-SSCP and DNA sequencing methods were employed to screen the genetic variation of VEGF gene in 671 individuals belonging to three Chinese indigenous cattle breeds including Nanyang, Jiaxian Red and Qinchuan. Three haplotypes (A, B and C), four observed genotypes (AA, AB, BB and AC) and three new SNPs (6765T>C ss130456744, 6860A>G ss130456745, 6893T>C ss130456746) were detected. The analysis suggested that one SNP (ss130456744) in the bovine VEGF gene had significant effects on birth weight, body weight and heart girth at 6 months old in the Nanyang breed (P < 0.05). The results showed that the SNP (ss130456744) in intron 2 of the VEGF gene is associated with early development and growth of Chinese cattle. These findings raise hope that this polymorphism can be a molecular breeding marker in breeding strategies through marker assisted selection (MAS) in Chinese domestic cattle.
90: Clinical and experimental medicine, 2010 Apr 8, 24(7)
VEGF expression and angiogenesis in oral squamous cell carcinoma: an immunohistochemical and morphometric study.
[Abstract]Angiogenesis is involved in tumor progression of oral squamous cell carcinoma (OSCC). In this study, we have investigated by immunohistochemistry vascular endothelial growth factor (VEGF) expression in tumor cells and we have correlated VEGF expression to microvessel area, evaluated by using CD105 as a marker of endothelial cells, in bioptic specimens of 54 human OSCC. Results demonstrated that VEGF is highly expressed in OSCC tumor specimens when compared to pre-neoplastic and normal tissues, without differences between the edge and inside the tumor. Moreover, VEGF expression is reduced in poor differentiated OSCC tumors when compared to moderate and good differentiated forms, and tumor microvessel area is higher in tumors when compared to pre-neoplastic lesions and normal tissues. Finally, VEGF and CD105 may be considered as reliable markers of tumor angiogenesis and progression in OSCC, even if we did not demonstrate any correlation between VEGF expression, tumor microvascular area, clinical stage, and lymph node status.
91: The Journal of biological chemistry, 2010 Apr 6, 24(7)
Activation of AP-1 transcription factors differentiates FGF2 and VEGF regulation of endothelial nitric oxide synthase expression in placental artery endothelial cells.
[Abstract]Fibroblast growth factor 2 (FGF2), but not vascular endothelial growth factor (VEGF), stimulates sustained activation of extracellular signal-regulated protein kinase 2/1 (ERK2/1) for endothelial nitric oxide (NO) synthase (NOS3) protein expression in ovine fetoplacental artery endothelial cells (oFPA EC). We deciphered herein the downstream signaling of ERK2/1 responsible for NOS3 expression by FGF2 in oFPAEC. FGF2, but not VEGF, increased NOS3 mRNA levels without altering its degradation. FGF2, but not VEGF, trans-activated sheep NOS3 promoter and this was dependent on ERK2/1 activation. FGF2 did not trans-activate NOS3 promoters with deletions upstream the consensus AP-1 site (TGAGTC A, -678 to -685). Trans-activation of wild-type NOS3 promoter by FGF2 was significantly inhibited when either the AP-1 or the cAMP response element (CRE) - like sequence (TGCGTCA, -752 to -758) was mutated and was completely blocked when both were mutated. EMSA analyses showed that FGF2, but not VEGF, stimulated AP-1 and CRE DNA/protein complexes primarily composed of JunB and Fra1. ChIP assays confirmed JunB/Fra1 binding to NOS3 promoter AP-1 and CRE elements in intact cells. FGF2, but not VEGF, stimulated JunB and Fra1 expressions; all preceded NOS3 upregulation and were inhibited by PD98059. Downregulation of JunB or Fra-1, but not c-Jun, blocked FGF2 stimulation of NOS3 expression and NO production. AP-1 inhibition suppressed FGF2 stimulation of NOS3 expression in human umbilical vein EC and uterine artery EC. Thus, FGF2 induction of NOS3 expression is mainly mediated by AP-1 dependent transcription involving JunB and Fra1 upregulation via sustained ERK2/1 activation in endothelial cells.
92: Nature, 2010 Apr 4, 7(2)
MicroRNA-mediated integration of haemodynamics and Vegf signalling during angiogenesis.
[Abstract]Within the circulatory system, blood flow regulates vascular remodelling, stimulates blood stem cell formation, and has a role in the pathology of vascular disease. During vertebrate embryogenesis, vascular patterning is initially guided by conserved genetic pathways that act before circulation. Subsequently, endothelial cells must incorporate the mechanosensory stimulus of blood flow with these early signals to shape the embryonic vascular system. However, few details are known about how these signals are integrated during development. To investigate this process, we focused on the aortic arch (AA) blood vessels, which are known to remodel in response to blood flow. By using two-photon imaging of live zebrafish embryos, we observe that flow is essential for angiogenesis during AA development. We further find that angiogenic sprouting of AA vessels requires a flow-induced genetic pathway in which the mechano-sensitive zinc finger transcription factor klf2a induces expression of an endothelial-specific microRNA, mir-126, to activate Vegf signalling. Taken together, our work describes a novel genetic mechanism in which a microRNA facilitates integration of a physiological stimulus with growth factor signalling in endothelial cells to guide angiogenesis.
93: Cell biology international, 2010 Apr 1, 86(1)
Effect of Budesonide on TGF-beta1 enhanced VEGF Production by Lung Fibroblasts.
[Abstract]ABSTRACT Vascular endothelial growth factor (VEGF) is a potent proangiogenic cytokine, and vascular change is one of the characteristic features of airway remodeling. Since the glucocorticoids have shown anti-fibrosis properties, we sought to investigate whether budesonide, a widely used glucocorticoid in clinical practice, could attenuate transforming growth factor-beta1 (TGF-beta1)-induced VEGF production by human lung fibroblasts (HFL-1). HFL-1 fibroblasts were treated with various concentrations of budesonide (10-11 M, 10-9 M and 10-7 M) in the absence or presence of TGF-beta1. Post-culture media were collected for enzyme-linked immunosorbent assay (ELISA) of VEGF at indicated time. The cell lysates were subjected to western blotting analysis to test TGF-beta1/Smad and mitogen-activated protein (MAP) kinase signaling activation respectively. The results suggested that budesonide pretreatment reduced the significant increase of VEGF release induced by TGF-beta1 in HFL-1 fibroblasts in a dose-dependent manner and suppressed the increase of phospho-Smad3 and phosphor-extracellular signal-regulated kinase (ERK) protein levels. In conclusion, budesonide may reduce TGF-beta1-induced VEGF production in the lung, probably through the Smad/ERK signaling pathway, and, thus, may provide new sight into the molecular mechanism underlying glucocorticoids therapy for airway inflammatory diseases.
94: Cancer research, 2010 Apr 15, 70(8)
A therapeutic anti-VEGF antibody with increased potency independent of pharmacokinetic half-life.
[Abstract]Bevacizumab [Avastin; anti-vascular endothelial growth factor (VEGF) antibody] is an antiangiogenic IgG approved for treating patients with certain types of colon, breast, and lung cancer. In these indications, bevacizumab is administered every 2 to 3 weeks, prompting us to study ways to reduce the frequency of administration. Increasing affinity to neonatal Fc receptor (FcRn) may extend the pharmacokinetic half-life of an antibody, but the quantitative effect of FcRn affinity on clearance has not been clearly elucidated. To gain further insight into this relationship, we engineered a series of anti-VEGF antibody variants with minimal amino acid substitutions and showed a range of half-life improvements in primates. These results suggest that, if proven clinically safe and effective, a modified version of bevacizumab could potentially provide clinical benefit to patients on long-term anti-VEGF therapy through less-frequent dosing and improved compliance with drug therapy. Moreover, despite having half-life similar to that of wild-type in mice due to the species-specific FcRn binding effects, the variant T307Q/N434A exhibited superior in vivo potency in slowing the growth of certain human tumor lines in mouse xenograft models. These results further suggest that FcRn variants may achieve increased potency through unidentified mechanisms in addition to increased systemic exposure.
95: American journal of physiology. Lung cellular and molecular physiology, 2010 Jun, 298(6)
Defective angiogenesis in hypoplastic human fetal lungs correlates with nitric oxide synthase deficiency that occurs despite enhanced angiopoietin-2 and VEGF.
[Abstract]Lung hypoplasia (LH) is a life-threatening congenital abnormality with various causes. It involves vascular bed underdevelopment with abnormal arterial muscularization leading to pulmonary hypertension. Because underlying molecular changes are imperfectly known and sometimes controversial, we determined key factors of angiogenesis along intrauterine development, focusing at the angiopoietin (ANG)/Tie-2 system. Lung specimens from medical terminations of pregnancy (9-37 wk) were used, including LH due to congenital diaphragmatic hernia (CDH) or other causes, and nonpulmonary disease samples were used as controls. ELISA determination indicated little ANG-1 change during pregnancy and no effect of LH, whereas Tie-2 declined similarly between 9 and 37 wk in LH and controls. By contrast, ANG-2 markedly increased in LH from 24 wk, whereas it remained stable in controls. Because VEGF increased also, this was interpreted as an attempt to overcome vascular underdevelopment. Hypothesizing that its inefficiency might be due to impaired downstream mechanism, endothelial nitric oxide synthase (eNOS) was determined by semiquantitative Western blot and found to be reduced by approximately 75%, mostly in the instance of CDH. In conclusion, angiogenesis remains defective in hypoplastic lungs despite reactive enhancement of VEGF and ANG-2 production, which could be due, at least in part, to insufficient eNOS expression.
96: American journal of physiology. Heart and circulatory physiology, 2010 Jun, 298(6)
Intermittent pneumatic leg compressions acutely upregulate VEGF and MCP-1 expression in skeletal muscle.
[Abstract]Application of intermittent pneumatic compressions (IPC) is an extensively used therapeutic strategy in vascular medicine, but the mechanisms by which this method works are unclear. We tested the hypothesis that acute application (150 min) of cyclic leg compressions in a rat model signals upregulation of angiogenic factors in skeletal muscle. To explore the impact of different pressures and frequency of compressions, we divided rats into four groups as follows: 120 mmHg (2 s inflation/2 s deflation), 200 mmHg (2 s/2 s), 120 mmHg (4 s/16 s), and control (no intervention). Blood flow and leg oxygenation (study 1) and the mRNA expression of angiogenic mediators in the rat tibialis anterior muscle (study 2) were assessed after a single session of IPC. In all three groups exposed to the intervention, a modest hyperemia (approximately 37% above baseline) between compressions and a slight, nonsignificant increase in leg oxygen consumption (approximately 30%) were observed during IPC. Compared with values in the control group, vascular endothelial growth factor (VEGF) and monocyte chemotactic protein-1 (MCP-1) mRNA increased significantly (P < 0.05) only in rats exposed to the higher frequency of compressions (2 s on/2 s off). Endothelial nitric oxide synthase, matrix metalloproteinase-2, and hypoxia-inducible factor-1alpha mRNA did not change significantly following the intervention. These findings show that IPC application augments the mRNA content of key angiogenic factors in skeletal muscle. Importantly, the magnitude of changes in mRNA expression appeared to be modulated by the frequency of compressions such that a higher frequency (15 cycles/min) evoked more robust changes in VEGF and MCP-1 compared with a lower frequency (3 cycles/min).
97: Chemico-biological interactions, 2010 May 14, 185(3)
(-)-Epigallocatechin gallate inhibits growth and activation of the VEGF/VEGFR axis in human colorectal cancer cells.
[Abstract](-)-Epigallocatechin gallate (EGCG), the major constituent of green tea, inhibits the growth of colorectal cancer cells by inhibiting the activation of various types of receptor tyrosine kinases (RTKs). The RTK vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) axis induces tumor angiogenesis in colorectal cancer. This study examined the effects of EGCG on the activity of the VEGF/VEGFR axis and the expression of hypoxia-inducible factor (HIF)-1alpha, which promotes angiogenesis by elevating VEGF levels, in human colorectal cancer cells. Total and phosphorylated (i.e., activated) form (p-VEGFR-2) of VEGFR-2 proteins were overexpressed in a series of human colorectal cancer cell lines. Within 3h, EGCG caused a decrease in the expression of HIF-1alpha protein and VEGF, HIF-1alpha, insulin-like growth factor (IGF)-1, IGF-2, epidermal growth factor (EGF), and heregulin mRNAs in SW837 colorectal cancer cells, which express a constitutively activated VEGF/VEGFR axis. A decrease was also observed in the expression of VEGFR-2, p-VEGFR-2, p-IGF-1 receptor, p-ERK, and p-Akt proteins within 6h after EGCG treatment. Drinking EGCG significantly inhibited the growth of SW837 xenografts in nude mice, and this was associated with the inhibition of the expression and activation of VEGFR-2. The consumption of EGCG also inhibited activation of ERK and Akt, both of which are downstream signaling molecules of the VEGF/VEGFR axis, and reduced the expression of VEGF mRNA in xenografts. These findings suggest that EGCG may exert, at least in part, growth-inhibitory effects on colorectal cancer cells by inhibiting the activation of the VEGF/VEGFR axis through suppressing the expression of HIF-1alpha and several major growth factors. EGCG may therefore be useful in the chemoprevention and/or treatment of colorectal cancer.
98: Cancer biology & therapy, 2010 May 5, 9(9)
Immunomodulatory effects of VEGF: Clinical implications of VEGF-targeted therapy in human cancer.
[Abstract]
99: American journal of physiology. Gastrointestinal and liver physiology, 2010 Jun, 298(6)
Ephrin B2/EphB4 pathway in hepatic stellate cells stimulates Erk-dependent VEGF production and sinusoidal endothelial cell recruitment.
[Abstract]Chemotaxis signals between hepatic stellate cells (HSC) and sinusoidal endothelial cells (SEC) maintain hepatic vascular homeostasis and integrity and also regulate changes in sinusoidal structure in response to liver injury. Our prior studies have demonstrated that the bidirectional chemotactic signaling molecules EphrinB2 and EphB4 are expressed in HSC. The aim of our present study was to explore whether and how the EphrinB2/EphB4 system in HSC could promote SEC recruitment, which is essential for sinusoidal structure and remodeling. Stimulation of human HSC (hHSC) with chimeric agonists (2 microg/ml) of either EphrinB2 or EphB4 (EphrinB2 Fc or EphB4 Fc, respectively) significantly increased VEGF mRNA levels in hHSC as assessed by quantitative PCR, with respective small interfering RNAs for EphrinB2 and EphB4 inhibiting this increase (P < 0.05, n = 3). EphrinB2 agonist-induced increase in VEGF mRNA levels in hHSC was associated with increased phosphorylation of Erk and was significantly blocked by U0126 (20 microM), an inhibitor of MEK, which is a kinase upstream from Erk (P < 0.05, n = 3). The EphB4 agonist also significantly increased human VEGF promoter activity (P < 0.05, n = 3) as assessed by promoter reporter luciferase assay in transfected LX2-HSC. This was associated with upregulation of the vasculoprotective transcription factor, Kruppel-like factor 2 (KLF2). In Boyden chamber assays, conditioned media from hHSC stimulated with agonists of EphrinB2 or EphB4 increased SEC chemotaxis in a VEGF-dependent manner, compared with control groups that included basal media with agonists of EphrinB2, EphB4, or HSC-conditioned media from HSC in absence of agonist stimulation (P < 0.05, n = 3). EphB4 expression was detected in situ within liver sinusoidal vessels of rats after carbon tetrachloride-induced liver injury. In summary, activation of the EphrinB2/EphB4 signaling pathway in HSC promotes chemotaxis of SEC through a pathway that involves Erk, KLF2, and VEGF. These studies identify EphrinB2 or EphB4 as a key intermediary that links HSC signal transduction pathways with angiogenesis and sinusoidal remodeling.
100: Protein expression and purification, 2010 Aug, 72(2)
High-yield expression of human vascular endothelial growth factor VEGF(165) in Escherichia coli and purification for therapeutic applications.
[Abstract]Vascular endothelial growth factor (VEGF(165)) is a potent mitogen that induces angiogenesis and vascular permeability in vivo and has demonstrated potential in therapeutic applications for accelerating wound healing. An industrial production method that provides high yield as well as high purity, quality, and potency is needed. The process described in this report involves a bacterial expression system capable of producing approximately 9g of rhVEGF per liter of broth and a downstream purification process consisting of protein refolding and three chromatography steps prior to formulation of the drug substance. A high cell density (HCD) fed-batch fermentation process was used to produce rhVEGF in periplasmic inclusion bodies. The inclusion bodies are harvested from the cell lysate and subjected to a single-step protein solubilization and refolding operation to extract the rhVEGF for purification. Overall recovery yields observed during development, including refolding and chromatography, were 30+/-6%. Host cell impurities are consistently cleared below target levels at both laboratory and large-scale demonstrating process robustness. The structure of the refolded and purified rhVEGF was confirmed by mass spectrometry, N-terminal sequencing, and tryptic peptide mapping while product variants were analyzed by multiple HPLC assays. Biological activity was verified by the proliferation of human umbilical vein derived endothelial cells.
101: Journal of controlled release : official journal of the Controlled Release Society, 2010 May 10, 143(3)
Efficient delivery of VEGF via heparin-functionalized nanoparticle-fibrin complex.
[Abstract](-)-Epigallocatechin gallate (EGCG), the major constituent of green tea, inhibits the growth of colorectal cancer cells by inhibiting the activation of various types of receptor tyrosine kinases (RTKs). The RTK vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) axis induces tumor angiogenesis in colorectal cancer. This study examined the effects of EGCG on the activity of the VEGF/VEGFR axis and the expression of hypoxia-inducible factor (HIF)-1alpha, which promotes angiogenesis by elevating VEGF levels, in human colorectal cancer cells. Total and phosphorylated (i.e., activated) form (p-VEGFR-2) of VEGFR-2 proteins were overexpressed in a series of human colorectal cancer cell lines. Within 3h, EGCG caused a decrease in the expression of HIF-1alpha protein and VEGF, HIF-1alpha, insulin-like growth factor (IGF)-1, IGF-2, epidermal growth factor (EGF), and heregulin mRNAs in SW837 colorectal cancer cells, which express a constitutively activated VEGF/VEGFR axis. A decrease was also observed in the expression of VEGFR-2, p-VEGFR-2, p-IGF-1 receptor, p-ERK, and p-Akt proteins within 6h after EGCG treatment. Drinking EGCG significantly inhibited the growth of SW837 xenografts in nude mice, and this was associated with the inhibition of the expression and activation of VEGFR-2. The consumption of EGCG also inhibited activation of ERK and Akt, both of which are downstream signaling molecules of the VEGF/VEGFR axis, and reduced the expression of VEGF mRNA in xenografts. These findings suggest that EGCG may exert, at least in part, growth-inhibitory effects on colorectal cancer cells by inhibiting the activation of the VEGF/VEGFR axis through suppressing the expression of HIF-1alpha and several major growth factors. EGCG may therefore be useful in the chemoprevention and/or treatment of colorectal cancer.
102: Investigative ophthalmology & visual science, 2010 Aug, 51(8)
VEGF-A165b Is Cytoprotective and Antiangiogenic in the Retina.
[Abstract]Purpose. A number of key ocular diseases, including diabetic retinopathy and age-related macular degeneration, are characterized by localized areas of epithelial or endothelial damage, which can ultimately result in the growth of fragile new blood vessels, vitreous hemorrhage, and retinal detachment. VEGF-A(165), the principal neovascular agent in ocular angiogenic conditions, is formed by proximal splice site selection in its terminal exon 8. Alternative splicing of this exon results in an antiangiogenic isoform, VEGF-A(165)b, which is downregulated in diabetic retinopathy. Here the authors investigate the antiangiogenic activity of VEGF(165)b and its effect on retinal epithelial and endothelial cell survival. Methods. VEGF-A(165)b was injected intraocularly in a mouse model of retinal neovascularization (oxygen-induced retinopathy [OIR]). Cytotoxicity and cell migration assays were used to determine the effect of VEGF-A(165)b. Results. VEGF-A(165)b dose dependently inhibited angiogenesis (IC(50), 12.6 pg/eye) and retinal endothelial migration induced by 1 nM VEGF-A(165) across monolayers in culture (IC(50), 1 nM). However, it also acts as a survival factor for endothelial cells and retinal epithelial cells through VEGFR2 and can stimulate downstream signaling. Furthermore, VEGF-A(165)b injection, while inhibiting neovascular proliferation in the eye, reduced the ischemic insult in OIR (IC(50), 2.6 pg/eye). Unlike bevacizumab, pegaptanib did not interact directly with VEGF-A(165)b. Conclusions. The survival effects of VEGF-A(165)b signaling can protect the retina from ischemic damage. These results suggest that VEGF-A(165)b may be a useful therapeutic agent in ischemia-induced angiogenesis and a cytoprotective agent for retinal pigment epithelial cells.
103: Journal of cellular physiology, 2010 Jul, 224(1)
Prolonged blockade of VEGF receptors does not damage retinal photoreceptors or ganglion cells.
[Abstract]It has recently been reported that relatively short-term inhibition of vascular endothelial growth factor (VEGF) signaling can cause photoreceptor cell death, a potentially clinically important finding since VEGF blockade has become an important modality of treatment of ocular neovascularization and macular edema. However, in a set of studies in which we achieved extended and complete blockage of VEGF-induced vascular leakage through retinal expression of a VEGF binding protein, we did not observe any toxicity to retinal neurons. To follow-up on these apparently discrepant findings, we designed a set of experiments with the kinase inhibitor SU4312, which blocks phosphorylation of VEGF receptors, to look directly for evidence of VEGF inhibition-related retinal toxicity. Using transgenic mice with sustained expression of VEGF in photoreceptors, we determined that periocular injection of 3 microg of SU4312 every 5 days markedly suppressed subretinal neovascularization, indicating effective blockade of VEGF signaling. Wild-type mice given periocular injections of 5 microg of SU4312 every 5 days for up to 12 weeks showed normal scotopic and photopic electroretinograms (ERGs), no TUNEL stained cells in the retina, and no reduction in outer nuclear layer thickness. Incubation of cultured ganglion cells or retinal cultures containing photoreceptors with high doses of SU4312 did not reduce cell viability. These data suggest that blocking VEGF signaling in the retina for up to 12 weeks does not damage photoreceptors nor alter ERG function and should reassure patients who are receiving frequent injections of VEGF antagonists for choroidal and retinal vascular diseases.
104: American journal of physiology. Heart and circulatory physiology, 2010 Jun, 298(6)
Sustained VEGF delivery via PLGA nanoparticles promotes vascular growth.
[Abstract]Technologies to increase tissue vascularity are critically important to the fields of tissue engineering and cardiovascular medicine. Currently, limited technologies exist to encourage angiogenesis and arteriogenesis in a controlled manner. In the present study, we describe an injectable controlled release system consisting of VEGF encapsulated in poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs). The majority of VEGF was released gradually over 2-4 days from the NPs as determined by an ELISA release kinetics experiment. An in vitro aortic ring bioassay was used to verify the bioactivity of VEGF-NPs compared with empty NPs and no treatment. A mouse femoral artery ischemia model was then used to measure revascularization in VEGF-NP-treated limbs compared with limbs treated with naked VEGF and saline. 129/Sv mice were anesthetized with isoflurane, and a region of the common femoral artery and vein was ligated and excised. Mice were then injected with VEGF-NPs, naked VEGF, or saline. After 4 days, three-dimensional microcomputed tomography angiography was used to quantify vessel growth and morphology. Mice that received VEGF-NP treatment showed a significant increase in total vessel volume and vessel connectivity compared with 5 microg VEGF, 2.5 microg VEGF, and saline treatment (all P < 0.001). When the yield of the fabrication process was taken into account, VEGF-NPs were over an order of magnitude more potent than naked VEGF in increasing blood vessel volume. Differences between the VEGF-NP group and all other groups were even greater when only small-sized vessels under 300 mum diameter were analyzed. In conclusion, sustained VEGF delivery via PLGA NPs shows promise for encouraging blood vessel growth in tissue engineering and cardiovascular medicine applications.
105: American journal of physiology. Lung cellular and molecular physiology, 2010 Jun, 298(6)
VEGF in the lung: a role for novel isoforms.
[Abstract]Vascular endothelial cell growth factor (VEGF) is a potent mitogen and permogen that increases in the plasma and decreases in the alveolar space in respiratory diseases such as acute respiratory distress syndrome (ARDS). This observation has led to controversy over the role of this potent molecule in lung physiology and disease. We hypothesized that some of the VEGF previously detected in normal lung may be of the anti-angiogenic family (VEGF(xxx)b) with significant potential effects on VEGF bioactivity. VEGF(xxx)b protein expression was assessed by indirect immunohistochemistry in normal and ARDS tissue. Expression of VEGF(xxx)b was also detected by immunoblotting in normal lung tissue, primary human alveolar type II (ATII) cells, and bronchoalveolar lavage (BAL) fluid in normal subjects and by ELISA in normal, "at risk," and ARDS subjects. The effect of VEGF(165) and VEGF(165)b on both human primary endothelial cells and alveolar epithelial cell proliferation was assessed by [(3)H]thymidine uptake. We found that VEGF(165)b was widely expressed in normal healthy lung tissue but is reduced in ARDS lung. VEGF(121)b and VEGF(165)b were present in whole lung, BAL, and ATII lysate. The proliferative effect of VEGF(165) on both human primary endothelial cells and human alveolar epithelial cells was significantly inhibited by VEGF(165)b (P < 0.01). These data demonstrate that the novel VEGF(xxx)b family members are expressed in normal lung and are reduced in ARDS. A specific functional effect on primary human endothelial and alveolar epithelial cells has also been shown. These data suggest that the VEGF(xxx)b family may have a role in repair after lung injury.
106: Biomaterials, 2010 Jun, 31(17)
A high-performance VEGF aptamer functionalized polypyrrole nanotube biosensor.
[Abstract]In this study, we examined the in vitro electrochemical detection of Vascular Endothelial Growth Factor (VEGF) as cancer biomarker using p-type field-effect transistor (FET) biosensor. We demonstrated the high-performance FET sensor, which could detect ca. 400 fM of VEGF concentration, based on anti-VEGF RNA aptamer conjugated carboxylated polypyrrole nanotubes (CPNTs). The CPNTs used as high-performance transducers of this FET system were successfully fabricated by cylindrical micelle templates in a water-in-oil emulsion system. The functional carboxyl group (-COOH) was effectively incorporated into the polymer backbone during the polymerization by using pyrrole-3-carboxylic acid (P3CA) as a co-monomer. Two types of CPNTs (CPNT1: ca. 200 nm in diameter, CPNT2: ca. 120 nm in diameter) demonstrated the excellent conductivity performance in this FET system. Based on CPNTs conjugated with anti-VEGF RNA aptamer (CPNTs-aptamer), VEGF (target molecule) acts as the gate dielectrics of p-type FET sensor and specifically interacts with anti-VEGF aptamer attached to CPNT surfaces. Importantly, the VEGF detection limit of the FET sensor based on CPNT2-aptamer was found to be near 400 fM in real-time. Moreover, the CPNTs-aptamer FET sensors can be repeatedly used for various concentrations of the target molecule (VEGFs) through the washing and rinsing processes.
107: The EMBO journal, 2010 Apr 21, 29(8)
VEGF receptor 2/-3 heterodimers detected in situ by proximity ligation on angiogenic sprouts.
[Abstract]The vascular endothelial growth factors VEGFA and VEGFC are crucial regulators of vascular development. They exert their effects by dimerization and activation of the cognate receptors VEGFR2 and VEGFR3. Here, we have used in situ proximity ligation to detect receptor complexes in intact endothelial cells. We show that both VEGFA and VEGFC potently induce formation of VEGFR2/-3 heterodimers. Receptor heterodimers were found in both developing blood vessels and immature lymphatic structures in embryoid bodies. We present evidence that heterodimers frequently localize to tip cell filopodia. Interestingly, in the presence of VEGFC, heterodimers were enriched in the leading tip cells as compared with trailing stalk cells of growing sprouts. Neutralization of VEGFR3 to prevent heterodimer formation in response to VEGFA decreased the extent of angiogenic sprouting. We conclude that VEGFR2/-3 heterodimers on angiogenic sprouts induced by VEGFA or VEGFC may serve to positively regulate angiogenic sprouting.
108: Experimental neurology, 2010 Jun, 223(2)
Bio-printing of collagen and VEGF-releasing fibrin gel scaffolds for neural stem cell culture.
[Abstract]Time-released delivery of soluble growth factors (GFs) in engineered hydrogel tissue constructs promotes the migration and proliferation of embedded cells, which is an important factor for designing scaffolds that ultimately aim for neural tissue regeneration. We report a tissue engineering technique to print murine neural stem cells (C17.2), collagen hydrogel, and GF (vascular endothelial growth factor: VEGF)-releasing fibrin gel to construct an artificial neural tissue. We examined the morphological changes of the printed C17.2 cells embedded in the collagen and its migration toward the fibrin gel. The cells showed high viability (92.89+/-2.32%) after printing, which was equivalent to that of manually-plated cells. C17.2 cells printed within 1mm from the border of VEGF-releasing fibrin gel showed GF-induced changes in their morphology. The cells printed in this range also migrated toward the fibrin gel, with the total migration distance of 102.4+/-76.1microm over 3days. The cells in the control samples (fibrin without the VEGF or VEGF printed directly in collagen) neither proliferated nor migrated. The results demonstrated that bio-printing of VEGF-containing fibrin gel supported sustained release of the GF in the collagen scaffold. The presented method can be gainfully used in the development of three-dimensional (3D) artificial tissue assays and neural tissue regeneration applications.
109: Oncology reports, 2010 Apr, 23(4)
Dexamethasone treatment inhibits VEGF production via suppression of STAT3 in a head and neck cancer cell line.
[Abstract]Glucocorticoids (GCs) modulate the synthesis of many pro-inflammatory cytokines and influence multiple transduction pathways. GCs negatively or positively influence the transcription factors of their target genes. All of these transcription signals are closely connected to cancer survival or death. We investigated the action of dexamethasone (DEX) on head and neck cancer cell lines. When SNU-1041 and SNU-1076 were treated with DEX, the cell lines showed different patterns of responses. DEX inhibition of cell growth depended on concentration in SNU-1041, but not in SNU-1076. Furthermore, DEX suppressed vascular endothelial growth factor (VEGF) secretion from SNU-1041, but not from SNU-1076. We explored the mechanism that explains these distinct differences. After DEX treatment, the differences of NF-kappaB (p65), glucocorticoid receptor and p-AKT were not observed between the cell lines. However, phospho-signal transducer and activator of transcription 3 (STAT3) decreased in SNU-1041 only. Moreover, STAT3 inhibition using si-RNA suppressed VEGF secretion. When STAT3 was overexpressed after DEX treatment, the level of VEGF in the culture media was restored. Taken together, we suggest that p-STAT3 can be a mediating factor which regulates VEGF secretion in the DEX treatment. Because the relationship between the three molecules DEX, STAT3 and VEGF is scarcely known, our findings clarified one of the signaling pathways of DEX, which is often used in clinical conditions.
110: Oncology reports, 2010 Apr, 23(4)
Intratumoral injection of pEGFC1-IGFBP7 inhibits malignant melanoma growth in C57BL/6J mice by inducing apoptosis and down-regulating VEGF expression.
[Abstract]Malignant melanoma (MM) is a type of aggressive skin cancer, and the effective therapy for MM is highly desired. Recently, genome-wide RNA interference screening study revealed that loss of expression of insulin-like growth factor binding protein 7 (IGFBP-7) is a critical step in development of MM, and this secreted protein plays a central role in apoptosis of MM. Furthermore, a prostatic carcinoma cell line stably transfected with IGFBP-7 cDNA showed poor tumorigenicity. Thus, we supposed it to be an efficacious agent for inhibiting melanomas. In this study, we constructed pEGFC1-IGFBP7 to try to obtain high expression of IGFPB7 and then we demonstrated that this plasmid inhibited proliferation of B16-F10 melanoma cells efficiently in vitro. Moreover, intratumoral injection of pEGFC1-IGFBP7 inhibited MM growth in C57BL/6J mice. The inhibition of MM growth is due to apoptosis and reduced expression of VEGF induced by pEGFC1-IGFBP7. These results suggest a potential new clinical strategy for MM treatment.
111: Vaccine, 2010 Apr 26, 28(19)
Immunogenicity and some safety features of a VEGF-based cancer therapeutic vaccine in rats, rabbits and non-human primates.
[Abstract]We have developed a cancer vaccine candidate (hereafter denominated CIGB-247), based on recombinant modified human vascular endothelial growth factor (VEGF) as antigen, and the adjuvant VSSP (very small sized proteoliposomes of Neisseria meningitidis outer membrane). In mice, previous work of our group had shown that vaccination with CIGB-247 extended tumor-take time, slowed tumor growth, and increased animal survival. Immunization elicited anti-human and murine VEGF-neutralizing antibodies, and spleen cells of vaccinated mice are cytotoxic in vitro to tumor cells that produce VEGF. We have now tested the immunogenicity of CIGB-247 in Wistar rats, New Zealand White rabbits and the non-human primate Chlorocebus aethiops sabaeus. Using weekly, biweekly and biweekly plus montanide immunization schemes, all three species develop antigen-specific IgG antibodies that can block the interaction of VEGF and VEGF receptor 2 in an ELISA assay. Antibody titers decline after vaccination stops, but can be boosted with new immunizations. In monkeys, DTH and direct cell cytotoxicity experiments suggest that specific T-cell responses are elicited by vaccination. Immunization with CIGB-247 had no effect on normal behavior, hematology, blood biochemistry and histology of critical organs, in the tested animals. Skin deep wound healing was not affected in vaccinated rats and monkeys.
112: Rapid communications in mass spectrometry : RCM, 2010 Apr 15, 24(7)
Development and validation of a NANOGold immunoassay for the detection of vascular endothelial growth factor (VEGF) in human serum using inductively coupled plasma mass spectrometry.
[Abstract]This work aimed to develop and validate a NANOGold based assay, quantified using inductively coupled plasma mass spectrometry (ICP-MS), for the detection of human vascular endothelial growth factor (hVEGF) in serum. The initial assay range based on calibration standards was 62.5-2000 pg/mL with a detection limit of approximately 30 pg/mL. After validation using spiked validation controls, a quantification range between 175 and 1928 pg/mL was obtained. The inter-assay precision was between 2.3 and 18.9% with accuracy between -8.8 and -3.1%. Additional performance parameters, including dilutional linearity, matrix specificity and time-factored drift, were within +/-20%, as defined by the validation acceptance criteria for the validation of macromolecule immunoassays used within our clinical environment. Serum samples from healthy donors were analysed to determine the endogenous levels of VEGF present; these ranged from 164 to 580 pg/mL with a mean of 273 pg/mL. The intra- and inter-assay precision obtained from the healthy donor samples were 1.3-10.7% and 4.2-17.5%, respectively. This demonstration of a validated immunoassay opens further possibilities, utilising the simultaneous detection capabilities of ICP-MS for the detection of multiple analytes in a single validated immunoassay, for routine use within a clinical environment. Copyright (c) 2010 John Wiley & Sons, Ltd.
113: Neuroscience letters, 2010 Apr 19, 474(1)
Differential brain, but not serum VEGF levels in a genetic rat model of depression.
[Abstract]Compared to the classical monoamine hypotheses focus on neuroplasticity is a major new approach in studies of depression and antidepressants. Recent studies have demonstrated that vascular endothelial growth factor (VEGF) is regulated by antidepressant treatment in rodents. However, in depressive patients no significant changes were found in the serum VEGF levels compared to control subjects. To our knowledge, brain and serum VEGF levels have never been reported in parallel for any psychiatric disease model. That prompted us to examine the levels of VEGF in serum, hippocampus, frontal cortex, corpus striatum, and hypothalamus in male Flinders Sensitive Line (FSL) and Flinders Resistant Line (FRL), a genetic rat model of depression. The VEGF levels were identical in the FSL and the FRL rats in serum, corpus striatum, and hypothalamus. In hippocampus and frontal cortex, the VEGF levels were significantly decreased in the FSL rats compared to the FRL rats. The results may add to the hypothesis that altered expression of growth factors/neurotrophic factors are related to the pathophysiology of depression.
114: mAbs, 2010 Mar 28, 2(2)
The VEGF family in cancer and antibody-based strategies for their inhibition.
[Abstract]Angiogenesis is required in normal physiological processes, but is also involved in tumor growth, progression and metastasis. Vascular endothelial growth factor (VEGF), a primary mediator of angiogenesis in normal physiology and in disease, and other VEGF family members and their receptors provide targets that have been explored extensively for cancer therapy. Small molecule inhibitors and antibody/protein-based strategies that target the VEGF pathway have been studied in multiple types of cancer. This review will focus on VEGF pathway targeting antibodies that are currently being evaluated in pre-clinical and clinical studies.
115: Arteriosclerosis, thrombosis, and vascular biology, 2010 May, 30(5)
Oxidized phospholipids regulate expression of ATF4 and VEGF in endothelial cells via NRF2-dependent mechanism: novel point of convergence between electrophilic and unfolded protein stress pathways.
[Abstract]OBJECTIVE: The ATF4 arm of the unfolded protein response is increasingly recognized for its relevance to pathology, and in particular to angiogenic reactions. Oxidized phospholipids (OxPLs), known to accumulate in atherosclerotic vessels, were shown to upregulate vascular endothelial growth factor (VEGF) and induce angiogenesis via an ATF4-dependent mechanism. In this study, we analyzed the mechanism of ATF4 upregulation by OxPLs and more specifically the involvement of NRF2, the major transcriptional mediator of electrophilic stress response. METHODS AND RESULTS: Using reverse transcription/real-time polymerase chain reaction and Western blotting, we found that OxPLs induced upregulation of ATF4 mRNA and protein in several types of endothelial cells and that these effects were suppressed by short interfering RNA (siRNA) against NRF2. Electrophilic (iso)prostaglandins and oxidized low-density lipoprotein, similarly to OxPLs, elevated ATF4 mRNA levels in an NRF2-dependent mode. Chromatin immunoprecipitation revealed OxPL-dependent binding of NRF2 to a putative antioxidant response element site in the ATF4 gene promoter. Knockdown of NRF2 inhibited OxPL-induced elevation of VEGF mRNA and endothelial cell sprout formation. CONCLUSION: Our data characterize NRF2 as a positive regulator of ATF4 and identify a novel cross-talk between electrophilic and unfolded protein responses, which may play a role in stress-induced angiogenesis.
116: Histology and histopathology, 2010 Apr, 25(4)
TGF-beta1 and VEGF after fresh frozen bone allograft insertion in oral-maxillo-facial surgery.
[Abstract]Bone regeneration technique using allografts is widely used in oral surgery to repair alveolar defects and to increase alveolar volume for endosseous implant insertions. Bone allografts promote the reabsorption and neo-synthesis of bone tissue, which are regulated by numerous cytokines, proteins and growth factors. In this study, six patients with insufficient alveolar volume for endosseous implant insertions, were treated with bone regeneration technique using Fresh Frozen Bone (FFB) allografts collected from the femoral head or the hip. Samples of bone graft collected during graft insertion surgery and biopsies collected six months later during implantology were fixed, decalcified and analyzed histomorphologically and morphometrically by haematoxylin-eosin staining. In addition, TGF-beta1 and VEGF were analyzed by immunohistochemistry. The histological analysis of FFBs showed wide areas of calcified bone organized in osteons intermingled with areas of non-calcified matrix containing osteoblasts. However, the regenerated alveolar bone, collected six months after the graft insertion surgery, showed wide areas of non-calcified matrix. TGF-beta1 and VEGF were less expressed in FFB than in regenerated alveolar bone.
117: Experimental & molecular medicine, 2010 Apr 30, 42(4)
Ovarian cancer-derived lysophosphatidic acid stimulates secretion of VEGF and stromal cell-derived factor-1 alpha from human mesenchymal stem cells.
[Abstract]Lysophosphatidic acid (LPA) stimulates growth and invasion of ovarian cancer cells and tumor angiogenesis. Cancer-derived LPA induces differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs) to alpha-smooth muscle actin (alpha-SMA)-positive cancer-associated fibroblasts. Presently, we explored whether cancer-derived LPA regulates secretion of pro-angiogenic factors from hASCs. Conditioned medium (CM) from the OVCAR-3 and SKOV3 ovarian cancer cell lines stimulated secretion angiogenic factors such as stromal-derived factor-1 alpha (SDF-1 alpha) and VEGF from hASCs. Pretreatment with the LPA receptor inhibitor Ki16425 or short hairpin RNA lentiviral silencing of the LPA((1)) receptor abrogated the cancer CM-stimulated expression of alpha-SMA, SDF-1, and VEGF from hASCs. LPA induced expression of myocardin and myocardin-related transcription factor-A, transcription factors involved in smooth muscle differentiation, in hASCs. siRNA-mediated depletion of endogenous myocardin and MRTF-A abrogated the expression of alpha-SMA, but not SDF-1 and VEGF. LPA activated RhoA in hASCs and pretreatment with the Rho kinase inhibitor Y27632 completely abrogated the LPA-induced expression of alpha-SMA, SDF-1, and VEGF in hASCs. Moreover, LPA-induced alpha-SMA expression was abrogated by treatment with the ERK inhibitor U0126 or the phosphoinositide-3-kinase inhibitor LY294002, but not the PLC inhibitor U73122. LPA-induced VEGF secretion was inhibited by LY294002, whereas LPA-induced SDF-1 secretion was markedly attenuated by U0126, U73122, and LY294002. These results suggest that cancer-secreted LPA induces differentiation of hASCs to cancer-associated fibroblasts through multiple signaling pathways involving Rho kinase, ERK, PLC, and phosphoinositide-3-kinase.
118: International journal of gynaecology and obstetrics: the official organ of the International Federation of Gynaecology and Obstetrics, 2010 May, 109(2)
Peritoneal VEGF burden as a predictor of cytoreductive surgery outcome in women with epithelial ovarian cancer.
[Abstract]OBJECTIVE: To determine whether peripheral plasma concentration, peritoneal fluid concentration, and/or peritoneal vascular endothelial growth factor (VEGF) burden can predict the possibility of optimal cytoreduction in women with epithelial ovarian carcinoma (EOC); and if so, to determine cutoff values below which optimal cytoreduction is likely to occur. METHODS: We measured plasma VEGF concentration, peritoneal VEGF concentration, and VEGF burden in 46 women undergoing cytoreductive surgery. Univariate analysis, bivariate analysis, correlation tests, and stepwise regression were performed with cytoreduction as the outcome. RESULTS: The VEGF burden best predicted the outcome. The area under the curve was 0.84 and the log-transformed cutoff value was 15.52 log pg. Overall, the chance of optimal cytoreduction was 11 times greater when the VEGF burden was less than 15.52 log pg. For women with advanced disease, the chance was 6 times greater below this value. CONCLUSION: The VEGF burden may quantify tumor activity, and it could be used when selecting patients likely to benefit from induction chemotherapy before undergoing cytoreductive surgery.
119: Growth factors (Chur, Switzerland), 2010 Jun, 28(3)
ZFP36L1 is regulated by growth factors and cytokines in keratinocytes and influences their VEGF production.
[Abstract]Keratinocyte-derived growth factors and cytokines play an important role in epidermal homeostasis and particularly in cutaneous wound repair. Thus, we analyzed a potential role of the ZFP36/tristetraprolin family of zinc finger proteins, which are targets of these factors, but also regulate their production, in keratinocytes. We show that expression of ZFP36, ZFP36L1, and ZFP36L2 is induced by a broad variety of growth factors and cytokines, and by scratch wounding. Since ZFP36L1 is a modulator of vascular endothelium growth factor (VEGF) mRNA stability, we subsequently used siRNA technology to inhibit ZFP36L1 gene expression. Notably, this treatment resulted in prolonged maintenance of elevated VEGF levels in HaCaT keratinocytes upon epidermal growth factor stimulation of these cells. Taken together, our results suggest an important role of ZFP36L1 in wound healing.
120: Neuro-oncology, 2010 Feb 14, 316(4)
Bevacizumab salvage therapy following progression in high-grade glioma patients treated with VEGF receptor tyrosine kinase inhibitors.
[Abstract]Agents targeting the vascular endothelial growth factor (VEGF) pathway are being used with increasing frequency in patients with recurrent high-grade glioma. The effect of more than one antiangiogenic therapy given in succession has not been established. We reviewed the efficacy of bevacizumab, a VEGF-A monoclonal antibody, in patients who progressed following prior therapy with VEGF receptor tyrosine kinase inhibitors (R-TKi). Seventy-three patients with recurrent high-grade gliomas received VEGF R-TKi (cediranib, sorafenib, pazopanib, or sunitinib) as part of phase I or II clinical trials. Twenty-four of these patients with glioblastoma progressed and received bevacizumab-containing regimens immediately after R-TKi. Those who stopped R-TKi therapy for reasons other than disease progression, or received a treatment that did not include bevacizumab, were excluded from the analysis. The efficacy of bevacizumab-containing regimens in these 24 patients was evaluated. During R-TKi therapy, 6 of 24 patients (25%) had a partial response (PR) to treatment. The 6-month progression-free survival (APF6) was 16.7% and median time-to-progression (TTP) was 14.3 weeks. Grade III/IV toxicities were seen in 13 of 24 patients (54%). Subsequently with bevacizumab salvage therapy, 5 of 24 patients (21%) had a PR, the APF6 was 12.5%, and the median TTP was 8 weeks. Five of 24 patients had grade III/IV toxicities (21%). The median overall survival (OS) from the start of R-TKi therapy was 9.2 months (range: 2.8-34.1+), whereas the median OS after bevacizumab was 5.2 months (range: 1.3-28.9+). Bevacizumab retains modest activity in high-grade glioma patients who progress on R-TKi. However, the APF6 of 12.5% in this cohort of patients indicates that durable tumor control is not achieved for most patients.
121: Investigational new drugs, 2010 Feb 12, 183(3)
The anti-proliferative side effects of AEE788, a tyrosine kinase inhibitor blocking both EGF- and VEGF-receptor, are liver-size-dependent after partial hepatectomy in rats.
[Abstract]Background Blocking of EGFR signaling by the tyrosine kinase inhibitor AEE788 was well tolerated and did not inhibit liver regeneration after standard 70% partial hepatectomy (PH) in a rat model, as demonstrated previously. However, serum levels of AEE788 at POW1 were 3-fold higher than in the non-resected control group. Therefore, we expanded theses studies to a model of extended 90%PH to investigate the role of liver size for the metabolism of AEE788 and its potential influence on side effects, liver regeneration and liver remodeling. Method Rats treated with 50 mg/kg AEE788 or solvent every other day orally were subjected to 90%PH. Animals were sacrificed at 1, 2, 7 and 28 days after PH. We measured plasma and liver levels of AEE788 and assessed anti-proliferative side effects, liver regeneration, and liver architecture. Result Liver regeneration and liver architecture were not impaired by AEE788 treatment after 90%PH. 90%PH caused a clinically relevant drug accumulation within 1 week of treatment (AEE788 serum and tissue levels: 90%PH*>70%PH*>normal control, *p < 0.05), suggesting a liver-size-dependent metabolism of the drug. Drug accumulation after 90%PH was associated with severe side effects (delayed body weight recovery, diarrhea, impaired hair growth) within 1 week of treatment. Conclusion Treatment with AEE788 could be a potential strategy for adjuvant treatment after oncological liver resection, as liver regeneration was not impaired. Our results suggest a liver-size-dependent metabolism of AEE788 leading to drug accumulation and subsequently to severe side effects. It calls for therapeutic drug monitoring in the early postoperative phase after extended resection.
122: Journal of immunotherapy (Hagerstown, Md. : 1997), 2010 Feb 6, 107(6)
Tumor Secretion of VEGF Induces Endothelial Cells to Suppress T cell Functions Through the Production of PGE2.
[Abstract]Endothelial cells are potent regulators of immune cell functions and have therefore been examined to determine their role in tumor-induced immune suppression. Previous studies by our laboratory showed that exposure to Lewis lung carcinoma (LLC)-secreted products induced endothelial cells to suppress T-cell functions in vitro. The current studies examined in vitro and in vivo the mechanism by which tumors induce the formation of suppressor endothelial cells and the means by which suppressor endothelial cells disrupt T-cell functions. In vitro studies demonstrated that inhibition of tumor-derived VEGF with neutralizing antibodies or treatment of endothelial cells with the VEGF receptor tyrosine kinase inhibitor, SU5416, prevented endothelial cells from being induced to suppress T-cell functions. Treatment of tumor-bearing mice with SU5416 blocked the development of endothelial cells that are suppressive to CD4 and CD8 T-cell functions. We next examined the role of suppressor endothelial cell-derived PGE2 in the inhibition of T-cell functions. Abrogation of endothelial cell PGE2 production in vitro with indomethacin prevented tumor-conditioned media from stimulating endothelial cell production of immune inhibitory activity toward T-cell functions. Similar treatment of endothelial cells from lungs of tumor-bearing mice blocked their capacity to produce T-cell-inhibitory mediators. These studies demonstrate that tumor-derived VEGF induces endothelial cells to upregulate production of PGE2 which, in turn, leads to suppression of T-cell functions.
123: Cancer research, 2010 Feb 9, 183(3)
A Placental Growth Factor Variant Unable to Recognize Vascular Endothelial Growth Factor (VEGF) Receptor-1 Inhibits VEGF-Dependent Tumor Angiogenesis via Heterodimerization.
[Abstract]Angiogenesis is one of the crucial events for cancer development and growth. Two members of the vascular endothelial growth factor (VEGF) family, VEGF-A and placental growth factor (PlGF), which are able to heterodimerize if coexpressed in the same cell, are both required for pathologic angiogenesis. We have generated a PlGF1 variant, named PlGF1-DE in which the residues Asp(72) and Glu(73) were substituted with Ala, which is unable to bind and activate VEGF receptor-1 but is still able to heterodimerize with VEGF. Here, we show that overexpression in tumor cells by adenoviral delivery or stable transfection of PlGF1-DE variant significantly reduces the production of VEGF homodimer via heterodimerization, determining a strong inhibition of xenograft tumor growth and neoangiogenesis, as well as significant reduction of vessel lumen and stabilization, and monocyte-macrophage infiltration. Conversely, the overexpression of PlGF1wt, also reducing the VEGF homodimer production comparably with PlGF1-DE variant through the generation of VEGF/PlGF heterodimer, does not inhibit tumor growth and vessel density compared with controls but induces increase of vessel lumen, vessel stabilization, and monocyte-macrophage infiltration. The property of PlGF and VEGF-A to generate heterodimer represents a successful strategy to inhibit VEGF-dependent angiogenesis. The PlGF1-DE variant, and not PlGF1wt as previously reported, acts as a "dominant negative" of VEGF and is a new candidate for antiangiogenic gene therapy in cancer treatment. Cancer Res; 70(5); OF1-10.
124: Proceedings of the National Academy of Sciences of the United States of America, 2010 Feb 9, 107(6)
Structural determinants of growth factor binding and specificity by VEGF receptor 2.
[Abstract]Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel formation through activation of three receptor tyrosine kinases, VEGFR-1, -2, and -3. The extracellular domain of VEGF receptors consists of seven immunoglobulin homology domains, which, upon ligand binding, promote receptor dimerization. Dimerization initiates transmembrane signaling, which activates the intracellular tyrosine kinase domain of the receptor. VEGF-C stimulates lymphangiogenesis and contributes to pathological angiogenesis via VEGFR-3. However, proteolytically processed VEGF-C also stimulates VEGFR-2, the predominant transducer of signals required for physiological and pathological angiogenesis. Here we present the crystal structure of VEGF-C bound to the VEGFR-2 high-affinity-binding site, which consists of immunoglobulin homology domains D2 and D3. This structure reveals a symmetrical 22 complex, in which left-handed twisted receptor domains wrap around the 2-fold axis of VEGF-C. In the VEGFs, receptor specificity is determined by an N-terminal alpha helix and three peptide loops. Our structure shows that two of these loops in VEGF-C bind to VEGFR-2 subdomains D2 and D3, while one interacts primarily with D3. Additionally, the N-terminal helix of VEGF-C interacts with D2, and the groove separating the two VEGF-C monomers binds to the D2/D3 linker. VEGF-C, unlike VEGF-A, does not bind VEGFR-1. We therefore created VEGFR-1/VEGFR-2 chimeric proteins to further study receptor specificity. This biochemical analysis, together with our structural data, defined VEGFR-2 residues critical for the binding of VEGF-A and VEGF-C. Our results provide significant insights into the structural features that determine the high affinity and specificity of VEGF/VEGFR interactions.
125: Stem cells (Dayton, Ohio), 2010 Feb 4, 9(3)
Hypoxia Influences the Vascular Expansion and Differentiation of Embryonic Stem Cell Cultures Through the Temporal Expression of VEGF- Receptors in an ARNT-Dependent Manner.
[Abstract]Adaptive responses to low oxygen (O(2)) tension (hypoxia) are mediated by the heterodimeric transcription factor Hypoxia Inducible Factor (HIF). When stabilized by hypoxia, bHLH-PAS alpha- and beta- (HIF-1beta or ARNT) HIF complex regulate the expression of multiple genes including vascular endothelial growth factor (VEGF). In order to address the mechanism(s) through which hypoxia contributes to blood vessel development, we utilized embryonic stem cell (ESC) differentiation cultures that develop into embryoid bodies (EBs) mimicking early embryonic development. Significantly, low O(2) levels promote vascular development and maturation in wild type (WT) ESC cultures measured by an increase in numbers of CD31(+) endothelial cells (ECs) and sprouting angiogenic EBs but refractory in Arnt(-/-) and Vegf(-/-) ESC cultures. Thus, we propose that hypoxia promotes the production of ECs and contributes to the development and maturation of vessels. Our findings further demonstrate that hypoxia alters the temporal expression of VEGF receptors Flk-1 (VEGFR-2) and the membrane and soluble forms of the antagonistic receptor Flt-1 (VEGFR-1). Moreover, these receptors are distinctly expressed in differentiating Arnt(-/-) and Vegf(-/-) EBs. These results support existing models wherein VEGF signaling is tightly regulated during specific biological events, but also provide important novel evidence that, in response to physiological hypoxia, HIF mediates a distinct stoichiometric pattern of VEGF receptors throughout EB differentiation analogous to the formation of vascular networks during embryogenesis.
126: Biochemical and biophysical research communications, 2010 Feb 1, 208(2)
Excess iodide decreases transcription of NIS and VEGF genes in rat FRTL-5 thyroid cells.
[Abstract]Although it is well known that an excess of iodide suppresses thyroid function and blood flow in vivo, the underlying molecular mechanisms are not fully known. The functional effect of iodide occurs at multiple steps, which include inhibition of sodium/iodide symporter (NIS) expression, transient block of organification, and inhibition of hormonal release. The vascular effect likely involves suppression of the vascular endothelial growth factor (VEGF) gene. In this report, we show that excess iodide coordinately suppresses the expression of the NIS and VEGF genes in FRTL-5 thyroid cells. We also demonstrate that the mechanism of iodide suppression of NIS gene expression is transcriptional, which is synergized by the addition of thyroglobulin. Based on the findings of reporter gene assays and electrophoretic gel mobility shift analysis, we also report two novel DNA binding proteins that responded specifically to iodide and modulated NIS promoter activity. The results suggest that excess iodide affects thyroid vascular function in addition to iodide uptake. This study provides additional insights into the mechanism of action of excess iodide on thyroid function.
127: Oncology, 2010 Feb 2, 77(6)
VEGF-Targeted Short Hairpin RNA Inhibits Intraperitoneal Ovarian Cancer Growth in Nude Mice.
[Abstract]Objective: In this study, we inhibited the expression of VEGF by short hairpin RNA (shRNA) in SKOV3 ovarian cancer cells in vitro and in vivo to explore the antitumor efficacy of shRNA in ovarian cancer cells. Methods: ShRNA targeting VEGF was cloned into pGenesil-2 plasmid vector and then transfected into SKOV3 ovarian cancer cells using liposome. Silencing of VEGF expression was measured by RT-PCR and ELISA assays. Furthermore, the growth inhibition capacity of shRNA on SKOV3 intraperitoneal ovarian carcinomatosis was tested in nude mice. Tumor weight was measured. Microvessel density, number of apoptotic cells and proliferation index in tumor tissues were assessed by CD31, TUNEL and PCNA immunostaining. Results: shRNA targeting VEGF significantly silenced VEGF expression in SKOV3 ovarian cancer cells, confirmed by RT-PCR and ELISA assays (p < 0.05). In vivo, the shRNA reduced tumor weight by approximately 60.3% compared with control groups (p < 0.05), accompanied with angiogenesis inhibition (p < 0.01) and apoptosis induction (p < 0.01). Conclusion: Our data indicated that shRNA-mediated silencing of VEGF might be a promising therapeutic strategy against ovarian cancer by reducing angiogenesis and inducing apoptosis.
128: Oncology reports, 2010 Mar, 23(3)
Expression of Efp, VEGF and bFGF in normal, hyperplastic and malignant endometrial tissue.
[Abstract]Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are two important angiogenic and prognostic factors for endometrial cancer. Estrogen is considered to be associated with increasing risk of endometrial adenocarcinoma. We investigated the expression of estrogen-responsive ring finger protein (Efp), VEGF and bFGF in endometrial cancer and correlated the results with clinicopathological features. We measured the expression of Efp, VEGF and bFGF in normal, hyperplastic and malignant endometrial tissues by quantitative RT-PCR, ELISA or Western blot analysis. The expression of Efp mRNA was significantly decreased in the groups of endometrial cancer (EC) in comparison with the groups of normal endometrium (NE) (P<0.05). Expression of VEGF mRNA in the groups of EC and atypical hyperplasia (AH) was significantly increased in comparison with the groups of NE (P<0.05). The expression of bFGF mRNA in groups of EC and AH was higher than that of NE groups (P<0.01 and P<0.05). There was positive relevance of bFGF expression and histologic grade and negative relevance of Efp expression and histologic grade. The expression of Efp, VEGF and bFGF protein was in agreement with the mRNA. The high expression of VEGF and bFGF indicate that both contribute in the angiogenesis of endometrial cancer. The regulation of Efp and bFGF expression during endometrial carcinogenesis suggests their potential utility as a prognostic biomarker.
129: Oncology reports, 2010 Mar, 23(3)
Constitutive NF-kappaB activity regulates the expression of VEGF and IL-8 and tumor angiogenesis of human glioblastoma.
[Abstract]Angiogenesis is a key pathologic feature of glioblastoma, which is the most common and most lethal primary brain tumor in adults. The degree of angiogenesis has been shown to be inversely related to patient survival. However, the molecular changes leading to angiogenesis in glioblastoma remain poorly understood. In the present study, we found a direct correlation between nuclear factor (NF)-kappaB activation and angiogenesis in glioblastomas. Blockade of NF-kappaB signaling significantly inhibited glioblastoma growth and angiogenesis in nude mice. These effects were consistent with significant inhibition of the expression of multiple angiogenic molecules, including vascular endothelial growth factor, and interleukin-8, in vitro and in vivo. Furthermore, blockade of NF-kappaB signaling also significantly inhibited the angiogenic potential of glioblastoma cells in vitro and angiogenesis of brain tumors in mouse xenograft models. Collectively, these results suggest that NF-kappaB activation plays a critical role in the growth and progression of glioblastoma and is a potential target for therapy for human glioblastoma.
130: Bone, 2010 Apr, 46(4)
Rho-kinase limits FGF-2-stimulated VEGF release in osteoblasts.
[Abstract]We previously reported that basic fibroblast growth factor (FGF-2) stimulates the release of vascular endothelial growth factor (VEGF) via p44/p42 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells and that FGF-2-activated p38 MAP kinase negatively regulates the VEGF release in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether Rho-kinase is involved in FGF-2-stimulated VEGF release in MC3T3-E1 cells. FGF-2 induced the phosphorylation of myosin phosphatase targeting subunit (MYPT-1), a substrate of Rho-kinase. Y27632, a specific inhibitor of Rho-kinase, which attenuated the MYPT-1 phosphorylation, significantly enhanced the FGF-2-stimulated VEGF release. Fasudil, another Rho-kinase inhibitor, also amplified the VEGF release. FGF-2 significantly stimulated VEGF accumulation and fasudil enhanced FGF-2-stimulated VEGF accumulation also in whole cell lysates. Neither Y27632 nor fasudil affected the phosphorylation levels of p44/p42 MAP kinase or p38 MAP kinase. Y27632 and fasudil markedly strengthened the FGF-2-induced phosphorylation of SAPK/JNK. Y27632 as well as fasudil enhanced FGF-2-stimulated VEGF release and Y27632 enhanced the FGF-2-induced phosphorylation levels of SAPK/JNK also in human osteoblasts. These results strongly suggest that Rho-kinase negatively regulates FGF-2-stimulated VEGF release in osteoblasts.
131: Journal of dental research, 2010 Jan 28, 115(4)
MAPK Signaling Is Required for LPS-induced VEGF in Pulp Stem Cells.
[Abstract]Caries-induced pulpitis is typically accompanied by an increase in dental pulp microvascular density. However, the mechanisms by which dental pulp cells recognize lipopolysaccharides (LPSs) remain unclear. We hypothesized that Porphyromonas endodontalis and Escherichia coli LPSs induce vascular endothelial growth factor (VEGF) expression in dental pulp stem cells (DPSC) and human dental pulp fibroblasts (HDPF) through mitogen-activated protein kinase (MAPK) signaling. ELISA, semi-quantitative RT-PCR, immunofluorescence, and Western blots were used. Here, we observed that LPSs induced VEGF expression in DPSC and HDPF cells, and both cell types express Toll-like receptor 4 (TLR4). Notably, LPS-induced VEGF is associated with phosphorylation of protein kinase C (PKC zeta) and extracellular signal-regulator kinase (ERK1/2) and is dependent upon MAPK activation. Analysis of these data, collectively, unveils a signaling pathway responsible for synthesis of VEGF by pulp cells and suggests a novel therapeutic target for the management of vascular responses in teeth with pulpitis.
132: The Journal of biological chemistry, 2010 Jan 28, 115(4)
Filamin B plays a key role in VEGF-induced endothelial cell motility through its interaction with Rac-1 and Vav-2.
[Abstract]Actin-binding proteins filamin A (FLNA) and B (FLNB) are expressed in endothelial cells and play an essential role during vascular development. In order to investigate their role in adult endothelial cell function, we initially confirmed their expression pattern in different adult mouse tissues and cultured cell lines and found that FLNB expression is concentrated mainly in endothelial cells while FLNA is more ubiquitously expressed. Functionally, siRNA-knockdown of endogenous FLNB in HUVEC inhibited Vascular Endothelial Growth Factor (VEGF)-induced in vitro angiogenesis by decreasing endothelial cell migration capacity, whereas FLNA ablation did not alter these parameters. Moreover, FLNB-depleted cells increased their substrate adhesion with more focal adhesions. The molecular mechanism underlying this effect implicates modulation of small GTP binding protein Rac-1 localization and activity, with altered activation of its downstream effectors p21 protein Cdc42/Rac-activated kinase (PAK)-4/5/6 and its activating guanine nucleotide exchange factor Vav-2. Moreover, our results suggest the existence of a signaling complex including FLNB, Rac-1 and Vav-2 under basal conditions that would further interact with VEGFR2 and integrin alphaVbeta5 after VEGF stimulation. In conclusion, our results reveal a crucial role for FLNB in endothelial cell migration and in the angiogenic process in adult endothelial cells.
133: Biomaterials, 2010 Apr, 31(11)
Dual delivery of VEGF and MCP-1 to support endothelial cell transplantation for therapeutic vascularization.
[Abstract]Transplantation of endothelial cells (EC) for therapeutic vascularization is a promising approach in tissue engineering but has yet to be proven effective in clinical trials. This cell-based therapy is hindered by significant apoptosis of EC upon transplantation as well as poor recruitment of host mural cells to stabilize nascent vessels. Here, we address these deficiencies by augmenting endothelial cell transplantation with dual delivery of vascular endothelial growth factor (VEGF) - to improve survival of transplanted EC - and monocyte chemotactic protein-1 (MCP-1) - to induce mural cell recruitment. We produced alginate microparticles that deliver VEGF and MCP-1 with distinct release kinetics and that can be integrated into a collagen/fibronectin (protein) gel construct for delivery of EC. Combined delivery of VEGF and MCP-1 increased functional vessel formation from transplanted EC and also led to a higher number of smooth muscle cell-invested vessels than did EC therapy alone. Despite the well-known role of MCP-1 in inflammation, these beneficial effects were accomplished without a long-term increase in monocyte/macrophage recruitment or a shift to a pro-inflammatory (M1) macrophage phenotype. Overall, these data suggest a potential benefit of combined delivery of MCP-1 and VEGF from EC-containing hydrogels as a strategy for therapeutic vascularization.
134: Molecular oncology, 2010 Apr, 4(2)
Targeting of VEGF-dependent transendothelial migration of cancer cells by bevacizumab.
[Abstract]Cancer progression is often associated with the formation of malignant effusions. Vascular endothelial growth factor (VEGF) is a major regulator of vascular permeability and has been implicated as mediator of tumor progression. We examined the production and secretion of VEGF(165) in various primary cancer cells derived from malignant effusions, and the role of exogenous VEGF(165) as a mediator of effusion formation. VEGF(165) was constantly secreted by all cultured tumor cells in an mTOR-dependent manner, as it was inhibited by the mTOR inhibitor rapamycin. Secreted VEGF(165) showed functional activity by inducing endothelial leakiness and tumor cell-transendothelial migration in vitro, effects which could be reverted by the anti-VEGF antibody bevacizumab. Thus, mTOR inhibitors as well as bevacizumab should be considered as potential agents in cancer patients suffering from malignant effusions.
135: The Journal of surgical research, 2010 Feb, 158(2)
Mesenchymal stem cells augment wound VEGF content, blood flow, and increase tensile strength.
[Abstract]PURPOSE: To determine whether long-term brimonidine (BRI) treatment prevents the hyperglycemia-induced increase in vitreoretinal vascular endothelial growth factor (VEGF) expression and breakdown of the blood-retinal barrier (BRB) in streptozotocin (STZ)-induced diabetic rats. METHODS: Brown Norway/Long-Evans rats were divided into three groups with similar distributions of blood glucose and body weight. Two groups received a single intravenous injection of STZ (65 mg/kg) and the remaining control group received vehicle. Drug treatment administered via miniosmotic pumps was initiated 1 or 6 weeks later. The STZ-induced diabetic rats were treated with BRI (1 mg/kg/d) or vehicle (VEH) and control nondiabetic rats were treated with VEH for 4 weeks. Vitreoretinal VEGF protein, vitreal glutamate, and BRB breakdown were then measured. RESULTS: At 5 weeks after STZ treatment, STZ-treated diabetic rats demonstrated significantly elevated vitreoretinal VEGF expression, vitreal glutamate concentrations, and BRB leakage compared with nondiabetic control rats. Chronic BRI treatment had no effect on vitreal glutamate concentrations in diabetic animals but significantly decreased vitreoretinal VEGF expression and BRB breakdown to levels similar to those observed in control rats. BRI also significantly reduced BRB breakdown in aged diabetic rats at 10 weeks after STZ treatment. CONCLUSIONS: BRI produced marked decreases in vitreoretinal VEGF and inhibition of BRB breakdown in diabetic rats. The mechanism for these effects may involve attenuation of retinal NMDA receptor activity by BRI. The results suggest that BRI would be useful for treatment of ocular diseases associated with BRB leakage, such as diabetic macular edema and retinopathy.
136: Growth factors (Chur, Switzerland), 2010 Jan 27, 115(4)
The skeletal muscle VEGF mRNA response to acute exercise in patients with chronic heart failure.
[Abstract]In seven patients with chronic heart failure (CHF) and six controls, we examined (a) resting and post-exercise muscle vascular endothelial growth factor (VEGF) mRNA levels and (b) their relationship with muscle structure and function. Muscle biopsies were taken after 30 min of single-leg knee-extensor exercise at 50% of maximum work rate (50% WR(max)) from both the exercised and rested legs. Muscle blood flow [Formula: see text] and O(2) uptake [Formula: see text] were measured during exercise. Resting VEGF mRNA levels were not different between patients and controls and both groups upregulated VEGF mRNA equally in response to acute exercise. Patients had lower [Formula: see text], [Formula: see text], and mitochondrial density but similar capillarity and fiber area. These findings reveal a normal basal level of muscle VEGF mRNA, its appropriate upregulation in response to acute exercise and, despite increased vascular resistance during exercise, a normal skeletal muscle vascular structure in patients with CHF.
137: The Journal of experimental medicine, 2010 Jan 18, 207(1)
Neuropilin-2 mediates VEGF-C-induced lymphatic sprouting together with VEGFR3.
[Abstract]Vascular sprouting is a key process-driving development of the vascular system. In this study, we show that neuropilin-2 (Nrp2), a transmembrane receptor for the lymphangiogenic vascular endothelial growth factor C (VEGF-C), plays an important role in lymphatic vessel sprouting. Blocking VEGF-C binding to Nrp2 using antibodies specifically inhibits sprouting of developing lymphatic endothelial tip cells in vivo. In vitro analyses show that Nrp2 modulates lymphatic endothelial tip cell extension and prevents tip cell stalling and retraction during vascular sprout formation. Genetic deletion of Nrp2 reproduces the sprouting defects seen after antibody treatment. To investigate whether this defect depends on Nrp2 interaction with VEGF receptor 2 (VEGFR2) and/or 3, we intercrossed heterozygous mice lacking one allele of these receptors. Double-heterozygous nrp2vegfr2 mice develop normally without detectable lymphatic sprouting defects. In contrast, double-heterozygote nrp2vegfr3 mice show a reduction of lymphatic vessel sprouting and decreased lymph vessel branching in adult organs. Thus, interaction between Nrp2 and VEGFR3 mediates proper lymphatic vessel sprouting in response to VEGF-C.
138: Human immunology, 2010 Jan 20, 29(2)
Synergistic effect and VEGF/HSP70-hom haplotype analysis: relationship to prostate cancer risk and clinical outcome.
[Abstract]Prostate cancer is a complex disorder resulting from the combined effects of multiple environmental and genetic factors. Our previous single locus analysis showed that VEGF and HSP70-hom polymorphisms were significantly associated with prostate cancer susceptibility and prognosis. Both genes encoding these proteins were located on chromosome 6p21, and combining the neighboring SNPs into haplotypes may increase the association with the disease. Three tagging polymorphisms, the HSP70-hom 2437 T/C, the VEGF-1154 G/A and the VEGF-634 G/C SNPs were genotyped in 101 cases and 80 controls. For the combined analysis of VEGF and HSP70-hom, we found a positive gradient in the ORs related to the number of high risk genotypes with a 3.53-fold increase of prostate carcinoma risk (OR= 3.53; P= 0.015). Furthermore, The TAG and CAG haplotypes at positions HSP70-hom, VEGF-1154 and VEGF-634 exhibited a twofold (OR= 0.46; P= 0.014) and a sevenfold (OR= 0.14; P= 0.00005) reduction in prostate cancer risk, respectively. Regarding prostate cancer prognosis, the TAG haplotype had a negative association with the aggressive phenotype as defined by the histopathological grade (OR= 0.28; P= 0.006). Our findings confirm the role of at-risk haplotype across the HSP70-hom/VEGF gene cluster in determining susceptibility to prostate cancer.
139: Experimental cell research, 2010 Jan 20, 29(2)
Autocrine effects of VEGF-D on endothelial cells after transduction with Ad-VEGF-D(DeltaNDeltaC).
[Abstract]Endothelial cells in tumor vessels display unusual characteristics in terms of survival and angiogenic properties which result from the increased expression of VEGF-D and its autocrine effect. To evaluate mechanisms by which VEGF-D leads to such abnormal phenotype we searched for proteins with modified expression in HUVECs enriched in the recombinant mature VEGF-D (VEGFD(DeltaNDeltaC)) delivered by adenovirus. Expression of membrane proteins in endothelial cells was characterized by FACS using anti-human IT-Box-135 antibodies. HUVECs transduced with Ad-VEGF-D(DeltaNDeltaC) revealed markedly increased expression of proteins involved in adhesion and migration such as (a) integrins (alphaVbeta5, alpha2beta1, alpha5beta1, alphaMbeta2, alphaLbeta2), (b) matrix metalloproteinases (MMP-2, MMP-9, and MMP-14), (c) components of fibrinolytic system (PAI-1, u-PAR), and (d) CD45, CD98, CD147. Interestingly, there also were numerous proteins with significantly reduced expression, particularly among surface exposed membrane proteins. Thus it can be concluded that to induce proangiogenic phenotype and facilitate migration of HUVECs, VEGF-D(DeltaNDeltaC) not only upregulates expression of proteins known to participate in the cell-matrix interactions but also silences some membrane proteins which could interfere with this process.
140: Retina (Philadelphia, Pa.), 2010 Jan 20, 29(2)
INTRAVITREAL ANTI-VEGF VERSUS PHOTODYNAMIC THERAPY WITH VERTEPORFIN FOR TREATMENT OF MYOPIC CHOROIDAL NEOVASCULARIZATION.
[Abstract]PURPOSE:: The purpose of this study was to compare visual outcomes after treatment with intravitreal anti-vascular endothelial growth factor (anti-VEGF) injection or photodynamic therapy (PDT) in patients with myopic choroidal neovascularization (CNV). METHODS:: One hundred and forty-two eyes of 128 consecutive patients treated with anti-VEGF (ranibizumab or bevacizumab) and/or PDT for myopic choroidal neovascularization were retrospectively reviewed. Patients were categorized into 3 groups: PDT (51 eyes), anti-VEGF (63 eyes), and a combination group (PDT with anti-VEGF) (28 eyes). Corrected visual acuity values at baseline and 3, 6, 9, and 12 months after treatment were compared. RESULTS:: The anti-VEGF group showed significant postoperative improvement in visual acuity compared with the PDT and combination groups (P = 0.01 and 0.04, respectively). The anti-VEGF group demonstrated visual improvement from baseline at every follow-up visit after treatment (P = 0.04, 0.02, 0.01, and 0.002, respectively). The anti-VEGF group showed visual improvement (Snellen equivalent) from 0.57 logarithm of the minimum angle of resolution (0.27) to 0.33 logarithm of the minimum angle of resolution (0.47) (P = 0.01). Furthermore, 98.4% of patients in the anti-VEGF group and 92.8% of those in the combination group lost <15 letters from baseline visual acuity compared with 72.6% in the PDT group (P = 0.001 and 0.02, respectively). In the anti-VEGF group, 39.7% of patients improved from baseline by 15 or more letters compared with 17.7% in the PDT group (P = 0.02) and 21.4% in the combination group (P = 0.07). CONCLUSION:: Intravitreal anti-VEGF injection is superior to PDT alone or a combination of PDT with anti-VEGF for treating myopic choroidal neovascularization.
141: Radiology, 2010 Feb, 254(2)
Noninvasive Contrast-enhanced US Quantitative Assessment of Tumor Microcirculation in a Murine Model: Effect of Discontinuing Anti-VEGF Therapy.
[Abstract]Purpose: To determine, by using contrast material-enhanced ultrasonography (US), how quickly renal tumors grafted in mice begin to revascularize after stopping bevacizumab treatment. Materials and Methods: All experiments were approved by the regional ethics committee. A human tumor cell line SK-NEP-1 was grafted at day 0 in the left kidney of 50 nude mice. Forty-two mice developed tumors and longitudinal follow-up was performed on 32 surviving mice. From day 13, 14 controls received biweekly saline; 11 mice received biweekly bevacizumab until day 35 (continuous); and seven received biweekly bevacizumab until day 22, then biweekly placebo until day 35 (discontinued). Contrast-enhanced US was performed on days 13, 14, 22, 27, and 35. Once the injected contrast material distribution reached an equilibrium phase, high-acoustic pressure pulses were applied to destroy microbubbles in the capillary bed in the imaged plane. Reperfusion was monitored, and time-signal intensity (SI) curves were obtained from the linear average of SIs in intratumoral and matched-depth renal cortex regions of interest. A kinetic parameter calculated from reperfusion curves reflects local perfusion, normalized with respect to adjacent renal cortex perfusion. Normalized perfusion obtained from each group was compared with that from the other groups and with necrosis percentages and microvascular density assessed histologically at day 35. Comparisons were made by using analyses of variance and Tukey-Kramer tests. Results: The lowest excised mean tumor weights (+/- standard deviation) corresponded to the longest bevacizumab-treatment duration: 1.4 g +/- 1.1 (continuous-treatment) compared with 2.3 g +/- 2.1 (discontinued) and 3.7 g +/- 1.9 (control) (P = .01). On day 35, the respective control and continuously treated groups had comparable and significantly larger necrotic areas: 37% +/- 14 and 32% +/- 17 larger than the discontinued-treatment group (15% +/- 9; P < .05). Normalized perfusion increased significantly with time (P = .02) in the discontinued-treatment group after therapy ceased (day 22). Conclusion: Noninvasively measured contrast-enhanced US parameters demonstrated tumor revascularization after stopping antiangiogenic therapy in this murine tumor model. (c) RSNA, 2010.
142: The Journal of endocrinology, 2010 Jan 21, 58(2)
Expression of vascular endothelial growth factor (VEGF) in the growth plate is stimulated by estradiol and increases during pubertal development.
[Abstract]Longitudinal bone growth is regulated in the growth plate. At the end of puberty, growth velocity diminishes and eventually ceases with fusion of the growth plate through mechanisms not yet completely understood. Vascular Endothelial Growth Factor (VEGF) has an important role in angiogenesis, but also in chondrocyte differentiation, survival and the final stages of endochondral ossification. Estrogen has been shown to up-regulate VEGF expression in the uterus and bone of rats. In this study we investigated the relation between estrogen and VEGF production in growth plate chondrocytes both in vivo and in vitro. The expression of VEGF protein was down-regulated upon ovariectomy and restored upon estradiol supplementation in rat growth plates. In cultured rat chondrocyte cell line RCJ3.1C5.18 estradiol dose-dependently stimulated 121 kDa and 189 kDa isoforms of VEGF but not the 164 kDa isoform. Finally, VEGF expression was observed at both protein and mRNA level in human growth plate specimens. The protein level increased during pubertal development supporting a link between estrogens and local VEGF production in the growth plate. We conclude that estrogens regulate VEGF expression in the epiphyseal growth plate although the precise role of VEGF in estrogen-mediated growth plate fusion remains to be clarified.
143: Journal of controlled release : official journal of the Controlled Release Society, 2010 Jan 18, 29(1)
Efficient revascularization by VEGF administration via heparin-functionalized nanoparticle-fibrin complex.
[Abstract]We investigated the angiogenic bioactivity and therapeutic angiogenic effect of vascular endothelial growth factor (VEGF) administration by the heparin-functionalized nanoparticle-fibrin gel complex. The markedly increased bioactivity was observed by the VEGF-loaded nanoparticle-fibrin gel complex, compared to the VEGF-loaded fibrin gel, the nanoparticle-fibrin gel complex without VEGF, or fibrin gel (control) in terms of the capillary density in a mouse subcutaneous implantation model. Furthermore, the VEGF-loaded nanoparticle-fibrin gel complex significantly enhanced the therapeutic angiogenic effect in a rabbit ischemic hind limb model: the noticeable increase in the recovered calf blood pressure, the angiographic score, and the density of collaterals, as well as the stable maintenance of the organized collaterals, compared to the VEGF-loaded fibrin gel. These results show the enhanced angiogenic potential of VEGF administration by the proposed heparin-functionalized nanoparticle-fibrin gel complex.
144: American journal of physiology. Cell physiology, 2010 Jan 20, 58(2)
Role of FAK phosphorylation in hypoxia-induced hMSCs migration: involvement of VEGF as well as MAPKs and eNOS pathways.
[Abstract]Here we show that the effect of hypoxia on human umbilical cord blood mesenchymal stem cell (hMSC) migration is via the modulation of focal adhesion kinase (FAK) and its related signaling pathways. Hypoxia increased hMSC migration and cell viability, while lactate dehydrogenase (LDH) release was not affected for up to 48 h (data not shown). In addition, hypoxia increased the level of reactive oxygen species (ROS) generation in a time-dependent manner. Hypoxia-induced phosphorylation of p38 mitogen-activated protein kinase (MAPK) and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) were inhibited by the antioxidant (NAC, 10(-6) M) and (taurine, 4x10(-6) M). Hypoxia-induced endothelial nitric oxide synthase (eNOS) phospholylation was regulated by p38 MAPK and SAPK/JNK activation. In addition, hypoxia increased the level of hypoxia inducible factor (HIF)-1alpha expression, which was blocked by inhibition of eNOS. Also, hypoxia-induced expression of Flk-1, vascular endothelial growth factor (VEGF) and its secreted form were inhibited by HIF-1alpha siRNA. In this hypoxic condition, FAK and Src phosphorylation were increased in a time-dependent manner. Inhibition of Src with specific inhibitor (PP2, 10(-8) M) blocked hypoxia-induced FAK activation. Subsequently, hypoxia-induced FAK phosphorylation was blocked by VEGF siRNA. Finally, hypoxia-induced increase of hMSC migration was inhibited by FAK siRNA. The results indicate that hypoxia increases migration of hMSCs via VEGF-mediated FAK phospholylation and involves the cooperative activity of the ROS, MAPK, eNOS and HIF-1alpha pathways.
145: Blood, 2010 Jan 19, 29(2)
Endothelial cell-specific chemotaxis receptor (ecscr) promotes angioblast migration during vasculogenesis and enhances VEGF receptor sensitivity.
[Abstract]Endothelial Cell-Specific Chemotaxis Receptor (ECSCR) is a cell surface protein expressed by blood endothelial cells with roles in endothelial cell migration and signal transduction. We investigated the function of ecscr in the development of the zebrafish vasculature. Zebrafish ecscr is expressed in angioblasts and in axial vessels during angioblast migration and vasculogenesis. Morpholino directed ecscr knockdown resulted in defective angioblast migration in the posterior lateral plate mesoderm, a process known to depend on VEGF. In cultured cells, transfected ECSCR localized to actin rich membrane protrusions, co-localizing with KDR/VEGF receptor 2 in these regions. ECSCR silenced cells show reduced VEGF-induced phosphorylation of KDR but not of FLT1/VEGF receptor 1. Finally, chemical inhibition of VEGF receptor activity in zebrafish resulted in angioblast deficiencies which partially overlap with those seen in ecscr morphants. We propose that ecscr promotes migration of zebrafish angioblasts by enhancing endothelial kdr sensitivity to VEGF.
146: Journal of experimental & clinical cancer research : CR, 2010 Jan 19, 29(1)
Higher expression of vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 (Flk-1) and metalloproteinase-9 (MMP-9) in a rat model of peritoneal endometriosis is similar to cancer diseases.
[Abstract]ABSTRACT: BACKGROUND: Endometriosis is a common disease characterized by the presence of a functional endometrium outside the uterine cavity, causing pelvic pain, dysmenorrheal, and infertility. This disease has been associated to development of different types of malignancies; therefore new blood vessels are essential for the survival of the endometrial implant. Our previous observations on humans showed that angiogenesis is predominantly found in rectosigmoid endometriosis, a deeply infiltrating disease. In this study, we have established the experimental model of rat peritoneal endometriosis to evaluate the process of angiogenesis and to compare with eutopic endometrium. METHODS: We have investigated the morphological characteristics of these lesions and the vascular density, VEGF and its receptor Flk-1 and MMP-9 expression, and activated macrophage distribution, using immunohistochemistry and RT-PCR. RESULTS: As expected, the auto-transplantation of endometrium pieces into the peritoneal cavity is a well-established method for endometriosis induction in rats. The lesions were cystic and vascularized, and demonstrated histological hallmarks of human pathology, such as endometrial glands and stroma. The vascular density and the presence of VEGF and Flk-1 and MMP-9 were significantly higher in endometriotic lesions than in eutopic endometrium, and confirmed the angiogenic potential of these lesions. We also observed an increase in the number of activated macrophages (ED-1 positive cells) in the endometriotic lesions, showing a positive correlation with VEGF. CONCLUSION: The present endometriosis model would be useful for investigation of the mechanisms of angiogenesis process involved in the peritoneal attachment of endometrial cells, as well as of the effects of therapeutic drugs, particularly with antiangiogenic activity.
147: Cancer biology & therapy, 2010 Feb 4, 9(3)
Two is better than one: Benefits of VEGF and PDGF inhibition in ovarian cancer.
[Abstract]
148: Proceedings of the National Academy of Sciences of the United States of America, 2010 Feb 2, 107(5)
Direct contacts between extracellular membrane-proximal domains are required for VEGF receptor activation and cell signaling.
[Abstract]Structural analyses of the extracellular region of stem cell factor (SCF) receptor (also designated KIT) in complex with SCF revealed a sequence motif in a loop in the fourth Ig-like domain (D4) that is responsible for forming homotypic receptor contacts and for ligand-induced KIT activation and cell signaling. An identical motif was identified in the most membrane-proximal seventh Ig-like domain (D7) of vascular endothelial growth factor receptor 1 (VEGFR1), VEGFR2, and VEGFR3. In this report we demonstrate that ligand-induced tyrosine autophosphorylation and cell signaling via VEGFR1 or VEGFR2 harboring mutations in critical residues (Arg726 or Asp731) in D7 are strongly impaired. We also describe the crystal structure of D7 of VEGFR2 to a resolution of 2.7 A. The structure shows that homotypic D7 contacts are mediated by salt bridges and van der Waals contacts formed between Arg726 of one protomer and Asp731 of the other protomer. The structure of D7 dimer is very similar to the structure of D4 dimers seen in the crystal structure of KIT extracellular region in complex with SCF. The high similarity between VEGFR D7 and KIT D4 in both structure and function provides further evidence for common ancestral origins of type III and type V RTKs. It also reveals a conserved mechanism for RTK activation and a novel target for pharmacological intervention of pathologically activated RTKs.
149: International journal of surgical pathology, 2010 Jan 13, 29(2)
Aberrant VEGF Expression Associated With Neoplasm-Induced Extramedullary Hematopoiesis in an Epithelioid Hemangioendothelioma: A Case Report.
[Abstract]The authors present the first report of extramedullary hematopoiesis occurring in association with an epithelioid hemangioendothelioma. A 64-year-old man sought medical attention for upper chest pain. CT evaluation identified a 2.8-cm mass involving the right hemithorax in the area of the right upper lobe and superior vena cava. A biopsy revealed the presence of an epithelioid malignant vascular neoplasm, which on subsequent resection was found to be an epithelioid hemangioendothelioma. The neoplasm was closely associated with an area of extramedullary hematopoiesis. VEGF immunohistochemical staining of the neoplasm revealed a ring of intensely positive staining cells around the periphery of the area of extramedullary hematopoiesis. This finding provides evidence for the role of aberrant VEGF expression in neoplasm-induced extramedullary hematopoiesis. This report discusses possible mechanisms by which extramedullary hematopoiesis may occur in vascular neoplasms.
150: Cancer investigation, 2010 Jan 14, 314(1)
The Role of NF-kappaB in Hepatitis B Virus X Protein-Mediated Upregulation of VEGF and MMPs.
[Abstract]ABSTRACT Hepatitis B virus X protein (HBx) promotes hepatocellular carcinoma (HCC) invasion and metastasis by a poorly understood mechanism. This study investigated the role of NF-kappa B in HBx-mediated upregulation of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs). In a stably expressing HBx cell line, NF-kappa B level was examined by laser scanning confocal microscopy before and after treatment with pyrrolidine dithiocarbamate (PDTC; an NF-kappa B inhibitor). VEGF, MMP2, MMP9, and MMP14 mRNA and protein levels were quantitated by real-time PCR and Western blotting, respectively. HBx stimulated NF-kappa B signaling and increased VEGF, MMP2, MMP9, and MMP14 mRNA and protein levels. PDTC treatment blocked HBx-mediated stimulation of NF-kappa B signaling and decreased VEGF, MMP9, and MMP14 (but not MMP2) mRNA and protein levels. In vivo studies, PDTC reduced angiogenesis in subcutaneous xenograft of nude mice which injected HepG2-HBx cells. This suggests that NF-kappa B is involved in upregulating these genes and in the HBx-mediated invasion and metastasis of HCC.
151: The Journal of biological chemistry, 2010 Jan 12, 314(1)
Engineering anti-VEGF disulfide-stabilized single chain antibody variable fragments (sc-dsFv) with phage displayed sc-dsFv libraries.
[Abstract]Phage display of antibody fragments from natural or synthetic antibody libraries with the single chain constructs combining the variable fragments (scFv) has been one of the most prominent technologies in antibody engineering. However, the nature of the artificial single chain constructs results in unstable proteins expressed on phage surface or as soluble proteins secreted in the bacterial culture medium. The stability of the variable domain structures can be enhanced with inter-domain disulfide bond, but the disulfide-stabilized single chain constructs (sc-dsFv) have yet to be established as a feasible format for bacterial phage display due to diminishing expression level on phage surface in known phage display systems. In this work, biological combinatorial searches were used to establish that the c-region of the signal sequence is critically responsible for effective expression and functional folding of the sc-dsFv on phage surface. The optimum signal sequences increase the expression of functional sc-dsFv by two orders of magnitude compared with wild-type signal sequences, enabling the construction of phage-displayed synthetic anti-VEGF sc-dsFv libraries. The comparison of the scFv and sc-dsFv variants selected from the phage-displayed libraries for VEGF-binding revealed the sequence preference differences resulting from the interdomain disulfide bond. These results underlie a new phage display format for antibody fragments with all the benefits from the scFv format, but without the downsides due to the instability of the dimeric interface in scFv.
152: Journal of neurochemistry, 2010 Jan 8, 188(1)
Temporal requirement of RPE-derived VEGF in the development of choroidal vasculature.
[Abstract]ABSTRACT Vascular endothelial growth factor (VEGF-A or VEGF) is a potent growth factor for the development of retinal and choroidal vasculatures. To define the temporal requirement of the retinal pigmented epithelium (RPE)-derived VEGF in choroidal vascular development, we generated conditional VEGF knockout mice using an inducible Cre/lox system. The loss of the RPE-derived VEGF was confirmed with immunoblotting and immunohistochemistry. Retinal function and structure were assessed with electroretinography and histology, respectively. Choroidal vascular density was analyzed with computer-assisted semi-quantitative assay using fluorescently labeled choroidal flat-mounts. Induction of RPE-specific VEGF disruption at embryonic day 10 (E10) or E13 for two days caused regulatable decreases in choroidal vascular density, photoreceptor function, and photoreceptor outer nuclear layer thickness. The loss of the RPE-produced VEGF after E15 did not cause detectable defects in choroidal vasculatures and photoreceptor function and morphology. These results suggest that the RPE-derived VEGF plays a critical role in choroidal vascular development during organogenesis before embryonic day 15.
153: mAbs, 2010 Jan 2, 2(1)
A dual-targeting PDGFRbeta/VEGF-A molecule assembled from stable antibody fragments demonstrates anti-angiogenic activity in vitro and in vivo.
[Abstract]Targeting angiogenesis is a promising approach to the treatment of solid tumors and age-related macular degeneration (AMD). Inhibition of vascularization has been validated by the successful marketing of monoclonal antibodies (mAbs) that target specific growth factors or their receptors, but there is considerable room for improvement in existing therapies. Combination of mAbs targeting both the VEGF and PDGF pathways has the potential to increase the efficacy of anti-angiogenic therapy without the accompanying toxicities of tyrosine kinase inhibitors and the inability to combine efficiently with traditional chemotherapeutics. However, development costs and regulatory issues have limited the use of combinatorial approaches for the generation of more efficacious treatments. The concept of mediating disease pathology by targeting two antigens with one therapeutic was proposed over two decades ago. While mAbs are particularly suitable candidates for a dual-targeting approach, engineering bispecificity into one molecule can be difficult due to issues with expression and stability, which play a significant role in manufacturability. Here, we address these issues upstream in the process of developing a bispecific antibody (bsAb). Single-chain antibody fragments (scFvs) targeting PDGFRbeta and VEGF-A were selected for superior stability. The scFvs were fused to both termini of human Fc to generate a bispecific, tetravalent molecule. The resulting molecule displays potent activity, binds both targets simultaneously, and is stable in serum. The assembly of a bsAb using stable monomeric units allowed development of an anti-PDGFRB/VEGF-A antibody capable of attenuating angiogenesis through two distinct pathways and represents an efficient method for rapid engineering of dual-targeting molecules.
154: The Journal of cell biology, 2010 Jan 11, 188(1)
Neuropilin-2 mediates VEGF-C-induced lymphatic sprouting together with VEGFR3.
[Abstract]Vascular sprouting is a key process-driving development of the vascular system. In this study, we show that neuropilin-2 (Nrp2), a transmembrane receptor for the lymphangiogenic vascular endothelial growth factor C (VEGF-C), plays an important role in lymphatic vessel sprouting. Blocking VEGF-C binding to Nrp2 using antibodies specifically inhibits sprouting of developing lymphatic endothelial tip cells in vivo. In vitro analyses show that Nrp2 modulates lymphatic endothelial tip cell extension and prevents tip cell stalling and retraction during vascular sprout formation. Genetic deletion of Nrp2 reproduces the sprouting defects seen after antibody treatment. To investigate whether this defect depends on Nrp2 interaction with VEGF receptor 2 (VEGFR2) and/or 3, we intercrossed heterozygous mice lacking one allele of these receptors. Double-heterozygous nrp2vegfr2 mice develop normally without detectable lymphatic sprouting defects. In contrast, double-heterozygote nrp2vegfr3 mice show a reduction of lymphatic vessel sprouting and decreased lymph vessel branching in adult organs. Thus, interaction between Nrp2 and VEGFR3 mediates proper lymphatic vessel sprouting in response to VEGF-C.
155: The American journal of tropical medicine and hygiene, 2010 Jan, 82(1)
Elevated levels of vascular endothelial growth factor (VEGF) and soluble vascular endothelial growth factor receptor (VEGFR)-2 in human malaria.
[Abstract]In cerebral malaria, the binding of parasitized erythrocytes to the cerebral endothelium and the consequent angiogenic dysregulation play a key role in pathogenesis. Because vascular endothelial growth factor (VEGF) is widely regarded as a potent stimulator of angiogenesis, edema, inflammation, and vascular remodeling, the plasma levels of VEGF and the soluble form of the VEGF receptor (sVEGFR)-1 and -2 in uncomplicated malaria patients and healthy adults were measured by enzyme-linked immunosorbent assay (ELISA) to examine their roles in malaria. The results showed that VEGF and sVEGFR-2 levels were significantly elevated in malaria patients compared with healthy adults. Moreover, it was confirmed that malarial parasite antigens induced VEGF secretion from the human mast cell lines HMC-1 or KU812 cell. This is the first report to suggest that the interaction of VEGF and sVEGFR-2 is involved in the host immune response to malarial infection and that malarial parasites induce VEGF secretion from human mast cells.
156: European journal of cancer (Oxford, England : 1990), 2010 Jan 9, 188(1)
VEGF-A promotes lymphoma tumour growth by activation of STAT proteins and inhibition of p27(KIP1) via paracrine mechanisms.
[Abstract]Increased levels of circulating VEGF-A have been demonstrated in patients with non-Hodgkin lymphoma (NHL) and are associated with progressive disease and poor clinical outcome. We investigated the role of VEGF-A in lymphoma tumour growth on a molecular level in order to identify the mechanism of VEGF-A-promoted tumour growth and to identify the potential targets for therapy. We used a model in which Daudi (human Burkitt lymphoma) tumour cells were transduced with VEGF-A165 or an empty vector (negative control) and subcutaneously injected in NOD/SCID mice. The weight of tumours overexpressing VEGF-A was increased 4-fold compared to that of control tumours (p<0.0001), whereas no in vitro growth advantage was demonstrated upon VEGF-A overexpression. VEGF-A-tumours were associated with increased microvessel densities (p=0.004) and increased tumour cell proliferation (Ki67; p<0.001) compared to control tumours. VEGF-A-tumours were characterised by upregulation of phosphorylated STAT-4 and STAT-6 and downregulation of phospho-p27(KIP1), a crucial cell cycle inhibitor (p<0.05). This was accompanied by increased levels of phosphorylated receptor tyrosine kinases, including EGFR (ErbB-2 and ErbB-4, p<0.05), an upstream regulator of STAT proteins. We demonstrated that various mouse-derived cytokines produced by mouse-derived tumour stromal cells are upregulated in VEGF-A-tumours compared to control tumours (p<0.05). These results indicate an important role for the tumour microenvironment in paracrine promotion of lymphoma tumour growth in response to tumour-derived VEGF-A. In conclusion, lymphoma-derived VEGF-A promoted lymphoma tumour growth in a paracrine loop by activation of tumour stromal cells. Our study reveals VEGF-A and STAT proteins as potential additional targets in the treatment of lymphoma.
157: Platelets, 2010 Jan 11, 188(1)
The effect of P2Y-mediated platelet activation on the release of VEGF and endostatin from platelets.
[Abstract]Vascular endothelial growth factor (VEGF) and endostatin are key protein modulators of angiogenesis found within platelets. The platelet activation pathways that control angiogenic protein release are incompletely elucidated. The differential release of pro-angiogenic and anti-angiogenic proteins from the platelet has been demonstrated for proteinase activated receptors (PARs). Given the ability of tumors to secrete ADP and the availability of ADP receptor antagonists clinically, we determined the influence of adenosine diphosphate (ADP) and the ADP receptors, P2Y(1) and P2Y(12), on platelet release of the angiogenic stimulator protein, VEGF, and the angiogenic inhibitor protein, endostatin. Minimally altered whole blood (WB) and platelet rich plasma (PRP) from healthy volunteers was stimulated with ADP alone (12.5 uM), in combination with a P2Y(1) antagonist (MRS2179) or a P2Y(12) antagonist (cangrelor). VEGF and endostatin protein concentrations were assessed by an ELISA assay. We report that maximally stimulating concentrations of ADP significantly increased VEGF release from platelets in both PRP and WB by 36+/-12% 36+/-12% 54+/-18% 36 +/- 12% (p < 0.05) respectively as compared to control. Both P2Y(1) and P2Y(12) receptor antagonism inhibited this release. Conversely, endostatin levels did not change following ADP stimulation in PRP, while a 4.7% (p = 0.03) increase was observed in WB. As compared to thrombin receptor activation, ADP activation was a weaker stimulus for VEGF release. We found that activation of platelets by ADP results in an increase in soluble VEGF concentrations with minimal effects on endostatin concentrations, suggesting ADP release in the tumor microenvironment may be, on balance, proangiogenic. P2Y receptor antagonism abrogates ADP mediated proangiogenic protein release and thus may represent a potential pharmacologic strategy for regulating platelet mediated angiogenesis.
158: The Journal of biological chemistry, 2010 Jan 8, 314(1)
Regulation of VEGF-induced endothelial cell migration by LIM kinase 1-mediated phosphorylation of annexin 1.
[Abstract]In this study we obtained evidence indicating that annexin 1 is a new target of the p38/MAPKAP kinase-2 pathway and that it regulates endothelial cell migration in response to vascular endothelial growth factor (VEGF). Firstly, by 2D-gel electrophoresis and mass spectrometry, we identified annexin 1 as a protein whose phosphorylation is induced by VEGF, and is impaired by inhibiting p38. Secondly, using in vitro kinase assays and in vivo phosphorylation assays, we found that VEGF-mediated activation of LIM kinase 1 downstream of the p38 pathway triggers the phosphorylation of annexin 1. Thirdly, VEGF-induced cell migration and tube formation in Matrigel are inhibited following small interfering RNA-mediated knockdown of annexin 1. Fourthly, both processes are rescued in cells expressing an annexin 1 construct insensitive to the siRNA knockdown. Finally, the VEGF/annexin 1-mediated cell migration is impaired by inhibiting p38. We conclude that phosphorylation of annexin 1 regulates the angiogenic effect associated with the activation of the p38/LIM kinase 1 axis by VEGF.
159: Gynecologic oncology, 2010 Jan 5, 102(1)
The relationship of VEGF polymorphisms with serum VEGF levels and progression-free survival in patients with epithelial ovarian cancer.
[Abstract]OBJECTIVE: The vascular endothelial growth factor (VEGF) is an important regulator of angiogenesis and vascular permeability of tumors. In the present study we evaluated the relation of five single nucleotide polymorphisms (SNPs) in the VEGF gene with progression-free survival. Furthermore, we evaluated the functional significance of the SNPs as determined by the influence on serum VEGF levels in ovarian cancer. METHODS: Serum from 143 consecutive ovarian cancer patients referred for first line platinum/paclitaxel treatment were analyzed for serum VEGF levels using commercially available enzyme-linked immunosorbent assay (ELISA). VEGF gene polymorphisms (-2578 C/A, -1154 G/A, -460 T/C, +405 G/C and +936C/T) were determined by real time PCR using genomic DNA extracted from whole blood samples. RESULTS: VEGF serum levels were significantly higher in carriers of the 2578C, 460T and 405C, alleles compared to non-carriers (p=0.003, p=0.003 and p=0.001, respectively). There was no significant correlation between VEGF SNP genotypes and progression-free survival. In haplotype analysis, the multivariate survival analysis showed that progression-free survival (PFS) for the patients with the AGCGC haplotype was significantly improved compared to patients with other haplotypes (HR 1.9, p=0.036). CONCLUSIONS: VEGF polymorphisms were found to be significantly related with serum VEGF levels. The AGCGC haplotype was found to be independently associated with improved PFS.
160: Molecular cancer therapeutics, 2010 Jan 6, 314(1)
A Human Monoclonal Anti-ANG2 Antibody Leads to Broad Antitumor Activity in Combination with VEGF Inhibitors and Chemotherapy Agents in Preclinical Models.
[Abstract]Localized angiopoietin-2 (Ang2) expression has been shown to function as a key regulator of blood vessel remodeling and tumor angiogenesis, making it an attractive candidate for antiangiogenic therapy. A fully human monoclonal antibody (3.19.3) was developed, which may have significant pharmaceutical advantages over synthetic peptide-based approaches in terms of reduced immunogenicity and increased half-life to block Ang2 function. The 3.19.3 antibody potently binds Ang2 with an equilibrium dissociation constant of 86 pmol/L, leading to inhibition of Tie2 receptor phosphorylation in cell-based assays. In preclinical models, 3.19.3 treatment blocked blood vessel formation in Matrigel plug assays and in human tumor xenografts. In vivo studies with 3.19.3 consistently showed broad antitumor activity as a single agent across a panel of diverse subcutaneous and orthotopic xenograft models. Combination studies of 3.19.3 with cytotoxic drugs or anti-vascular endothelial growth factor agents showed significant improvements in antitumor activity over single-agent treatments alone with no apparent evidence of increased toxicity. Initial pharmacokinetic profiling studies in mice and nonhuman primates suggested that 3.19.3 has a predicted human half-life of 10 to 14 days. These studies provide preclinical data for 3.19.3 as a potential new antiangiogenic therapy as a single agent or in combination with chemotherapy or vascular endothelial growth factor inhibitors for the treatment of cancer. Mol Cancer Ther; 9(1); 145-56.
161: Biosensors & bioelectronics, 2010 Mar 15, 25(7)
Electrochemical detection of vascular endothelial growth factors (VEGFs) using VEGF antibody fragments modified Au NPs/ITO electrode.
[Abstract]A new electrochemical technique for the detection of vascular endothelial growth factors (VEGFs) as a cancer-related biomarker is presented in this paper. Gold nanoparticles (Au NPs) were self-assembled onto an indium tin oxide (ITO) electrode to prepare a modified sandwich type electrochemical immunoassay platform. VEGF antibodies were cleaved into two half-fragments by 2-mercaptoethylamine-HCl (2-MEA) and the fragments were immobilized onto the Au NP substrates by their thiol groups. Through this strategy, randomly oriented attachment of antibodies was prevented which frequently occurs in a general use of whole antibody and reduces the number of available sites for the attachment of target molecules. VEGF target molecules were applied to the immunoelectrodes and they combined with the antibody fragments covering the Au NP electrode, forming antigen-antibody complexes. Then, ferrocene-tagged antibodies, which release electrons under a proper applied potential, were added to the system and they combined with the VEGF molecules pre-attached to the antibody fragments. The redox current of ferrocene measured by the differential pulse voltammetry (DPV) increased almost linearly from 1.27 x 10(-4) to 4.17 x 10(-4)A according to the increase in the concentration of the VEGF target molecules from 100 to 600 pg/ml. The measured current values represent the concentration of the VEGF since they are proportional to the number of ferrocene molecules which is in turn proportional to the concentration of VEGF target molecules. Using this modified sandwich immunoassay with the Au NP/ITO electrode, VEGFs as low as 100 pg/ml were detected with high specificity.
162: Molecular pharmaceutics, 2010 Jan 4, 584(1)
Inhibition of breast cancer cell growth and invasiveness by dual silencing of HER-2 and VEGF.
[Abstract]Over-expression of HER-2 accounts for ~25% of all breast cancer cases, while 87.7% of HER-2 positive breast cancers are associated with up-regulated VEGF. The objective of this study is to explore the combination therapy of blocking HER-2 and VEGF expressions simultaneously using siRNA. This is the first report to examine the effect of dual silencing of HER-2 and VEGF genes on tumor growth and invasiveness. We have designed nine HER-2 siRNAs and ten VEGF siRNAs, and identified potent siRNA which can silence the target gene up to 75~83.5%. The most potent HER-2 and VEGF siRNAs were used to conduct functional studies in HER-2 positive breast cancer cells. Tumor invasiveness properties including cell morphology change, in vitro migration, cell spreading, and adhesion to ECM were evaluated. In addition, cell proliferation and apoptosis were examined after the siRNA treatment. Our data demonstrated for the first time that HER-2 siRNA could inhibit cell migration and invasion abilities. Combination of HER-2 and VEGF siRNAs exhibited synergistic silencing effect on VEGF. Both HER-2 siRNA and VEGF siRNA showed significant inhibition on cell migration and proliferation. HER-2 siRNA also demonstrated dramatic suppression on cell spreading and adhesion to ECM, as well as induction of apoptosis. Dual silencing of HER-2 and VEGF exhibited significant cell morphology change, and substantial suppression on migration, spreading, cell adhesion, and proliferation. Our observations suggested that HER-2 positive breast cancer may be more effectively treated by dual inhibition of HER-2 and VEGF gene expressions using siRNA.
163: Biomaterials, 2010 Apr, 31(10)
The ability of a collagen/calcium phosphate scaffold to act as its own vector for gene delivery and to promote bone formation via transfection with VEGF(165).
[Abstract]Collagen/calcium phosphate scaffolds have been used for bone reconstruction due to their inherent similarities to the bone extracellular matrix. Calcium phosphate alone has also been used as a non-viral vector for gene delivery. The aim of this study was to determine the capability of a collagen/calcium phosphate scaffold to deliver naked plasmid DNA and mediate transfection in vivo. The second goal of the study was to deliver a plasmid encoding vascular endothelial growth factor(165) (pVEGF(165)) to promote angiogenesis, and hence bone formation, in a mouse intra-femoral model. The delivery of naked plasmid DNA resulted in a 7.6-fold increase in mRNA levels of beta-Galactosidase compared to the delivery of plasmid DNA complexed with a partially degraded PAMAM dendrimer (dPAMAM) in a subcutaneous murine model. When implanted in a muirne intra-femoral model, the delivery of pVEGF(165) resulted in a 2-fold increase in bone volume at the defect site relative to control scaffolds without pVEGF(165). It was concluded that a collagen/calcium phosphate scaffold can mediate transfection without the use of additional transfection vectors and can promote bone formation in a mouse model via the delivery of pVEGF(165).
164: Investigative ophthalmology & visual science, 2009 Dec 30, 180(1)
Regulation of VEGF-A in uveal melanoma.
[Abstract]Purpose: Blood vessels are important constituents of intraocular uveal melanoma (UM), but whether angiogenesis is regulated by environmental factors such as ischemia, or by genetic mechanisms is not known. We examined regulation of the pro-angiogenic factor Vascular Endothelial Growth Factor (VEGF-A). Material and Methods: Cell lines and primary tumours was tested for expression of VEGF-A, under normoxic and hypoxic conditions, using quantitative RT-PCR, ELISA, WST-1 viability, and in-cell Western experiments. VEGF-A serum levels were determined by ELISA. Results: Hypoxia induced HIF-1alpha and VEGF-A in UM cell lines and primary tumour cultures. Hypoxia did not influence proliferation. VEGF-A expression in primary tumours was variable, demonstrating no correlation with specific histological markers or prognosis. However, VEGF-A levels were significantly raised in UM patients with metastases compared with those without metastases (p<0.001). Conclusion: VEGF-A expression by UM cells is mainly controlled by hypoxia and involves the HIF-1alpha pathway, thus indicating an important role for the tumour cell environment. Metastases led to increased serum VEGF-A levels, indicating that VEGF-A may be involved in the growth of metastases.
165: Investigative ophthalmology & visual science, 2009 Dec 30, 180(1)
VEGF and Anti-VEGF drugs have minimal effects on the permeability or selectivity of RPE tight junctions.
[Abstract]Purpose: Bevacizumab and ranibizumab are currently used to treat age-related macular degeneration by neutralizing vascular endothelial growth factor (VEGF). This study examines potential side effects on the outer blood-retinal barrier. Methods: Human fetal RPE (hfRPE) was used because they are highly differentiated in culture. The claudin composition of RPE tight junctions was determined by RT-PCR, immunoblotting and immunofluorescene. ELISA assays monitored the secretion and trafficking of VEGF and a fluid-phase marker, methylpolyethylene glycol (mPEG). Tight junction functions were assessed by the conductance of K(+) and Na(+) (derived from the transepithelial electrical resistance, TER) and the flux of NaCl and mPEG. Results: Claudins 3, 10 and 19 were detected in RPE tight junctions. VEGF was secreted in equal amounts across the apical and basolateral membranes, but the apical membrane was more active in endocytosing and degrading VEGF. Exogenous VEGF and mPEG crossed the RPE monolayer by transcytosis predominantly in the apical-to-basal direction. RPE tight junctions were selective for K(+), but did not discriminate between Na(+) and Cl(-). VEGF, bevacizumab and ranibizumab had minimal effect on the TER, permeation of mPEG, and selectivity for K(+), Na(+) and Cl(-). They had minimal effect on the expression and distribution of the claudins. Conclusions: RPE has mechanisms for maintaining low concentrations of VEGF in the subretinal space that includes endocytosis and degradation and fluid phase transcytosis in the apical-to-basal directions. RPE tight junctions are selective for K(+) over Na(+) and Cl(-). Permeability and selectivity of the junctions are not affected by VEGF, bevacizumab or ranibizumab.
166: Current opinion in ophthalmology, 2010 Mar, 21(2)
Anti-VEGF therapy for glaucoma.
[Abstract]PURPOSE OF REVIEW: The role of antivascular endothelial growth factor (anti-VEGF) agents in treating various ophthalmic diseases is currently being investigated. There have been many advances in the understanding of how anti-VEGF agents work and speculation on when to implement them clinically for neovascular glaucoma. Recent studies exploring the utility of anti-VEGF agents for wound modulation after trabeculectomy reveal promising results. RECENT FINDINGS: Anti-VEGF agents have been shown to be beneficial in treating neovascular glaucoma. Their use leads to regression of both iris and angle neovascularization, intraocular pressure control when the angle remains open and, in many cases, prompts symptomatic improvement. In addition, research on the wound modulatory properties of anti-VEGF agents has revealed a dose-dependent inhibition of fibroblast proliferation. Studies exploring the use of anti-VEGF agents at time of trabeculectomy or in bleb revision procedures suggest a beneficial effect on bleb survival and subsequent improvement in intraocular pressure control. Prospective randomized clinical trials are still needed. SUMMARY: The recent use of anti-VEGF agents for neovascular glaucoma as well as wound modulation after trabeculectomy has shown great promise. Through future research, the antiangiogenic and antifibroblastic properties of anti-VEGF agents may prove to be beneficial in patients being treated for various forms of glaucoma.
167: Development (Cambridge, England), 2010 Jan, 137(2)
VEGF is required for dendritogenesis of newly born olfactory bulb interneurons.
[Abstract]The angiogenic factor vascular endothelial growth factor A (VEGF) has been shown to have a role in neurogenesis, but how it affects adult neurogenesis is not fully understood. To delineate a role for VEGF in successive stages of olfactory bulb (OB) neurogenesis, we used a conditional transgenic system to suppress VEGF signaling at the adult mouse sub-ventricular zone (SVZ), rostral migratory stream (RMS) and OB, which constitute the respective sites of birth, the migration route, and sites where newly born interneurons mature and integrate within the existing OB circuitry. Following the development of fluorescently tagged adult-born neurons, we show that sequestration of VEGF that is constitutively expressed by distinct types of resident OB neurons greatly impaired dendrite development in incoming SVZ-born neurons. This was evidenced by reduced dendritic spine density of granule cells and significantly shorter and less branched dendrites in periglomerular neurons. Notably, the vasculature and perfusion of the SVZ, RMS and OB were not adversely affected when VEGF suppression was delayed until after birth, thus uncoupling the effect of VEGF on dendritogenesis from its known role in vascular maintenance. Furthermore, a requirement for VEGF was specific to newly born neurons, as already established OB neurons were not damaged by VEGF inhibition. This study thus uncovered a surprising perfusion-independent role of VEGF in the adult brain, namely, an essential role in the maturation of adult-born neurons.
168: Archives of dermatological research, 2009 Dec 25, 180(1)
Human skin-derived mesenchymal stem cells as a source of VEGF and nitric oxide.
[Abstract]Researches on stem cells bring promise to functional skin repair. In particular, it has been recently suggested that mesenchymal stem cells (MSCs) could positively affect cutaneous wound healing through differentiation and paracrine action. The molecular mechanisms are not clear, even if there is increasing evidence for an important action of nitric oxide (NO), probably mediated by the regulation of the gene encoding for vascular endothelial growth factor (VEGF). The aim of our study was to investigate the immunohistochemical expression of VEGF and nitric oxide synthase (NOS) isoforms in human skin-derived MSCs, as well as the production of VEGF and NO, because these cells are less well characterized than bone marrow MSCs. MSCs were obtained from skin biopsies of healthy adult patients undergoing cosmetic plastic surgery, expanded and characterized for specific surface antigens. The cells were then evaluated for the immunohistochemical expression of VEGF, and NOS isoforms, as well as for VEGF and NO secretion in cell culture medium. Our immunohistochemical analysis showed that proliferating MSCs derived from human skin exhibit VEGF expression at cytoplasmic level as well as cytosolic and nuclear localization of all the three isoforms of NOS, even if with different patterns. In addition, our data evidenced the release of both VEGF and NO in cell culture supernatants. In conclusion, our results suggest that a therapeutic approach based on the human skin-derived MSCs may have a positive effect in wound healing conditions, through their ability to provide VEGF and NO to the damaged area.
169: The Journal of reproduction and development, 2009 Dec 25, 180(1)
Expression of VEGF And Its Receptors in the Bovine Endometrium Throughout the Estrous Cycle: Effects of VEGF on Prostaglandin Production in Endometrial Cells.
[Abstract]Vascular endothelial growth factor (VEGF) is a well known angiogenic factor that has been suggested to play some physiological roles in reproductive organs. To clarify whether VEGF is involved in regulating bovine endometrial function locally, in experiment 1, we determined the expression of VEGF, VEGF receptor (VEGFR) 1 and VEGFR2 throughout the estrous cycle in endometrial tissues. Endometrial tissue was collected at estrus (Day 0), the early I (Days 2-3), early II (Days 5-6), mid (Days 8-12) and late luteal stages (Days 15-17) and the follicular stage (Days 19-21). RT-PCR and Western blotting analysis revealed that VEGF mRNA expression at estrus was higher than at the early I, early II and late luteal stages (P<0.05), whereas VEGF protein content was greatest at the early I luteal stage and decreased thereafter. VEGFR1 mRNA expression was lower at estrus and at the early I and early II luteal stages than at the other stages, whereas VEGFR1 protein expression did not change significantly throughout the estrous cycle (P<0.05). VEGFR2 mRNA expression was higher at the mid and late luteal stages than at the early I and early II luteal stages, and VEGFR2 protein was higher at the mid and late luteal stages than at estrus (P<0.05). In experiment 2, to determine the effect of VEGF on prostaglandin (PG) F2alpha and PGE2 production by endometrial cells, cultured endometrial epithelial and stromal cells were exposed to VEGF (0, 5, 50, 100 and 200 ng/ml) for 24 h. VEGF (200 ng/ml) stimulated PGF2alpha production by stromal cells (P<0.05), but not PGE2 production. VEGF did not affect PG production by endometrial epithelial cells. The overall results suggest that VEGF and its receptors are regulated throughout the estrous cycle and that VEGF participates in the local regulation of bovine endometrial function by a selective modulation of PGF2alpha production in stromal cells in an auto- and/or paracrine manner.
170: Cancer chemotherapy and pharmacology, 2009 Dec 24, 114(27)
Biodistribution of humanized anti-VEGF monoclonal antibody/bevacizumab on peritoneal metastatic models with subcutaneous xenograft of gastric cancer in mice.
[Abstract]PURPOSE: Vascular endothelial growth factor (VEGF) is correlated with peritoneal metastasis of gastric cancer, increasing vascular permeability accompanied by accumulation of ascites. The aim of the current study is to investigate the biodistribution of bevacizumab in a peritoneal metastatic model of gastric cancer and to clarify which is more suited to treatment of peritoneal metastasis, systemic or regional therapy. METHODS: A highly peritoneal-seeding cell line of gastric cancer, OCUM-2MD3, which exhibited high production and release of VEGF was used in this study. The biodistribution of bevacizumab was investigated using peritoneal metastatic models together with subcutaneous xenografts, and (125)I-radiolabelled bevacizumab was administrated to these models subcutaneously (s.c.) or intraperitoneally (i.p.), respectively. In addition, the anti-tumor response of bevacizumab and paclitaxel was assessed as single agents or in combination using peritoneal metastatic models. RESULTS: In the analysis of biodistribution, (125)I-bevacizumab administrated i.p. indicated low peritoneal clearance. On the other hand, s.c. administration of (125)I-bevacizumab showed preferential accumulation in subcutaneous tumors and peritoneal nodules, with a high blood concentration. In peritoneal metastatic models, the effects of bevacizumab were found for both the growth inhibition of peritoneal nodules (P < 0.01) and the reduction of ascites (P < 0.05). These effects were more prominent by s.c. administration compared with i.p. administration and were increased in combination with i.p. paclitaxel. CONCLUSION: Bevacizumab should be administrated systemically compared to regionally, and the combination with i.p. paclitaxel has a potential to be useful for patients with peritoneal metastasis of gastric cancer.
171: Clinical and experimental medicine, 2009 Dec 24, 114(27)
VEGF-A/HGF induce Prox-1 expression in the chick embryo chorioallantoic membrane lymphatic vasculature.
[Abstract]Vascular endothelial growth factor-A (VEGF-A) and hepatocyte growth factor (HGF) are well known angiogenesis inductors and promoters in normal and pathologic conditions. Recent data showed that VEGF-A and HGF could also influence lymphangiogenesis but this matter has not been completely elucidated. Administration of VEGF-A and HGF in combination has been used to improve the angiogenic response in different experimental models, but their effects on lymphangiogenesis have not been investigated. The aim of this study was to characterize blood and lymphatic vascular response to VEGF-A/HGF administration. To this purpose, we built a pBlast VEGF-A/HGF combination suitable for in vivo research. By using as an experimental in vivo model the chick embryo chorioallantoic membrane (CAM) assay, we applied pBlast VEGF-A/HGF combination for 7 days. Results showed that VEGF-A/HGF combination was able to induce a strong angiogenic response and the expression of Prox-1 in the lymphatic endothelial cells of the CAM. The possible mechanisms involved have been speculated.
172: Recent results in cancer research. Fortschritte der Krebsforschung. Progr¨¨s dans les recherches sur le cancer, 2010, 180(1)
Targeting Inflammatory Cells to Improve Anti-VEGF Therapies in Oncology.
[Abstract]Vascular endothelial growth factor A (VEGF-A) is a well-characterized regulator of physiological and pathological angiogenesis. Multiple therapeutic compounds interfering with VEGF-A-regulated signal transduction pathways are currently being developed for the treatment of neoplasias and other malignancies associated with pathological angiogenesis. A major challenge in developing anti-VEGF therapies are tumor intrinsic refractoriness and the emergence of treatment-induced resistance. A variety of molecular and cellular mechanisms contribute to tumor angiogenesis, including the recruitment of bone marrow (BM)-derived endothelial cell progenitors (EPCs) and inflammatory cells to the tumor mass. Among the latter, two types of tumor infiltrating, inflammatory cells were recently identified to mediate refractoriness to anti-VEGF treatment: CD11b + Gr1+ myeloid derived suppressor cells (MDSC) and tumor-associated macrophages (TAMs). In this chapter, we review some of the inflammatory components regulating tumor angiogenesis and their roles in mediating refractoriness toward anti-VEGF treatment. In addition, we discuss potential therapeutic strategies targeting angiogenic pathways regulated by inflammatory cells. A better understanding of the biological and molecular events involved in mediating refractoriness to anti-VEGF treatment may help to further improve therapeutic strategies targeting tumor angiogenesis.
173: Recent results in cancer research. Fortschritte der Krebsforschung. Progr¨¨s dans les recherches sur le cancer, 2010, 180(1)
Angiogenesis Inhibition in Cancer Therapy : Platelet-Derived Growth Factor (PDGF) and Vascular Endothelial Growth Factor (VEGF) and their Receptors: Biological Functions and Role in Malignancy.
[Abstract]Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen in vitro and an angiogenic inducer in a variety of in vivo models. VEGF gene transcription is induced in particular in hypoxic cells. In developmental angiogenesis, the role of VEGF is demonstrated by the finding that the loss of a single VEGF allele results in defective vascularization and early embryonic lethality. Substantial evidence also implicates VEGF as a mediator of pathological angiogenesis. In situ hybridization studies demonstrate expression of VEGF mRNA in the majority of human tumors. Platelet-derived growth factor (PDGF) is mainly believed to be an important mitogen for connective tissue, and also has important roles during embryonal development. Its overexpression has been linked to different types of malignancies. Thus, it is important to understand the physiology of VEGF and PDGF and their receptors as well as their roles in malignancies in order to develop antiangiogenic strategies for the treatment of malignant disease.
174: International journal of cancer. Journal international du cancer, 2009 Dec 21, 67(1)
MMP-2 alters VEGF expression via alphaVbeta3 integrin-mediated PI3K/AKT signaling in A549 lung cancer cells.
[Abstract]Vascular endothelial growth factor (VEGF) is one of the most important angiogenic growth factors for tumor angiogenesis. Here, we sought to explore whether RNA interference (RNAi) targeting Matrix metalloproteinase-2 (MMP-2) could disrupt VEGF mediated angiogenesis in lung cancer. MMP-2 siRNA inhibited lung cancer cell-induced tube formation of endothelial cells in vitro; addition of recombinant human-MMP-2 restored angiogenesis. MMP-2 transcriptional suppression decreased VEGF, PI3K protein levels, and AKT phosphorylation in lung cancer cells. In addition, MMP-2 suppression decreased Hypoxia inducible factor-1alpha (HIF-1alpha), a transcription factor for VEGF, as determined by Electrophoretic mobility shift assay (EMSA). We also show that MMP-2 suppression disrupted phosphatidylinositol 3-kinase (PI3K) dependent VEGF expression; ectopic expression of myr-AKT restored VEGF inhibition. Further, MMP-2 suppression decreased the interaction of integrin-alphaVbeta3 and MMP-2 as confirmed by immunoprecipitation analyses. Studies with either function blocking integrin-alphaVbeta3 antibody or MMP-2 specific inhibitor (ARP-100) indicate that suppression of MMP-2 decreased integrin-alphaVbeta3-mediated induction of PI3K/AKT leading to decreased VEGF expression. Moreover, A549 xenograft tissue sections from mice that treated with MMP-2 siRNA showed reduced expression of VEGF, and the angiogenic marker, Factor-VIII. The inhibition of tumor angiogenesis in MMP-2 suppressed tumor sections was associated with decreased co-localization of integrin-alphaVbeta3 and MMP-2. In summary, these data provide new insights into the mechanisms underlying MMP-2-mediated VEGF expression in lung tumor angiogenesis. (c) 2009 UICC.
175: The Journal of experimental medicine, 2009 Dec 21, 67(1)
VEGF-A expression by HSV-1-infected cells drives corneal lymphangiogenesis.
[Abstract]Inflammatory lymphangiogenesis plays a crucial role in the development of inflammation and transplant rejection. The mechanisms of inflammatory lymphangiogenesis during bacterial infection, toll-like receptor ligand administration, and wound healing are well characterized and depend on ligands for the vascular endothelial grow factor receptor (VEGFR) 3 that are produced by infiltrating macrophages. But inflammatory lymphangiogenesis in nonlymphoid tissues during chronic viral infection is unstudied. Herpes simplex virus 1 (HSV-1) infection of the cornea is a leading cause of blindness and depends on aberrant host immune responses to antigen within the normally immunologically privileged cornea. We report that corneal HSV-1 infection drives lymphangiogenesis and that corneal lymphatics persist past the resolution of infection. The mechanism of HSV-1-induced lymphangiogenesis was distinct from the described mechanisms of inflammatory lymphangiogenesis. HSV-1-elicited lymphangiogenesis was strictly dependent on VEGF-A/VEGFR-2 signaling but not on VEGFR-3 ligands. Macrophages played no role in the induction of lymphangiogenesis and were not a detectable source of VEGF-A. Rather, using VEGF-A reporter transgenic mice, we have identified infected epithelial cells as the primary source of VEGF-A during HSV-1 infection. Our results indicate that HSV-1 directly induces vascularization of the cornea through up-regulation of VEGF-A expression.
176: Experimental cell research, 2009 Dec 17, 390(3)
Impairment of VEGF-A-stimulated lamellipodial extensions and motility of vascular endothelial cells by Chondromodulin-I, a cartilage-derived angiogenesis inhibitor.
[Abstract]Chondromodulin-I (ChM-I) is a cartilage-derived angiogenesis inhibitor that has been identified as inhibitory to the growth activity of vascular endothelial cells. In our present study, we demonstrate the anti-angiogenic activity of recombinant human ChM-I (rhChM-I) in mouse corneal angiogenesis and examine its action. We focus on the VEGF-A-induced migration of vascular endothelial cells, a critical regulatory step in angiogenesis. In a modified Boyden chamber assay, nanomolar concentrations of rhChM-I inhibited the chemotactic migration of human umbilical vein endothelial cells (HUVECs) induced by VEGF-A, as well as by FGF-2 and IGF-I. The ChM-I action was found to be endothelial cell-specific and independent of cell adhesions. Time-lapse analysis further revealed that rhChM-I markedly reduces VEGF-A-stimulated motility of HUVECs and causes frequent alterations of the moving front due to the appearance of multiple transient protrusions. This action involved the inhibition of cell spreading and the disrupted reorganization of the actin cytoskeleton upon VEGF-A stimulation. Consistent with these observations, rhChM-I was found to significantly reduce the activity of Rac1/Cdc42 during cell spreading, and the VEGF-A-induced Rac1 activity, but not its basal activity in quiescent cells. Taken together, our present data suggest that ChM-I impairs the VEGF-A-stimulated motility of endothelial cells by destabilizing lamellipodial extensions.
177: IDrugs : the investigational drugs journal, 2010 Jan, 13(1)
Current drug patenting for retinal diseases: Beyond VEGF inhibitors.
[Abstract]An analysis of patent applications that address strategies for the pharmacological treatment of retinal diseases that are not directly related to VEGF inhibition, published under the PCT during the 18-month period from January 2008 to June 2009, is presented. The largest number of therapeutic patent applications focused on attempts to correct visual cycle dysfunctions, complement overactivation or beta-amyloid deposition in drusen to control age-related macular degeneration (AMD). Biomarker-based and genetic diagnostic modalities that assess AMD risk were also frequently claimed in the patent applications, and have become a significant factor in patenting for ocular disorders. The fields of both visual cycle therapy and AMD biomarkers were dominated by non-corporate patent assignees. Diabetic retinopathy has not received as much attention from inventors compared with AMD; retinopathy of prematurity remains a field in which little specific patenting occurs.
178: Blood, 2009 Dec 18, 390(3)
AlphaB crystallin regulation of angiogenesis by modulation of VEGF.
[Abstract]alphaB-Crystallin is a chaperone belonging to the small heat shock protein family. Herein we show attenuation of intraocular angiogenesis in alphaB-crystallin knockout (-/-) mice in two models of intraocular disease: oxygen-induced retinopathy and laser-induced choroidal neovascularization (CNV). Vascular endothelial growth factor-A (VEGF) mRNA and hypoxia inducible factor-1alpha protein expression were induced during retinal angiogenesis, but VEGF protein expression remained low in alphaB-crystallin (-/-) retina vs wild-type mice, while VEGF-R2 expression was not affected. Both alphaB-crystallin and its phosphorylated form (pSer59) were expressed, and immunoprecipitation revealed alphaB-crystallin binding to VEGF, but not transforming growth factor-beta in cultured retinal pigment epithelial cells (RPE). alphaB-crystallin and VEGF are colocalized in the endoplasmic reticulum in RPE cells under chemical hypoxia. alphaB-crystallin (-/-) RPE showed low VEGF secretion under serum-starved condition compared to wild-type cells. VEGF is polyubiquitinated in control and alphaB-crystallin siRNA treated RPE; however, mono-tetra ubiquitinated VEGF increases with alphaB-crystallin knockdown. Endothelial cell apoptosis in newly formed vessels was greater in alphaB-crystallin (-/-) than wild-type mice. Proteasomal inhibition in alphaB-crystallin (-/-) mice partially restores VEGF secretion and angiogenic phenotype in CNV. Our studies indicate an important role for alphaB-crystallin as a chaperone for VEGF in angiogenesis and its potential as a therapeutic target.
179: Gynecologic oncology, 2010 Mar, 116(3)
RhoC expression level is correlated with the clinicopathological characteristics of ovarian cancer and the expression levels of ROCK-I, VEGF, and MMP9.
[Abstract]OBJECTIVE: To determine the clinicopathological significance of RhoC expression in human ovarian cancer and its effect on the expression of vascular endothelial growth factor (VEGF), Rho-associated coiled-coil-forming kinase (ROCK), and metal matrix proteinases (MMPs). METHODS: Tissue samples from normal ovaries, benign ovarian tumors, and epithelial ovarian cancer were collected. The mRNA and protein expression levels of RhoC, ROCK-I, VEGF, and MMP9 were assessed using reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blot, and compared to the clinicopathological characteristics of the sample of origin using the Pearson method of correlation analysis. Small interfering RNA (siRNA) was also used to target RhoC expression in the OVCAR3 and CaOV3 ovarian cancer cell lines, after which cell invasion and migration assays were performed, and the expression of ROCK-I, VEGF, and MMP9 was evaluated. RESULTS: The expression levels of RhoC, ROCK-I, VEGF, and MMP9 mRNA and protein were significantly higher in ovarian cancer, showing a correlation with clinical stage but not histological type. RhoC expression was positively correlated with ROCK-I, VEGF, and MMP9 expression. Decreased RhoC expression in siRNA-targeted cells inhibited their ability to invade and migrate, as well as inhibiting ROCK-I, VEGF, and MMP9 expression. CONCLUSION: The expression level of RhoC is correlated to clinical stage and vascularization in ovarian cancer.
180: Expert opinion on therapeutic patents, 2010 Jan, 20(1)
Anti-VEGF agents for age-related macular degeneration.
[Abstract]Age-related macular degeneration (AMD) is a leading cause of legal blindness in the elderly in the industrialized world and the third major cause of blindness around the globe. Although neovascular AMD is less prevalent than atrophic AMD, it accounts for most cases with severe visual loss from AMD. VEGF seems to be a key contributary factor in the pathophysiology underlying neovascular AMD. Until recently, treatment options for neovascular AMD were limited. With the recent development of anti-VEGF therapies that have demonstrated efficacy in studies with broad eligibility criteria, the repertoire of treatments for neovascular AMD has been significantly expanded to now include the various recognized angiographic lesion subtypes. To discuss recent anti-VEGF agents in the management of AMD. Although therapy with anti-VEGF agents is the gold standard with promising results, many intravitreal injections are often required, and they do not cure all cases of wet AMD. With the recent advances in the medical therapy of exudative AMD, there is reason to be optimistic about future management of AMD as well.
181: The FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2009 Dec 17, 390(3)
Cardiomyocyte VEGFR-1 activation by VEGF-B induces compensatory hypertrophy and preserves cardiac function after myocardial infarction.
[Abstract]Mounting evidence indicates that the function of members of the vascular endothelial growth factor (VEGF) family extends beyond blood vessel formation. Here, we show that the prolonged intramyocardial expression of VEGF-A165 and VEGF-B167 on adeno-associated virus-mediated gene delivery determined a marked improvement in cardiac function after myocardial infarction in rats, by promoting cardiac contractility, preserving viable cardiac tissue, and preventing remodeling of the left ventricle (LV) over time. Consistent with this functional outcome, animals treated with both factors showed diminished fibrosis and increased contractile myocardium, which were more pronounced after expression of the selective VEGF receptor-1 (VEGFR-1) ligand VEGF-B, in the absence of significant induction of angiogenesis. We found that cardiomyocytes expressed VEGFR-1, VEGFR-2, and neuropilin-1 and that, in particular, VEGFR-1 was specifically up-regulated in hypoxia and on exposure to oxidative stress. VEGF-B exerted powerful antiapoptotic effect in both cultured cardiomyocytes and after myocardial infarction in vivo. Finally, VEGFR-1 activation by VEGF-B was found to elicit a peculiar gene expression profile proper of the compensatory, hypertrophic response, consisting in activation of alphaMHC and repression of betaMHC and skeletal alpha-actin, and an increase in SERCA2a, RYR, PGC1alpha, and cardiac natriuretic peptide transcripts, both in cultured cardiomyocytes and in infarcted hearts. The finding that VEGFR-1 activation by VEGF-B prevents loss of cardiac mass and promotes maintenance of cardiac contractility over time has obvious therapeutic implications.-Zentilin, L., Puligadda, U., Lionetti, V., Zacchigna, S., Collesi, C., Pattarini, L., Ruozi, G., Camporesi, S., Sinagra, G., Pepe, M., Recchia, F. A., Giacca, M. Cardiomyocyte VEGFR-1 activation by VEGF-B induces compensatory hypertrophy and preserves cardiac function after myocardial infarction.
182: Journal of biomedical materials research. Part A, 2009 Dec 14, 106(50)
Biocompatibility of porous polyethylene implants tissue-engineered by extracellular matrix and VEGF.
[Abstract]Rapid ingrowth of blood vessels and low inflammatory response are considered major prerequisites for successful implantation of biomaterials in reconstructive surgery. Aim of the present study was to evaluate whether tissue-engineered porous polyethylene (PPE) implants providing extracellular matrix components (ECM) and vascular endothelial growth factor (VEGF) in vivo improve microvascular ingrowth and mechanical integration with regard to initial inflammatory responses. PPE implants (3 x 3 x 0.1 mm(3), pore size approximately 100-200 mum) were tissue-engineered by incorporation of ECM components (GFR-Matrigel) adding recombinant murine VEGF (1 mug/mL) and grafted into dorsal skinfold chamber preparations of C57BL/6 mice. Control animals received uncoated implants or implants coated with ECM components alone (n = 6 per group). Using in vivo fluorescence microscopy angiogenic activity and inflammatory leukocyte-endothelial cell interactions were analyzed for 2weeks. Finally, mechanical integration was quantified by measurement of dynamic desintegration strengths at the host-implant border. Functional vessel density, red blood cell velocity, and vessel diameters increased continuously in all groups indicating that rapid microvascular integration of PPE occurred even without incorporation of ECM or VEGF. However, a transient initial inflammatory response with increased leukocyte-endothelial cell adherence on day 7 in uncoated control implants was efficiently reduced by incorporation of ECM and VEGF. Measurement of dynamic breaking strengths revealed no significant differences between the groups although there was a tendency to improved mechanical integration in tissue-engineered implants. Therefore, novel tissue- engineered constructs of PPE implants providing ECM and VEGF in high local concentrations can increase biocompatibility especially under unfavorable conditions for implantation. (c) 2009 Wiley Periodicals, Inc. J Biomed Mater Res 2010.
183: British journal of cancer, 2009 Dec 15, 390(3)
Biomarkers of angiogenesis and their role in the development of VEGF inhibitors.
[Abstract]Vascular endothelial growth factor (VEGF) has been confirmed as an important therapeutic target in randomised clinical trials in multiple disease settings. However, the extent to which individual patients benefit from VEGF inhibitors is unclear. If we are to optimise the use of these drugs or develop combination regimens that build on this efficacy, it is critical to identify those patients who are likely to benefit, particularly as these agents can be toxic and are expensive. To this end, biomarkers have been evaluated in tissue, in circulation and by imaging. Consistent drug-induced increases in plasma VEGF-A and blood pressure, as well as reductions in soluble VEGF-R2 and dynamic contrast-enhanced MRI parameters have been reported. In some clinical trials, biomarker changes were statistically significant and associated with clinical end points, but there is considerable heterogeneity between studies that are to some extent attributable to methodological issues. On the basis of observations with these biomarkers, it is now appropriate to conduct detailed prospective studies to define a suite of predictive, pharmacodynamic and surrogate response biomarkers that identify those patients most likely to benefit from and monitor their response to this novel class of drugs.British Journal of Cancer advance online publication, 15 December 2009; doi:10.1038/sj.bjc.6605483 www.bjcancer.com.
184: The EMBO journal, 2010 Jan 20, 29(2)
Increased skeletal VEGF enhances beta-catenin activity and results in excessively ossified bones.
[Abstract]Vascular endothelial growth factor (VEGF) and beta-catenin both act broadly in embryogenesis and adulthood, including in the skeletal and vascular systems. Increased or deregulated activity of these molecules has been linked to cancer and bone-related pathologies. By using novel mouse models to locally increase VEGF levels in the skeleton, we found that embryonic VEGF over-expression in osteo-chondroprogenitors and their progeny largely pheno-copied constitutive beta-catenin activation. Adult induction of VEGF in these cell populations dramatically increased bone mass, associated with aberrant vascularization, bone marrow fibrosis and haematological anomalies. Genetic and pharmacological interventions showed that VEGF increased bone mass through a VEGF receptor 2- and phosphatidyl inositol 3-kinase-mediated pathway inducing beta-catenin transcriptional activity in endothelial and osteoblastic cells, likely through modulation of glycogen synthase kinase 3-beta phosphorylation. These insights into the actions of VEGF in the bone and marrow environment underscore its power as pleiotropic bone anabolic agent but also warn for caution in its therapeutic use. Moreover, the finding that VEGF can modulate beta-catenin activity may have widespread physiological and clinical ramifications.
185: Experimental cell research, 2010 Feb 15, 316(4)
VEGF elicits epithelial-mesenchymal transition (EMT) in prostate intraepithelial neoplasia (PIN)-like cells via an autocrine loop.
[Abstract]Vascular endothelial growth factor (VEGF) is overexpressed during the transition from prostate intraepithelial neoplasia (PIN) to invasive carcinoma. We have mimicked such a process in vitro using the PIN-like C3(1)/Tag-derived Pr-111 cell line, which expresses low levels of VEGF and exhibits very low tumorigenicity in vivo. Elevated expression of VEGF164 in Pr-111 cells led to a significant increase in tumorigenicity, invasiveness, proliferation rates and angiogenesis. Moreover, VEGF164 induced strong changes in cell morphology and cell transcriptome through an autocrine mechanism, with changes in TGF-beta1- and cytoskeleton-related pathways, among others. Further analysis of VEGF-overexpressing Pr-111 cells or following exogenous addition of recombinant VEGF shows acquisition of epithelial-mesenchymal transition (EMT) features, with an increased expression of mesenchymal markers, such as N-cadherin, Snail1, Snail2 (Slug) and vimentin, and a decrease in E-cadherin. Administration of VEGF led to changes in TGF-beta1 signaling, including reduction of Smad7 (TGF-beta inhibitory Smad), increase in TGF-betaR-II, and translocation of phospho-Smad3 to the nucleus. Our results suggest that increased expression of VEGF in malignant cells during the transition from PIN to invasive carcinoma leads to EMT through an autocrine loop, which would promote tumor cell invasion and motility. Therapeutic blockade of VEGF/TGF-beta1 in PIN lesions might impair not only tumor angiogenesis, but also the early dissemination of malignant cells outside the epithelial layer.
186: Journal of neurotrauma, 2009 Dec 14, 106(50)
VEGF IS INVOLVED IN MEDIATING INCREASED DE NOVO HIPPOCAMPAL NEUROGENESIS IN RESPONSE TO TRAUMATIC BRAIN INJURY.
[Abstract]Stimulating the endogenous repair process after traumatic brain injury (TBI) can be an important approach in neuroregenerative medicine. Vascular endothelial growth factor (VEGF) is one of the molecules that can increase de novo hippocampal neurogenesis. Here we tested whether VEGF signaling through Flk1 (VEGF receptor 2) is involved in the neurogenic process after experimental TBI. We found that Flk1 is expressed both by neuroblasts in the subgranular layer (SGL) and by maturing granule neurons in the adult dentate gyrus (DG) of the hippocampus. After lateral fluid percussion TBI (LFP-TBI) in the rat, we detected elevated VEGF levels and also increased numbers of de novo neurons in the ipsilateral DG. To directly test the involvement of VEGF and Flk1 in the neurogenic process, we delivered recombinant VEGF or SU5416, an inhibitor to Flk1, into the ipsilateral cerebral ventricle of injured animals. We found that VEGF infusion significantly increased the number of BrdU+/Prox1+ new neurons, decreased the number of TUNEL positive cells but did not change the number of BrdU+ newborn cells per se. Infusion with SU5416 caused no significant changes. Our results suggest that small i, Ukrainian) VEGF is a part of the molecular signaling network that mediates de novo hippocampal neurogenesis after TBI; small i, Ukrainiansmall i, Ukrainian) VEGF predominantly mediates survival of de novo granule neurons rather than proliferation of neuroblasts in the injured brain; small i, Ukrainiansmall i, Ukrainiansmall i, Ukrainian) additional VEGF receptor(s) and/or other molecular mechanism(s) are also involved in mediating increased neurogenesis following injury.
187: Proceedings of the National Academy of Sciences of the United States of America, 2009 Dec 15, 106(50)
Systemic anti-VEGF treatment strongly reduces skin inflammation in a mouse model of psoriasis.
[Abstract]Although(,) vascular remodeling is a hallmark of many chronic inflammatory disorders such as rheumatoid arthritis, inflammatory bowel disease, and psoriasis, anti-vascular strategies to treat these conditions have received little attention to date. We investigated the anti-inflammatory activity of systemic blockade of VEGF-A by the inhibitory monoclonal antibody G6-31, employing a therapeutic trial in a mouse model of psoriasis. Simultaneous deletion of JunB and c-Jun (DKO*) in the epidermis of adult mice leads to a psoriasis-like phenotype with hyper- and parakeratosis and increased subepidermal vascularization. Moreover, an inflammatory infiltrate and elevated levels of cytokines/chemokines including TNFalpha, IL-1alpha/beta, IL-6, and the innate immune mediators IL-22, IL-23, IL-23R, and IL-12p40 are detected. Here we show that anti-VEGF antibody treatment of mice already displaying disease symptoms resulted in an overall improvement of the psoriatic lesions leading to a reduction in the number of blood vessels and a significant decrease in the size of dermal blood and lymphatic vessels. Importantly, anti-VEGF-treated mice showed a pronounced reduction of inflammatory cells within the dermis and a normalization of epidermal differentiation. These results demonstrate that systemic blockade of VEGF by an inhibitory antibody might be used to treat patients who have inflammatory skin disorders such as psoriasis.
188: Cancer investigation, 2010 May, 28(4)
Vascular endothelial growth factor (VEGF) serum levels are associated with survival in early stages of lung cancer patients.
[Abstract]AIM: Evaluate the serum vascular endothelial growth factor (VEGF) levels in the prognosis of lung cancer patients. METHODS: Fifty-four serum samples were analyzed for VEGF concentrations (79.3% nonsmall cell lung cancer (NSCLC) and 20.7% small cell lung cancer). RESULTS: Patients with serum VEGF-A levels higher than the mean of the patients studied (434.93 pg/mL) presented a shorter median survival time than those with lower levels (p =.04), as in patients with NSCLC tumors (p =.04) and in those with stages I-II (p <.05), and high serum VEGF-A levels. CONCLUSION: Elevated VEGF serum levels have a negative prognostic impact on survival in NSCLC and early stages of lung cancer patients.
189: Blood, 2010 Jan 28, 115(4)
VEGF/VEGFR2 interaction down-regulates matrix metalloproteinase-9 via STAT1 activation and inhibits B chronic lymphocytic leukemia cell migration.
[Abstract]B-cell chronic lymphocytic leukemia (B-CLL) migration involves several molecules, including matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF). We have studied whether VEGF regulates MMP-9. VEGF significantly reduced MMP-9 protein expression in a dose-dependent manner, measured by gelatin zymography. Blocking the VEGFR2 receptor restored MMP-9 levels, implicating this receptor in the observed effect. Down-regulation of MMP-9 by VEGF resulted in significant inhibition of B-CLL cell migration through Matrigel or human umbilical vein endothelial cells, confirming the crucial role of MMP-9 in these processes. Reverse-transcription polymerase chain reaction analyses revealed that VEGF regulated MMP-9 at the transcriptional level. Indeed, VEGF induced STAT1 tyrosine phosphorylation, and this was blocked by inhibiting VEGFR2. STAT1 was responsible for MMP-9 down-regulation, as STAT1 gene silencing restored MMP-9 production and B-CLL cell migration in the presence of VEGF. Thus, the levels of VEGF and MMP-9 influence B-CLL cell expansion and both molecules could constitute therapeutic targets for this disease.
190: Biomaterials, 2009 Dec 4, 28(12)
The effect of VEGF functionalization of titanium on endothelial cells in vitro.
[Abstract]One of the key challenges in bone healing and regeneration is the engineering of an implant with surface properties that can enhance revascularization to meet the metabolic demands of recovery. Successful implant integration into the surrounding tissue is highly dependent on the crucial role of blood supply in driving bone repair and development. Therapeutic application of vascular endothelial growth factor (VEGF) is a promising approach to enhance blood supply and healing through revascularization around an engineered implant in a regulated manner. In this in vitro study, we investigated the effects of immobilized VEGF on titanium alloy substrates coated with thin adherent polydopamine film. X-ray photoelectron spectroscopy (XPS) was used to determine the chemical composition of the surfaces at various stages of surface functionalization to verify the successful deposition of polydopamine and VEGF on the metal surface. Surface topography was evaluated from the surface profile determined by atomic force microscopy (AFM). The functionalized surfaces showed a significant increase in human dermal microvascular endothelial cells (HDMECs) attachment, viability and proliferation compared to the pristine substrate. Furthermore the immobilized VEGF was able to induce the differentiation of human mesenchymal stem cells (hMSCs) into endothelial cells. Therefore utilizing the reactivity of polydopamine films to immobilize VEGF onto metal substrates may provide a promising approach for application in situations where revascularization around implants would be beneficial in improving bone healing and implant integration.
191: Journal of controlled release : official journal of the Controlled Release Society, 2009 Dec 3, 28(12)
Local controlled release of VEGF and PDGF from a combined brushite- chitosan system enhances bone regeneration.
[Abstract]The two growth factors VEGF and PDGF are involved in the process of bone regeneration. For this reason, we developed a brushite-chitosan system which controls the release kinetics of incorporated VEGF and PDGF to enhance bone healing. PDGF (250ng) was incorporated in the liquid phase. Alginate microsphere-encapsulated VEGF (350ng) was pre-included in small cylindrical chitosan sponges. VEGF and PDGF release kinetics and tissue distribution were determined using iodinated ((125)I) growth factor. In vivo, PDGF was more rapidly delivered from these systems implanted in rabbit femurs than VEGF. 80% of PDGF were released by the end of two weeks while only 70% of VEGF were delivered after a period of three weeks. Both GFs released from the brushite-chitosan constructs remained located around the implantation site (5cm) with negligible systemic exposure. A PDGF bone peak concentration of approximately 5ng/g was achieved on the 4th day. Thereafter, PDGF concentrations stayed higher than 2ng/g during the first week. These scaffolds also provided a local VEGF bone concentration above 3ng/g during a total of 4weeks, with a peak concentration of 5.5ng/g on the 7th day. The present work demonstrates that our brushite-chitosan system is capable of controlling the release rate and localization of the both GFs within a bone defect. The effect on bone formation was considerably enhanced with PDGF loaded brushite-chitosan scaffolds as well as with the PDGF/VEGF combination.
192: Comparative biochemistry and physiology. Part A, Molecular & integrative physiology, 2009 Dec 2, 28(12)
Investigating expression profiles of VEGF-Flk, and Angpt1 during development of gas glands in Japanese eel (Anguilla japonica).
[Abstract]Angiogenesis is a highly regulated physiological process in animals. Angiopoietin-1 (Angpt1) induces the signaling pathways related to vessel maturation in late phase of angiogenesis, which recruits pericytes supplements to make compact interaction with vessel tubes. There are only few data showing Angpt1 functions in fish. By using degenerate primers, partial sequence (812bp) of Angpt1 was cloned from Anguilla japonica, and deduced amino acids showed 80% similarity to those of zebrafish. Physiological functions of cloned eel Angpt1 were studied by in vitro and in vivo manipulations with gas glands (rete mirabile) taken as the tested target tissues. RT-PCR and immunofluorescent staining techniques were performed to examine the expression patterns of Angpt1 as well as VEGF-Flk. Experimental data showed that, in vitro, bFGF, PPAR beta agonist, and estradiol affected Angpt1 expression; while cobalt ions, a VEGF expression-inducer, did not affect Angpt1 expression. In vivo, expression levels of Angpt1 increased with body growth. Furthermore, Angpt1 expressions increased significantly in the late stage of gas glands in the stimulated eel. Successive expression patterns on VEGF-Flk, and Angpt1 on different development stages of gas glands were observed. Our results suggest that the original function of angiopoietin-1 on angiogenesis is conserved during evolution.
193: Neuropeptides, 2009 Dec 2, 28(12)
Bradykinin B(1) receptor antagonist R954 inhibits eosinophil activation/proliferation/migration and increases TGF-beta and VEGF in a murine model of asthma.
[Abstract]In the present study the effects of bradykinin receptor antagonists were investigated in a murine model of asthma using BALB/c mice immunized with ovalbumin/alum and challenged twice with aerosolized ovalbumin. Twenty four hours later eosinophil proliferation in the bone marrow, activation (lipid bodies formation), migration to lung parenchyma and airways and the contents of the pro-angiogenic and pro-fibrotic cytokines TGF-beta and VEGF were determined. The antagonists of the constitutive B(2) (HOE 140) and inducible B(1) (R954) receptors were administered intraperitoneally 30min before each challenge. In sensitized mice, the antigen challenge induced eosinophil proliferation in the bone marrow, their migration into the lungs and increased the number of lipid bodies in these cells. These events were reduced by treatment of the mice with the B(1) receptor antagonist. The B(2) antagonist increased the number of eosinophils and lipid bodies in the airways without affecting eosinophil counts in the other compartments. After challenge the airway levels of VEGF and TGF-beta significantly increased and the B(1) receptor antagonist caused a further increase. By immunohistochemistry techniques TGF-beta was found to be expressed in the muscular layer of small blood vessels and VEGF in bronchial epithelial cells. The B(1) receptors were expressed in the endothelial cells. These results showed that in a murine model of asthma the B(1) receptor antagonist has an inhibitory effect on eosinophils in selected compartments and increases the production of cytokines involved in tissue repair. It remains to be determined whether this effects of the B(1) antagonist would modify the progression of the allergic inflammation towards resolution or rather towards fibrosis.
194: Chinese journal of cancer, 2009 Dec 5, 28(12)
Vascular endothelial growth factor (VEGF)-D in association with VEGF receptor-3 in lymphatic metastasis of breast cancer.
[Abstract]Breast carcinoma is the most common malignant tumor in women. For these patients, lymph node metastasis is one of the most important prognostic factors. Recent studies suggest that lymphangiogenesis can contribute to the lymphatic metastasis in tumors. Several members of vascular endothelial growth factor (VEGF) family, such as VEGF-C, VEGF-D, and VEGF receptor-3 (VEGFR-3), have been found to promote lymphangiogenesis in breast cancer. However, there are still some controversy about the prognostic value of VEGF-D and the relation between VEGFR-3 and lymphangiogenesis. This article tried to provide an overview of the research progress of lymphangiogenic factor VEGF-D and its receptor VEGFR-3 in lymphatic metastasis of breast cancer.
195: Gene therapy, 2009 Dec 3, 28(12)
Electroporation-mediated delivery of a naked DNA plasmid expressing VEGF to the porcine heart enhances protein expression.
[Abstract]Gene therapy is an attractive method for the treatment of cardiovascular disease. However, using current strategies, induction of gene expression at therapeutic levels is often inefficient. In this study, we show a novel electroporation (EP) method to enhance the delivery of a plasmid expressing an angiogenic growth factor (vascular endothelial growth factor, VEGF), which is a molecule previously documented to stimulate revascularization in coronary artery disease. DNA expression plasmids were delivered in vivo to the porcine heart with or without coadministered EP to determine the potential effect of electrically mediated delivery. The results showed that plasmid delivery through EP significantly increased cardiac expression of VEGF compared with injection of plasmid alone. This is the first report showing successful intracardiac delivery, through in vivo EP, of a protein expressing plasmid in a large animal.Gene Therapy advance online publication, 3 December 2009; doi:10.1038/gt.2009.153.
196: Cardiovascular research, 2009 Dec 2, 28(12)
The effects of VEGF-R1 and VEGF-R2 ligands on angiogenic responses and left ventricular function in mice.
[Abstract]AIMS Vascular endothelial growth factors (VEGFs) and their receptors (VEGF-Rs) are among the most powerful factors regulating vascular growth. However, it has remained unknown whether stimulation of VEGF-R1, VEGF-R2 or both of the receptors produces the best angiogenic responses in myocardium. The aim of this study was to compare VEGF-R1 specific ligand VEGF-B(186), VEGF-R2 specific ligand VEGF-E and VEGF-A(165,) which stimulates both receptors, regarding their effects on angiogenesis and left ventricular function in mice. METHODS High-resolution echocardiography was used to guide closed-chest injections of adenoviral vectors expressing VEGF-B(186,) VEGF-E and VEGF-A(165) into the anterior wall of the left ventricle in C57Bl/6J mice. Angiogenic and functional effects were analyzed using histology, ultrasound and perfusion analyses 6 (D6) and 14 (D14) days after the adenoviral (Ad) injection. RESULTS Adenoviral (Ad)VEGF-A(165) induced a strong angiogenic response seen as an enlargement of myocardial capillaries while angiogenesis induced by AdVEGF-B(186) and AdVEGF-E seemed more physiological. The increase in capillary area was accompanied with an increase in myocardial perfusion at D6 after the gene injection. AdVEGF-A(165) and AdVEGF-E induced endothelial-specific proliferation while AdVEGF-B(186) mostly induced proliferation of cardiomyocytes. AdVEGF-A(165) induced more pronounced tissue damage than AdVEGF-B(186) and AdVEGF-E. Left ventricular function measured as ejection fraction did not change during the follow-up. AdVEGF-A(165) increased both VEGF-R1 and VEGF-R2 protein expression while AdVEGF-B(186) and AdVEGF-E did not affect endogenous receptor expression levels. CONCLUSIONS AdVEGF-B(186) and AdVEGF-E are equally potent in inducing therapeutic angiogenesis in mouse myocardium and produce less side effects than AdVEGF-A(165).
197: Brain research bulletin, 2010 Mar 16, 81(4-5)
Adenoviral vector-mediated transduction of VEGF improves neural functional recovery after hypoxia-ischemic brain damage in neonatal rats.
[Abstract]Previous studies have showed that vascular endothelial growth factor (VEGF) displayed neurotrophic and neuroprotective activities. To examine whether target delivery of VEGF gene directly into brain may prevent ischemic brain damage, the VEGF expression adenoviral vectors, AVHP.VEGF-with 476bp of the human preproendothelin-1 (ppET-1) promoter and 35bp of the hypoxia-reponse element (HRE) driving VEGF expression and CMV.VEGF were transferred into hypoxic-induced ischemic (HI) rat brains. Seven-day-old rats that were underwent left carotid ligation followed by 2h of hypoxic stress (8% O(2) at 37 degrees C) were received VEGF adenoviral vectors or buffer (PBS) injection 3 days after HI. The body weight, VEGF expression, neuronal apoptosis, cerebral morphology and brain functional assays were performed between 7 and 28 days after HI. There were remarkable increases in the body weight and VEGF protein expression, and decrease in the number of TUNEL-positive cells in the VEGF vector groups as compared with PBS group. The VEGF vector groups also had better brain functional performs than PBS group. The better performs by the animals that received VEGF vectors may be directly linked to the inhibitory effect of VEGF on neuronal apoptosis because the animals had less neural loss in the cortex and hippocampal CA1 region as compared with PBS group. Overall, these results indicated that over-expression of VEGF in the brain exerted a neuroprotective effect and promoted neural functional recovery in neonatal rats after hypoxic-ischemic brain damage, suggesting that in vivo target VEGF gene transfer to brain may be a promising approch for the treatment of such implications.
198: Bone, 2010 Mar, 46(3)
Plasma VEGF determination in disseminated lymphangiomatosis-Gorham-Stout syndrome: a marker of activity? A case report with a 5-year follow-up.
[Abstract]Disseminated lymphangiomatosis and Gorham-Stout disease are being considered as two forms of a single rare disease, characterized by a proliferation of lymphatic vessels, triggered by lymphangiogenic factors. There is no biological marker of the disease. Plasma VEGF might be a useful tool since the recent demonstration of its pivotal role in the mechanism of this disease. A 45-year-old woman with a history of disseminated lymphangiomatosis involving mediastinum, retroperitoneum, spleen and systemic bones for 29 years was treated with Interferon alpha 2b at a dosage of 7.5 to 15 million IU 3 times a week for 5 years. Plasma VEGF quantification was performed twice a year and showed a marked increase before therapy, which normalize after 18 months of treatment with Interferon. The normalization of plasma VEGF is correlated with the clinical improvement objectively appraised by a marked reduction of spleen lesions and significant improvement of the other damages in soft tissues and bones. Thus, we conclude that plasma VEGF determination should be considered for diagnosis and follow-up of the course and the treatment of disseminated lymphangiomatosis-Gorham-Stout disease.
199: Antioxidants & redox signaling, 2010 Jun 15, 12(12)
Cigarette smoke-induced oxidative/nitrosative stress impairs VEGF- and fluid shear stress-mediated signaling in endothelial cells.
[Abstract]VEGF receptor 2 (VEGFR2), a tyrosine kinase receptor, is activated by VEGF and fluid shear stress (FSS), and its downstream signaling is important in the regulation of endothelial functions, such as cell migration, endothelium-dependent relaxation, and angiogenesis. Cigarette smoke (CS) is known to cause oxidative/nitrosative stress, leading to modifications of tyrosine kinase receptors and impaired downstream signaling. We hypothesized that CS-induced oxidative/nitrosative stress impairs VEGF- and FSS-mediated VEGFR2 activation, leading to endothelial dysfunction. Human lung microvascular endothelial cells and human umbilical vein endothelial cells were treated with different concentrations of cigarette smoke extract (CSE) to investigate the VEGF- or FSS-mediated VEGFR2 phosphorylation and its downstream signaling involved in endothelial function. CSE treatment impaired both VEGF- and FSS-mediated VEGFR2 phosphorylation, resulting in impaired endothelial nitric oxide synthase (eNOS) phosphorylation by Akt. CS-derived reactive oxygen/nitrogen species react with VEGFR2, rendering VEGFR2 inactive for its downstream signaling. Pretreatment with nitric oxide scavenger (PTIO), reactive oxygen species scavengers (combination of SOD with catalase), and N-acetyl-L-cysteine, significantly attenuated the CSE-induced impairment of VEGF-mediated Akt and eNOS phosphorylation. These findings suggest that CSE-induced oxidative/nitrosative stress impairs VEGF- and FSS-mediated endothelial cell function and has important implications in the pathogenesis of CS-induced pulmonary and cardiovascular diseases associated with endothelial dysfunction.
200: Journal of agricultural and food chemistry, 2010 Jan 27, 58(2)
Dietary compound quercitrin dampens VEGF induction and PPARgamma activation in oxidized LDL-exposed murine macrophages: association with scavenger receptor CD36.
[Abstract]Oxidized LDL (oxLDL) has been implicated in the pathogenesis of atherosclerosis accompanying lipid-laden cell appearance, inflammatory responses, and vascular dysfunction. This study examined the potentials of polyphenol quercitrin to inhibit oxLDL induction of scavenger receptor A (SR-A) and CD36 involving activation of peroxisome proliferator-activated receptor gamma (PPARgamma). J774A1 murine macrophages were cultured with 10 microg/mL Cu(2+)-oxLDL for various times in the presence of 1-10 micromol/L quercitrin. Cu(2+)-oxLDL at the given concentration facilitated macrophage proliferation and enhanced oxLDL uptake. Quercitrin dampened oxLDL uptake and lipid accumulation elevated in macrophages exposed to oxLDL. Western blot analysis revealed that 10 microg/mL oxLDL upregulated expression of SR-A and CD36, which was rapidly abolished at the transcriptional levels by 10 micromol/L quercitrin within 4 h. Quercitrin diminished production of proinflammatory and proatherogenic vascular endothelial growth factor that augmented through the oxLDL binding to CD36. Similarly, quercitrin repressed expression of macrophage inflammatory protein-2 and monocyte chemoattractant protein-1 involved in monocyte trafficking and macropahage migration. In addition, quercitrin attenuated oxLDL-induced transcriptional activation of PPARgamma leading to CD36 induction. Furthermore, quercitrin alleviated macrophage uptake of oxLDL through interfering with PKC-PPAR signaling cascades. These results demonstrate that quercitrin blocked oxLDL uptake, cholesterol influx and lipid-laden foam cell formation through inhibiting induction of SR and VEGF linked to PKCalpha-PPARgamma-responsive pathways. Therefore, quercitrin may be an antiatherogenic agent blocking foam cell formation pertaining to induction of SR and VEGF.
201: Cancer biology & therapy, 2009 Dec 19, 8(23)
Caveolin-1 regulates VEGF-stimulated angiogenic activities in prostate cancer and endothelial cells.
[Abstract]Caveolin-1 (cav-1) is a multifunctional protein and major component of caveolae membranes serving important functions related to signal transduction, endocytosis, transcytosis, and molecular transport. We previously showed that cav-1 is overexpressed and secreted by metastatic prostate cancer cells. We now report that cav-1 gene transduction (Adcav-1) or recombinant cav-1 (rcav-1) protein treatment of cav-1-negative prostate cancer cell line LP-LNCaP or cav-1(-/-) endothelial cells potentiated VEGF-stimulated angiogenic signaling. Downregulation of cav-1 in prostate cancer cell line PC-3 or human umbilical vein endothelial cells (HUVECs) through cav-1 siRNA significantly reduced basal and VEGF-stimulated phosphorylation of VEGFR2 (Y951), PLCgamma1 (Y783) and/or Akt (S473 & T308) relative to those in control siRNA treated cells. Additionally rcav-1 stimulation of cav-1 siRNA treated HUVECs restored this signaling pathway. Confocal microscopy and immunoprecipitation analysis revealed association and colocalization of VEGFR2 and PLCgamma1 with cav-1 following VEGF stimulation in HUVECs. Interestingly, treatment of HUVECs with cav-1 scaffolding domain (CSD) caused significant reduction in the VEGF-stimulated phosphorylation of VEGFR2, PLCgamma1 and Akt suggesting that CSD inhibits cav-1-mediated angiogenic signaling. VEGF stimulation of HUVECs significantly increased tubule length and cell migration, but this stimulatory effect was significantly reduced by cav-1 siRNA and/or CSD treatment. The present study demonstrates that cav-1 regulates VEGF-stimulated VEGFR2 autophosphorylation and activation of downstream angiogenic signaling, possibly through compartmentalization of specific signaling molecules. Our results provide mechanistic insight into the role of cav-1 in prostate cancer and suggest the use of CSD as a therapeutic tool to suppress angiogenic signaling in prostate cancer.
202: Growth factors (Chur, Switzerland), 2009 Dec, 27(6)
Hypoxia but not cigarette smoke modulates VEGF secretion from human T cells.
[Abstract]Vascular endothelial growth factor (VEGF) is an important mitogen with multiple functions. In the present study we investigated whether T cell secreted VEGF and inflammatory cytokines were modulated by cigarette smoke and by a hypoxic microenvironment. T cells from peripheral blood of healthy donors were activated under normoxia (21% O(2)) or hypoxia (1-2% O(2)) with or without exposure to cigarette smoke extract. T cells were also obtained from patients with chronic obstructive pulmonary disease (COPD), a smoking-related disease characterized by accumulation of both CD4+ and CD8+T cells. Hypoxia stimulated VEGF secretion from activated T cells, whereas the release of IL-4, IL-6, IL-10, IL-13, IFN-gamma and tumour necrosis factor were not altered. Cigarette smoke extract did not affect VEGF secretion neither in hypoxia nor in normoxia, whereas the secretion of all cytokines was inhibited by the extract in both conditions. When recombinant VEGF was added the smoke-induced inhibition of the IFN-gamma and IL-13 was not observed. Activated T cells from COPD-patients secreted significantly (p < 0.05) more VEGF compared to T cells from healthy individuals. Our data suggest that both cigarette smoke extract and hypoxia modulate the T cell response. This may be of importance in diseases characterized by T cell accumulation, such as COPD.
203: Laboratory investigation; a journal of technical methods and pathology, 2010 Jan, 90(1)
Nephron-deficient Fvb mice develop rapidly progressive renal failure and heavy albuminuria involving excess glomerular GLUT1 and VEGF.
[Abstract]Reduced nephron numbers may predispose to renal failure. We hypothesized that glucose transporters (GLUTs) may contribute to progression of the renal disease, as GLUTs have been implicated in diabetic glomerulosclerosis and hypertensive renal disease with mesangial cell (MC) stretch. The Os (oligosyndactyly) allele that typically reduces nephron number by approximately 50%, was repeatedly backcrossed from ROP (Ra/+ (ragged), Os/+ (oligosyndactyly), and Pt/+ (pintail)) Os/+ mice more than six times into the Fvb mouse background to obtain Os/+ and +/+ mice with the Fvb background for study. Glomerular function, GLUT1, signaling, albumin excretion, and structural and ultrastructural changes were assessed. The FvbROP Os/+ mice (Fvb background) exhibited increased glomerular GLUT1, glucose uptake, VEGF, glomerular hypertrophy, hyperfiltration, extensive podocyte foot process effacement, marked albuminuria, severe extracellular matrix (ECM) protein deposition, and rapidly progressive renal failure leading to their early demise. Glomerular GLUT1 was increased 2.7-fold in the FvbROP Os/+ mice vs controls at 4 weeks of age, and glucose uptake was increased 2.7-fold. These changes were associated with the activation of glomerular PKCbeta1 and NF-kappaB p50 which contribute to ECM accumulation. The cyclic mechanical stretch of MCs in vitro, used as a model for increased MC stretch in vivo, reproduced increased GLUT1 at 48 h, a stimulus for increased VEGF expression which followed at 72 h. VEGF was also shown to act in a positive feedback manner on MC GLUT1, increasing GLUT1 expression, glucose uptake and fibronectin (FN) accumulation in vitro, whereas antisense suppression of GLUT1 largely blocked FN upregulation by VEGF. The FvbROP Os/+ mice exhibited an early increase in glomerular GLUT1 leading to increased glomerular glucose uptake PKCbeta1, and NF-kappaB activation, with excess ECM accumulation. A GLUT1-VEGF-GLUT1 positive feedback loop may play a key role in contributing to renal disease in this model of nondiabetic glomerulosclerosis.
204: Biomaterials, 2010 Feb, 31(6)
The effect of VEGF on the myogenic differentiation of adipose tissue derived stem cells within thermosensitive hydrogel matrices.
[Abstract]We investigated the combination of human adipose tissue derived stem cells (ADSC) and in vivo gel-forming methoxy poly (ethyleneglycol)-poly (varepsilon-caprolactone) (MPEG-PCL) as a muscle regeneration matrix, with and without inclusion of vascular endothelial cell growth factor (VEGF). VEGF(165)-treated stem cell grafts showed significant proliferation and differentiation into muscle tissue in vivo. Importantly, the inclusion of VEGF enhanced vascularization. This scaffold supported preconditioned ADSC, and allowed them to differentiate into mature muscle tissues in vivo, indicating that ADSC of human origin and MPEG-PCL scaffolds provided an appropriate environment for cellular growth and expansion. Our results thus provide a potential solution to the major obstacle encountered in the engineering of thick complex tissues, which require an adequate blood supply to maintain cell viability during tissue growth and to induce appropriate structural organization. Therefore, the combination of ADSC and in vivo gel-forming MPEG-PCL with VEGF(165) might serve as a suitable non-invasive biomaterial for clinical muscle regeneration applications.
205: FEBS letters, 2010 Jan 4, 584(1)
Differential functions of genes regulated by VEGF-NFATc1 signaling pathway in the migration of pulmonary valve endothelial cells.
[Abstract]We have reported that vascular endothelial growth factor (VEGF)-A induces the proliferation of human pulmonary valve endothelial cells (HPVECs) through nuclear factor in activated T cells (NFAT)c1 activation [1]. Here we show that VEGF-A increases the migration of HPVECs through NFATc1 activation, suggesting that VEGF-A/NFATc1 regulates the migration of HPVECs. To learn how this pathway may be involved in post-natal valvular repair, HPVECs were treated with VEGF-A, with or without cyclosporine A to selectively block VEGF-NFATc1 signaling. Down Syndrome critical region 1 (DSCR1) and heparin-binding EGF-like growth factor (HB-EGF) are two genes identified by DNA microarray as being up-regulated by VEGF-A in a cyclosporine-A-sensitive manner. DSCR1 silencing increased the migration of ovine valve endothelial cells, whereas HB-EGF silencing inhibited migration. This differential effect suggests that VEGF-A/NFATc1 signaling might be a crucial coordinator of endothelial cell migration in post-natal valves.
206: Biochemical Society transactions, 2009 Dec, 37(Pt 6)
The anti-angiogenic isoforms of VEGF in health and disease.
[Abstract]Anti-angiogenic VEGF (vascular endothelial growth factor) isoforms, generated from differential splicing of exon 8, are widely expressed in normal human tissues but down-regulated in cancers and other pathologies associated with abnormal angiogenesis (cancer, diabetic retinopathy, retinal vein occlusion, the Denys-Drash syndrome and pre-eclampsia). Administration of recombinant VEGF(165)b inhibits ocular angiogenesis in mouse models of retinopathy and age-related macular degeneration, and colorectal carcinoma and metastatic melanoma. Splicing factors and their regulatory molecules alter splice site selection, such that cells can switch from the anti-angiogenic VEGF(xxx)b isoforms to the pro-angiogenic VEGF(xxx) isoforms, including SRp55 (serine/arginine protein 55), ASF/SF2 (alternative splicing factor/splicing factor 2) and SRPK (serine arginine domain protein kinase), and inhibitors of these molecules can inhibit angiogenesis in the eye, and splice site selection in cancer cells, opening up the possibility of using splicing factor inhibitors as novel anti-angiogenic therapeutics. Endogenous anti-angiogenic VEGF(xxx)b isoforms are cytoprotective for endothelial, epithelial and neuronal cells in vitro and in vivo, suggesting both an improved safety profile and an explanation for unpredicted anti-VEGF side effects. In summary, C-terminal distal splicing is a key component of VEGF biology, overlooked by the vast majority of publications in the field, and these findings require a radical revision of our understanding of VEGF biology in normal human physiology.
207: Biochemical Society transactions, 2009 Dec, 37(Pt 6)
The heparin-binding domain confers diverse functions of VEGF-A in development and disease: a structure-function study.
[Abstract]The longer splice isoforms of VEGF (vascular endothelial growth factor)-A, including VEGF(164(165)), contain a highly basic HBD (heparin-binding domain). This domain allows these isoforms to interact with and localize to the HS (heparan sulfate)-rich extracellular matrix, and bind to the co-receptor Nrp-1 (neuropilin-1). Heparin-binding VEGF-A isoforms are critical for survival: mice engineered to express exclusively the non-heparin-binding VEGF(120) have diminished vascular branching during embryonic development and die from postnatal angiogenesis defects shortly after birth. Although it is thought that the HBD contributes to the diverse functions of VEGF-A in both physiological and pathological processes, little is known about the molecular features within this domain that enable these functions. In the present paper, we discuss the roles of the VEGF HBD in normal and disease conditions, with a particular focus on the VEGF(164(165)) isoform.
208: Biochemical Society transactions, 2009 Dec, 37(Pt 6)
VEGF-A-stimulated signalling in endothelial cells via a dual receptor tyrosine kinase system is dependent on co-ordinated trafficking and proteolysis.
[Abstract]The mammalian endothelium expresses two related but distinct receptor tyrosine kinases, VEGFR1 and VEGFR2 [VEGF (vascular endothelial growth factor) receptor 1 and 2], that regulate the vascular response to a key cytokine, VEGF-A. In the present review, we suggest a model for integrating the signals from these receptor tyrosine kinases by co-ordinating the spatial and temporal segregation of these membrane proteins linked to distinct signalling outputs associated with each intracellular location. Activation of pro-angiogenic VEGFR2 stimulates a programme of tyrosine phosphorylation, ubiquitination and proteolysis. This is linked to ESCRT (endosomal sorting complex required for transport)-mediated recognition of activated VEGFR2 and sorting in endosomes before arrival in lysosomes for terminal degradation. In addition, Rab GTPases regulate key events in VEGFR2 trafficking between the plasma membrane, early and late endosomes, with distinct roles for Rab4a, Rab5a and Rab7a. Manipulation of GTPase levels affects not only VEGFR2 activation and intracellular signalling, but also functional outputs such as VEGF-A-stimulated endothelial cell migration. In contrast, VEGFR1 displays stable Golgi localization that can be perturbed by cell stimuli that elevate cytosolic Ca(2+) ion levels. One model is that VEGFR1 translocates from the trans-Golgi network to the plasma membrane via a calcium-sensitive trafficking step. This allows rapid and preferential sequestration of VEGF-A by the higher-affinity VEGFR1, thus blocking further VEGFR2 activation. Recycling or degradation of VEGFR1 allows resensitization of the VEGFR2-dependent signalling pathway. Thus a dual VEGFR system with a built-in negative-feedback loop is utilized by endothelial cells to sense a key cytokine in vascular tissues.
209: Biochemical Society transactions, 2009 Dec, 37(Pt 6)
VEGF receptor trafficking in angiogenesis.
[Abstract]The intracellular trafficking of receptors provides a way to control the overall sensitivity of a cell to receptor stimulation. These sorting pathways are also used to shape the balance of signals that are generated in response to receptor activation. The major pro-angiogenic growth factor receptor is VEGFR2 (vascular endothelial growth factor 2). VEGFR2 activates a very similar set of signalling pathways to other RTKs (receptor tyrosine kinases); however, its intracellular trafficking is very different. Furthermore, VEGFR2 can form a complex with a range of different angiogenic regulators that in turn regulate the trafficking of VEGFR2 through the endosomal pathway. This regulated trafficking of VEGFR2 has important consequences for angiogenic signalling and is a clear demonstration of how the endosomal pathway plays a critical role in connecting receptor signalling pathways to cellular events.
210: Biochemical Society transactions, 2009 Dec, 37(Pt 6)
VEGF resistance as a molecular basis to explain the angiogenesis paradox in diabetes mellitus.
[Abstract]The action of VEGF (vascular endothelial growth factor) is essential to maintain proper endothelial and vascular function. VEGF stimulates virtually all aspects of endothelial function, namely proliferation, migration, permeability and nitric oxide production and release. In addition, the action of VEGF makes the endothelium anti-apoptotic. In turn, the inhibition of VEGF action is associated with endothelial dysfunction. Likewise, endothelial dysfunction can be found in the presence of several cardiovascular risk factors, including diabetes mellitus, hypercholesterolaemia and smoking. As circulating monocytes express functionally active VEGFR-1 (VEGF receptor 1) on their surface, monocytes and the related VEGFR-1-mediated signal transduction cascades have come into focus. The function of monocytes is negatively affected by diabetes mellitus, resulting in monocyte dysfunction. More specifically, a VEGF-related signal transduction defect can be detected in monocytes isolated from diabetic individuals. This reduced monocyte response to VEGF, demonstrated by a reduced chemotactic response, can be regarded as VEGF resistance. It is based on the pre-activation of certain intracellular pathways secondary to the diabetes mellitus-related RAGE (receptor for advanced glycation end-products) activation, ROS (reactive oxygen species) activation and inhibition of PTPs (protein tyrosine phosphatases). This unspecific pre-activation of intracellular pathways represents the molecular basis of VEGF resistance in diabetes mellitus.
211: Biochemical Society transactions, 2009 Dec, 37(Pt 6)
Unique signal transduction of the VEGF family members VEGF-A and VEGF-E.
[Abstract]Both VEGF (vascular endothelial growth factor)-A and Orf-virus-encoded VEGF-E bind and activate VEGFR (VEGF receptor)-2; however, only VEGF-A binds VEGFR-1. To understand the biological differences between VEGF-A and VEGF-E in vivo, we established transgenic mouse models. K14 (keratin-14)-promoter-driven VEGF-E transgenic mice showed a significant increase in mature blood vessels. However, K14-VEGF-A transgenic mice exhibited severe inflammation and oedema with increased angiogenesis, as well as lymphangiogenesis and lymph vessel dilatation. K14-VEGF-A transgenic mice deficient in VEGFR-1 signalling (K14-VEGF-A-tg/VEGFR-1 TK(-/-) mice) showed decreases in oedema and inflammation with less recruitment of macrophage-lineage cells, suggesting an involvement of VEGFR-1 in these adverse effects. VEGFE might be more useful than VEGFA for pro-angiogenic therapy.
212: The Prostate, 2009 Nov 11, 285(2)
Interaction among variant vascular endothelial growth factor (VEGF) and its receptor in relation to prostate cancer risk.
[Abstract]BACKGROUND: Prostate cancer (PCa) incidence and mortality are disproportionately high among African-American (AA) men. Its detection and perhaps its disparities could be improved through the identification of genetic susceptibility biomarkers within essential biological pathways. Interactions among highly variant genes, central to angiogenesis, may modulate susceptibility for prostate cancer, as previous demonstrated. This study evaluates the interplay among three highly variant genes (i.e., IL-10, TGFbetaR-1, VEGF), their receptors and their influence on PCa within a case-control study consisting of an under-served population. METHODS: This study evaluated single gene and joint modifying effects on PCa risk in a case-control study comprised of 859 AA men (193 cases and 666 controls) using TaqMan qPCR. Interaction among polymorphic IL-10, TGFbetaR-1 and VEGF was analyzed using conventional logistic regression analysis (LR) models, multi-dimensionality reduction (MDR) and interaction entropy graphs. Symbolic modeling allowed validation of gene-gene interaction findings identified by MDR. RESULTS: No significant single gene effects were demonstrated in relation to PCa risk. However, carriers of the VEGF 2482T allele had a threefold increase in the risk of developing aggressive PCa. The presence of VEGF 2482T combined with VEGFR IVS6 + 54 loci were highly significant for the risk of PCa based on MDR and symbolic modeling analyses. These findings were substantiated by 1,000-fold cross validation permutation testing (P = 0.04), respectively. CONCLUSION: These findings suggest the inheritance of VEGF and VEGFR IVS6 + 54 sequence variants may jointly modify PCa susceptibility through their influence on angiogenesis. Larger sub-population studies are needed to validate these findings and evaluate whether the VEGF-VEGR axis may serve as predictors of disease prognosis and ultimately clinical response to available treatment strategies. Prostate (c) 2009 Wiley-Liss, Inc.
213: The Journal of biological chemistry, 2009 Nov 11, 285(2)
Regulation of vascular endothelial growth factor (VEGF) splicing from pro-angiogenic to anti-angiogenic isoforms - a novel therapeutic strategy for angiogenesis.
[Abstract]VEGF is produced either as pro-angiogenic or anti-angiogenic proteins depending upon splice site choice in the terminal, eighth exon. Proximal splice site selection (PSS) in exon 8 generates pro-angiogenic isoforms such as VEGF165, and distal splice site selection (DSS) results in anti-angiogenic isoforms such as VEGF165b. Cellular decisions on splice site selection depend upon the activity of RNA binding splice factors, such as ASF/SF2, which have previously been shown to regulate VEGF splice site choice. To determine the mechanism by which the pro-angiogenic splice site choice is mediated, we investigated the effect of inhibition of ASF/SF2 phosphorylation by SR protein kinases (SRPK1/2) on splice site choice in epithelial cells and in in vivo angiogenesis models. Epithelial cells treated with insulin like growth factor-1 (IGF-1) increased PSS and produced more VEGF165 and less VEGF165b. This down-regulation of DSS and increased PSS was blocked by protein kinase C inhibition and SRPK1/2 inhibition. IGF-1 treatment resulted in nuclear localisation of ASF/SF2, which was blocked by SPRK1/2 inhibition. Pull down assay and RNA immunoprecipitation using VEGF mRNA sequences identified an 11 nucleotide sequence required for ASF/SF2 binding. Injection of an SRPK1/2 inhibitor reduced angiogenesis in a mouse model of retinal neovascularisation, suggesting that regulation of alternative splicing could be a potential therapeutic strategy in angiogenic pathologies.
214: Biomaterials, 2009 Nov 9, 285(2)
Effects of VEGF temporal and spatial presentation on angiogenesis.
[Abstract]Therapeutic angiogenesis relies on the delivery of angiogenic factors capable of reversing tissue ischemia. Polymeric materials that can provide spatial and temporal over vascular endothelial growth factor (VEGF) presentation provide clear benefit, but the influence of VEGF dose, temporal, and spatial presentation on the resultant angiogenic process are largely unknown. The influence of the temporal profile of VEGF concentration, dose, and the impact of VEGF spatial distribution on angiogenesis in in vitro models of angiogenesis and ischemic murine limbs was analyzed in this study. Importantly, a profile consisting of a high VEGF concentration initially, followed by a decreasing concentration over time was found to yield optimal angiogenic sprouting. A total VEGF dose 0.1mug/g, when delivered with kinetics found to be optimal in vitro, provided a favorable therapeutic dose in murine hindlimb ischemia model, and distributing this VEGF dose in two spatial locations induces a higher level of vascularization and perfusion than a single location. These findings suggest that material systems capable of controlling and regulating the temporal and spatial presentation of VEGF maybe useful to achieve a robust and potent therapeutic angiogenic effect in vivo.
215: Eye (London, England), 2009 Nov 6, 285(2)
Anti-VEGF therapy for choroidal neovascularisation previously treated with photodynamic therapy.
[Abstract]PurposeThis interventional, non-comparative case series assessed the outcome of intravitreal pan-anti-vascular endothelial growth factor (VEGF) agents in eyes with persistent or reactivated choroidal neovascularisation (CNV) following previous treatment with photodynamic therapy (PDT).MethodsBaseline assessments including best-corrected visual acuity, fluorescein angiography (FFA), and optical coherent tomography (OCT) were performed. Intravitreal ranibizumab and/ or bevacizumab were administered on a PRN basis, guided by changes in visual outcome and OCT findings. The follow-up period was at least 6 months.ResultsTwenty-five subjects with predominantly classic CNV, previously treated with PDT (mean 1.84 PDT sessions) showed reactivation or persistent CNV. The mean interval between PDT and intravitreal anti-VEGF treatment was 18.32 months (1-48 months); and patients received an average of 3.2 injections over a 6-month period. The mean change of visual acuity following PDT was -10.12 Early Treatment Diabetic Retinopathy Study (ETDRS) letters (54.36+/-15.79-44.24+/-17.32 letters). Following anti-VEGF therapy, the mean change in visual acuity at 3 and 6 months were +1.76 and +0.72, respectively. The proportion of subjects with stable vision (loss of /=15 letters) was 8% at 3 months and 4% at 6 months.ConclusionsAnti-VEGF agents stabilised the visual outcomes of eyes previously treated with PDT. However, the proportion of patients who showed improved vision in this group was smaller than the proportion reported in subjects with treatment-naive lesions.Eye advance online publication, 6 November 2009; doi:10.1038/eye.2009.266.
216: Placenta, 2009 Nov 4, 285(2)
Hypoxia Enhances FGF2- and VEGF-Stimulated Human Placental Artery Endothelial Cell Proliferation: Roles of MEK1/2/ERK1/2 and PI3K/AKT1 Pathways.
[Abstract]Placental development occurs under a low oxygen (2-8% O(2)) environment, which is critical for placental development and angiogenesis. In this study, we examined if hypoxia affected fibroblast growth factor-2 (FGF2)- and vascular endothelial growth factor (VEGF)-stimulated cell proliferation via the mitogen-activated protein kinase kinase 1/2 (MEK1/2)/extracellular signal-regulated kinases 1/2 (ERK1/2) and phosphatidylinositol-3 kinase (PI3K)/v-akt murine thymomaviral oncogene homologue (AKT1) pathways in human placental artery endothelial (HPAE) cells. We observed that under normoxia ( approximately 20% O(2)), FGF2 and VEGF dose-dependently stimulated cell proliferation. Hypoxia (3% O(2)) significantly promoted FGF2- and VEGF-stimulated cell proliferation as compared to normoxia. Under both normoxia and hypoxia, FGF2 rapidly induced ERK1/2 and AKT1 phosphorylation, while VEGF-induced ERK1/2, but not AKT1 phosphorylation. However, hypoxia did not significantly alter FGF2- and VEGF-induced ERK1/2 and AKT1 phosphorylation as compared to normoxia. PD98059 (a MEK1/2 inhibitor) at 20muM and LY294002 (a PI3K inhibitor) at 5muM attenuated FGF2- and VEGF-induced phosphorylation of ERK1/2 and AKT1, respectively. PD98059, even at doses that drastically inhibited FGF2-induced ERK1/2 phosphorylation (20muM) and caused cell loss (40muM), did not affect FGF2-stimulated cell proliferation, which was confirmed by U0126 (another potent MEK1/2 inhibitor). PD98059, however, dose-dependently inhibited VEGF-stimulated cell proliferation. Conversely, LY294002 dose-dependently inhibited FGF2-, but not VEGF-stimulated cell proliferation. These data suggest that in the MEK1/2/ERK1/2 and PI3K/AKT1 pathways differentially mediate FGF2- and VEGF-stimulated HPAE cell proliferation. These results also indicate that hypoxia promotes FGF2- and VEGF-stimulated cell proliferation without further activation of the PI3K/AKT1 and MEK1/2/ERK1/2, respectively.
217: Journal of orthopaedic research : official publication of the Orthopaedic Research Society, 2009 Nov 4, 285(2)
Hyaluronic acid modulates gene expression of connective tissue growth factor (CTGF), transforming growth factor-beta1 (TGF-beta1), and vascular endothelial growth factor (VEGF) in human fibroblast-like synovial cells from advanced-stage osteoarthritis in
[Abstract]Intraarticular injection of hyaluronan (hyaluronic acid; HA) is the common way to treat osteoarthritis (OA) of knees. This treatment cannot only maintain the viscoelastic properties of knee but also release the OA pain. However, the exact molecular mechanism is unknown. In this study, after human synovial cells were stimulated with HA and Hylan (Synvisc(R)) for 24 h, real-time polymerase chain reaction (real-time PCR) was used to detect the alteration of connective tissue growth factor (CTGF), transforming growth factor-beta1 (TGF-beta1), and vascular endothelial growth factor (VEGF) gene expression, which were specific genes related to pathogenesis of OA knees. Our results illustrated that both HA and Hylan might not cause cytotoxicity or apoptosis of synovial cells in serum deprivation environment. The gene expressions of TGF-beta1 and VEGF were significantly increased at the concentration of 0.1 mg/mL HA and 0.1 mg/mL Hylan, respectively (alpha < 0.05). The synovial cells with treatment of 0.1 mg/mL Hylan decreased the CTGF gene expression (0.66-fold) and VEGF (0.78-fold) compared to 0.1 mg/mL HA (alpha < 0.05). We suggested that the profile of CTGF, TGF-beta1, and VEGF gene expressions in our study might provide the rational mechanism for the therapeutic effect of hyaluronan on OA knees. (c) 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.
218: Colorectal disease : the official journal of the Association of Coloproctology of Great Britain and Ireland, 2009 Nov 3, 285(2)
ROLE OF GELATINASES (MMP-2 AND MMP-9), VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) AND ENDOSTATIN ON CLINICOPATHOLOGICAL BEHAVIOR OF RECTAL CANCER.
[Abstract]Abstract Aim: The aim of this study was to evaluate the role of metalloproteinases (MMPs), their tissue inhibitor (TIMP) and activator (MT1-MMP), vascular endothelial growth factor (VEGF) and endostatin on clinicopathological variables and prognosis in patients with rectal cancer. Method: Tumour and paired normal tissue samples were obtained from patients with rectal cancer who underwent curative surgery (n=34). Gelatin zymography (for MMP-2 and MMP-9), activity assay (for MT1-MMP) and enzyme-linked immunoassay (ELISA) (for TIMP-2, VEGF, and endostatin) analysis were performed. Results: Active MMP-9 showed statistically significant relationships with metastatic disease and perineural invasion (p=0.002 and p=0.042). A significant relation was observed between tumoural proMMP-2, proMMP-9 levels and the presence of lymph node metastasis (p=0.012 and p=0.021, respectively). Tumoural TIMP-2 levels showed a significant relationship with tumour recurrence (p=0.011). A significant relation was also observed between tumour VEGF and the presence of perineural invasion (p=0.044), and VEGF values were correlated with size of the tumour (p=0.009 r=0.454). Conclusion: These results might contribute to further investigation of a possible prognostic significance in rectal cancer.
219: PloS one, 2009, 4(11)
Cytokine levels correlate with immune cell infiltration after anti-VEGF therapy in preclinical mouse models of breast cancer.
[Abstract]The effect of blocking VEGF activity in solid tumors extends beyond inhibition of angiogenesis. However, no studies have compared the effectiveness of mechanistically different anti-VEGF inhibitors with respect to changes in tumor growth and alterations in the tumor microenvironment. In this study we use three distinct breast cancer models, a MDA-MB-231 xenograft model, a 4T1 syngenic model, and a transgenic model using MMTV-PyMT mice, to explore the effects of various anti-VEGF therapies on tumor vasculature, immune cell infiltration, and cytokine levels. Tumor vasculature and immune cell infiltration were evaluated using immunohistochemistry. Cytokine levels were evaluated using ELISA and electrochemiluminescence. We found that blocking the activation of VEGF receptor resulted in changes in intra-tumoral cytokine levels, specifically IL-1beta, IL-6 and CXCL1. Modulation of the level these cytokines is important for controlling immune cell infiltration and ultimately tumor growth. Furthermore, we demonstrate that selective inhibition of VEGF binding to VEGFR2 with r84 is more effective at controlling tumor growth and inhibiting the infiltration of suppressive immune cells (MDSC, Treg, macrophages) while increasing the mature dendritic cell fraction than other anti-VEGF strategies. In addition, we found that changes in serum IL-1beta and IL-6 levels correlated with response to therapy, identifying two possible biomarkers for assessing the effectiveness of anti-VEGF therapy in breast cancer patients.
220: Oncology reports, 2009 Dec, 22(6)
Effect of 5-ALA-PDT on VEGF and PCNA expression in human NPC-bearing nude mice.
[Abstract]Photodynamic therapy (PDT) is a promising treatment for nasopharyngeal carcinoma. However, recurrence and metastasis of the tumor after PDT remains problematic. In this study we investigated VEGF and PCNA expression in tumor tissues after 5-ALA-PDT. BALB/c nude mice with NPC tumors of similar size were randomly assigned to three groups (n=10 each). In the two treatment groups, mice were administrated 5-ALA intratumorally at a dose of 100 mg/kg and 3-3.5 h prior to laser irradiation (630 nm, 100 J/cm(2), 100 mW/cm(2)). The mice in one of the treatment groups were sacrificed at 24 h after PDT. The other treatment group and control group mice were sacrificed 14 days after PDT, and the tumor weights were determined for all three groups. Mean tumor weights at 14 days after PDT were 1.353+/-0.204 g in the treatment group and 3.124+/-0.380 g in the control group (p<0.001). Results showed the VEGF level in tumor tissues at 24 h after PDT was slightly higher than that in the control group, while it was down-regulated at 14 days after PDT. The PCNA level was not significantly different in tumor tissues among the three groups, but it was lower in degenerated tumor cells 24 h after PDT. Our results suggest that VEGF may play a role in tumor recurrence and metastasis following PDT. Residual tumor cells escaped from PDT is the main reason for tumor recurrence.
221: European journal of ophthalmology, 2010 Jan-Feb, 20(1)
VEGF TrapR1R2 suppresses experimental corneal angiogenesis.
[Abstract]Purpose. To determine the effect of vascular endothelial growth factor (VEGF) TrapR1R2 on bFGF-induced experimental corneal neovascularization (NV). Methods. Control pellets or pellets containing 80 ng bFGF were surgically implanted into wild-type C57BL/6 and VEGF-LacZ mouse corneas. The corneas were photographed, harvested, and the percentage of corneal NV was calculated. The harvested corneas were evaluated for VEGF expression. VEGF-LacZ mice received tail vein injections of an endothelial-specific lectin after pellet implantation to determine the temporal and spatial relationship between VEGF expression and corneal NV. Intraperitoneal injections of VEGF TrapR1R2 or a human IgG Fc domain control protein were administered, and bFGF pellet-induced corneal NV was evaluated. Results. NV of the corneal stroma began on day 4 and was sustained through day 21 following bFGF pellet implantation. Progression of vascular endothelial cells correlated with increased VEGF-LacZ expression. Western blot analysis showed increased VEGF expression in the corneal NV zone. Following bFGF pellet implantation, the area of corneal NV in untreated controls was 1.05+/-0.12 mm2 and 1.53+/-0.27 mm2 at days 4 and 7, respectively. This was significantly greater than that of mice treated with VEGF Trap (0.24+/-0.11 mm2 and 0.35+/-0.16 mm2 at days 4 and 7, respectively; p<0.05). Conclusions. Corneal keratocytes express VEGF after bFGF stimulation and bFGF-induced corneal NV is blocked by intraperitoneal VEGF TrapR1R2 administration. Systemic administration of VEGF TrapR1R2 may have potential therapeutic applications in the management of corneal NV.
222: International journal of cancer. Journal international du cancer, 2010 Jun 15, 126(12)
A gastrin precursor, gastrin-gly, upregulates VEGF expression in colonic epithelial cells through an HIF-1-independent mechanism.
[Abstract]One of the major angiogenic factor released by tumor cells is VEGF. Its high expression is correlated with poor prognosis in colorectal tumors. In colon cancer, gastrin gene expression is also upregulated. In these tumors, gastrin precursors are mainly produced and act as growth factors. Recently, a study has also shown that the gastrin precursor, G-gly induced in vitro tubules formation by vascular endothelial cells suggesting a potential proangiogenic role. Here, we demonstrate that stimulation of human colorectal cancer cell lines with G-gly increases the expression of the proangiogenic factor VEGF at the mRNA and protein levels. In addition, blocking the progastrin autocrine loop leads to a downregulation of VEGF. Although HIF-1 is a major transcriptional activator for VEGF our results suggest an alternative mechanism for VEGF regulation in normoxic conditions, independent of HIF-1 that involves the PI3K/AKT pathway. Indeed we show that G-gly does not lead to HIF-1 accumulation in colon cancer cells. Moreover, we found that G-gly activates the PI3K/AKT pathway and inhibition of this pathway reverses the effects of G-gly observed on VEGF mRNA and protein levels. In correlation with these results, we observed in vivo, on colon tissue sections from transgenic mice overexpressing G-gly, an increase in VEGF expression in absence of HIF-1 accumulation. In conclusion, our study demonstrates that gastrin precursors, known to promote colon epithelial cells proliferation and survival can also contribute to the angiogenesis process by stimulating the expression of the proangiogenic factor VEGF via the PI3K pathway and independently of hypoxia conditions.
223: Arteriosclerosis, thrombosis, and vascular biology, 2010 Jan, 30(1)
VEGF induces differentiation of functional endothelium from human embryonic stem cells: implications for tissue engineering.
[Abstract]OBJECTIVE: Human embryonic stem cells (hESCs) offer a sustainable source of endothelial cells for therapeutic vascularization and tissue engineering, but current techniques for generating these cells remain inefficient. We endeavored to induce and isolate functional endothelial cells from differentiating hESCs. METHODS AND RESULTS: To enhance endothelial cell differentiation above a baseline of approximately 2% in embryoid body (EB) spontaneous differentiation, 3 alternate culture conditions were compared. Vascular endothelial growth factor (VEGF) treatment of EBs showed the best induction, with markedly increased expression of endothelial cell proteins CD31, VE-Cadherin, and von Willebrand Factor, but not the hematopoietic cell marker CD45. CD31 expression peaked around days 10 to 14. Continuous VEGF treatment resulted in a 4- to 5-fold enrichment of CD31(+) cells but did not increase endothelial proliferation rates, suggesting a primary effect on differentiation. CD31(+) cells purified from differentiating EBs upregulated ICAM-1 and VCAM-1 in response to TNFalpha, confirming their ability to function as endothelial cells. These cells also expressed multiple endothelial genes and formed lumenized vessels when seeded onto porous poly(2-hydroxyethyl methacrylate) scaffolds and implanted in vivo subcutaneously in athymic rats. Collagen gel constructs containing hESC-derived endothelial cells and implanted into infarcted nude rat hearts formed robust networks of patent vessels filled with host blood cells. CONCLUSIONS: VEGF induces functional endothelial cells from hESCs independent of endothelial cell proliferation. This enrichment method increases endothelial cell yield, enabling applications for revascularization as well as basic studies of human endothelial biology. We demonstrate the ability of hESC-derived endothelial cells to facilitate vascularization of tissue-engineered implants.
224: Oncogene, 2009 Oct 26, 90(1-2)
Overexpression of myosin VI in prostate cancer cells enhances PSA and VEGF secretion, but has no effect on endocytosis.
[Abstract]Tissue expression microarrays, employed to determine the players and mechanisms leading to prostate cancer development, have consistently shown that myosin VI, a unique actin-based motor, is upregulated in medium-grade human prostate cancers. Thus, to understand the role of myosin VI in prostate cancer development, we have characterized its intracellular localization and function in the prostate cancer cell line LNCaP. Using light and electron microscopy, we identified myosin VI on Rab5-positive early endosomes, as well as on recycling endosomes and the trans-Golgi network. Intracellular targeting seems to involve two myosin VI-interacting proteins, GIPC and LMTK2, both of which can be co-immunoprecipitated with myosin VI from LNCaP cells. The absence of Disabled-2 (Dab2), a tumour suppressor and myosin VI-binding partner, inhibits recruitment of myosin VI to endocytic structures at the plasma membrane in LNCaP cells, but interestingly has no effect on endocytosis. Small interfering RNA-mediated downregulation of myosin VI expression results in a significant reduction in prostate-specific antigen (PSA) and vascular endothelial growth factor (VEGF) secretion in LNCaP cells. Our results suggest that in prostate cancer cells, myosin VI regulates protein secretion, but the overexpression of myosin VI has no major impact on clathrin-mediated endocytosis.Oncogene advance online publication, 26 October 2009; doi:10.1038/onc.2009.328.
225: The British journal of ophthalmology, 2009 Oct 23, 90(1-2)
Long-Term Visual and Anatomic Outcomes Following Anti-VEGF Monotherapy for Retinal Angiomatous Proliferation.
[Abstract]PURPOSE: To study the long-term visual and anatomic outcomes of anti-vascular endothelial growth factor (VEGF) monotherapy for the treatment of patients with retinal angiomatous proliferation (RAP). METHODS: Retrospective review of patients who were diagnosed with AMD and RAP lesions, and who received anti-VEGF injections as the only mode of therapy. RESULTS: 20 eyes (15 patients; 9 women, 6 men) with RAP lesions treated by anti- VEGF were encountered. Mean patient age was 85.8 years (SD +/- 4.54). Nine eyes were treated with intravitreal ranibizumab alone, 8 eyes with bevacizumab alone, and 3 eyes received both drugs. At the 1, 3 and 6 month follow-up the median VA had improved from baseline (20/72) to 20/52, (range: 20/25 to 20/400), 20/45 (range: 20/20 to 20/400), and 20/56 (range: 20/20 to 20/400), respectively, (P> 0.001, P= 0.001, and P= 0.05, respectively). At 24 month follow-up, the improvement of VA, defined as halving of the visual angle, occurred in 37.5% of the cases. CONCLUSIONS: Anti-VEGF monotherapy represents a useful treatment option for RAP, with stable or improved visual acuity in 62.5% of patients at 2 years. Although 25% of eyes only required a single injection, in most cases (75%) repeated treatments were required, therefore long-term follow-up is recommended.
226: Developmental dynamics : an official publication of the American Association of Anatomists, 2009 Nov, 238(11)
Role of VEGF and tissue hypoxia in patterning of neural and vascular cells recruited to the embryonic heart.
[Abstract]We hypothesized that oxygen gradients and hypoxia-responsive signaling may play a role in the patterning of neural or vascular cells recruited to the developing heart. Endothelial progenitor and neural cells are recruited to and form branched structures adjacent to the relatively hypoxic outflow tract (OFT) myocardium from stages 27-32 (ED6.5-7.5) of chick development. As determined by whole mount confocal microscopy, the neural and vascular structures were not anatomically associated. Adenoviral delivery of a VEGF trap dramatically affected the remodeling of the vascular plexus into a coronary tree while neuronal branching was normal. Both neuronal and vascular branching was diminished in the hearts of embryos incubated under hyperoxic conditions. Quantitative analysis of the vascular defects using our recently developed VESGEN program demonstrated reduced small vessel branching and increased vessel diameters. We propose that vascular and neural patterning in the developing heart share dependence on tissue oxygen gradients but are not interdependent.
227: Journal of nuclear medicine : official publication, Society of Nuclear Medicine, 2009 Nov, 50(11)
Reporter gene PET for monitoring survival of transplanted endothelial progenitor cells in the rat heart after pretreatment with VEGF and atorvastatin.
[Abstract]It has been suggested that vascular endothelial growth factor (VEGF) and statins enhance the survival, proliferation, and function of endothelial progenitor cells (EPCs). We investigated whether reporter gene PET can be used to detect the effects of atorvastatin and VEGF on survival of EPCs after transplantation in the rat heart. METHODS: Healthy nude rats received an intramyocardial injection of 4 million human EPCs retrovirally transduced with the sodium/iodide symporter gene for reporter gene imaging. Reporter gene expression was imaged at days 1 and 3 after injection on a small-animal PET scanner with (124)I, and the presence of EPCs was confirmed by immunohistochemistry with human CD31 antibodies. The control group received EPCs transduced only with the reporter gene, whereas treatment groups received oral atorvastatin (10 mg/kg/d) and EPCs cotransduced with adenoviral vectors encoding VEGF in addition to sodium/iodide symporter. RESULTS: Immunohistochemistry showed more EPCs at the site of injection after atorvastatin treatment and in the presence of VEGF expression in EPCs than in controls. PET successfully visualized EPCs as focal (124)I accumulation at the site of injection. The quantitative amount of (124)I accumulation assessed by PET was significantly higher in the pretreatment than control group. Autoradiography confirmed (124)I accumulation in the myocardium that correlated with the number of EPCs. CONCLUSION: Early survival of transplanted EPCs in the rat myocardium is prolonged by pretreatment with a combination of atorvastatin and VEGF. Reporter gene PET, by successfully quantifying the effect, is an attractive tool for monitoring stem cell survival in vivo.
228: Biochemical and biophysical research communications, 2009 Dec 18, 390(3)
Vascular endothelial growth factor (VEGF) as a key therapeutic trophic factor in bone marrow mesenchymal stem cell-mediated cardiac repair.
[Abstract]We recently demonstrated a novel effective therapeutic regimen for treating hamster heart failure based on injection of bone marrow mesenchymal stem cells (MSCs) or MSC-conditioned medium into the skeletal muscle. The work highlights an important cardiac repair mechanism mediated by the myriad of trophic factors derived from the injected MSCs and local musculature that can be explored for non-invasive stem cell therapy. While this therapeutic regimen provides the ultimate proof that MSC-based cardiac repair is mediated by the trophic actions independent of MSC differentiation or stemness, the trophic factors responsible for cardiac regeneration after MSC therapy remain largely undefined. Toward this aim, we took advantage of the finding that human and porcine MSCs exhibit species-related differences in expression of trophic factors. We demonstrate that human MSCs when compared to porcine MSCs express and secrete 5-fold less vascular endothelial growth factor (VEGF) in conditioned medium (40+/-5 and 225+/-17 pg/ml VEGF, respectively). This deficit in VEGF output was associated with compromised cardiac therapeutic efficacy of human MSC-conditioned medium. Over-expression of VEGF in human MSCs however completely restored the therapeutic potency of the conditioned medium. This finding indicates VEGF as a key therapeutic trophic factor in MSC-mediated myocardial regeneration, and demonstrates the feasibility of human MSC therapy using trophic factor-based cell-free strategies, which can eliminate the concern of potential stem cell transformation.
229: Cell death and differentiation, 2010 Mar, 17(3)
VEGF-dependent tumor angiogenesis requires inverse and reciprocal regulation of VEGFR1 and VEGFR2.
[Abstract]Vascular endothelial growth factor (VEGF) signaling is critical for tumor angiogenesis. However, therapies based on inhibition of VEGF receptors (VEGFRs) have shown modest results for patients with cancer. Surprisingly little is known about mechanisms underlying the regulation of VEGFR1 and VEGFR2 expression, the main targets of these drugs. Here, analysis of tissue microarrays revealed an inversely reciprocal pattern of VEGFR regulation in the endothelium of human squamous-cell carcinomas (high VEGFR1, low VEGFR2), as compared with the endothelium of control tissues (low VEGFR1, high VEGFR2). Mechanistic studies demonstrated that VEGF signals through the Akt/ERK pathway to inhibit constitutive ubiquitination and induce rapid VEGFR1 accumulation in endothelial cells. Surprisingly, VEGFR1 is primarily localized in the nucleus of endothelial cells. In contrast, VEGF signals through the JNK/c-Jun pathway to induce endocytosis, nuclear translocation, and downregulation of VEGFR2 via ubiquitination. VEGFR1 signaling is required for endothelial-cell survival, while VEGFR2 regulates capillary tube formation. Notably, the antiangiogenic effect of bevacizumab (anti-VEGF antibody) requires normalization of VEGFR1 and VEGFR2 levels in human squamous-cell carcinomas vascularized with human blood vessels in immunodeficient mice. Collectively, this work demonstrates that VEGF-induced angiogenesis requires inverse regulation of VEGFR1 and VEGFR2 in tumor-associated endothelial cells.
230: The American journal of pathology, 2009 Nov, 175(5)
Effects of increased renal tubular vascular endothelial growth factor (VEGF) on fibrosis, cyst formation, and glomerular disease.
[Abstract]The role of vascular endothelial growth factor (VEGF) in renal fibrosis, tubular cyst formation, and glomerular diseases is incompletely understood. We studied a new conditional transgenic mouse system [Pax8-rtTA/(tetO)(7)VEGF], which allows increased tubular VEGF production in adult mice. The following pathology was observed. The interstitial changes consisted of a ubiquitous proliferation of peritubular capillaries and fibroblasts, followed by deposition of matrix leading to a unique kind of fibrosis, ie, healthy tubules amid a capillary-rich dense fibrotic tissue. In tubular segments with high expression of VEGF, cysts developed that were surrounded by a dense network of peritubular capillaries. The glomerular effects consisted of a proliferative enlargement of glomerular capillaries, followed by mesangial proliferation. This resulted in enlarged glomeruli with loss of the characteristic lobular structure. Capillaries became randomly embedded into mesangial nodules, losing their filtration surface. Serum VEGF levels were increased, whereas endogenous VEGF production by podocytes was down-regulated. Taken together, this study shows that systemic VEGF interferes with the intraglomerular cross-talk between podocytes and the endocapillary compartment. It suppresses VEGF secretion by podocytes but cannot compensate for the deficit. VEGF from podocytes induces a directional effect, attracting the capillaries to the lobular surface, a relevant mechanism to optimize filtration surface. Systemic VEGF lacks this effect, leading to severe deterioration in glomerular architecture, similar to that seen in diabetic nephropathy.
231: Investigative ophthalmology & visual science, 2010 Feb, 51(2)
Vascular endothelial growth factor as an autocrine survival factor for retinal pigment epithelial cells under oxidative stress via the VEGF-R2/PI3K/Akt.
[Abstract]PURPOSE: Vascular endothelial cell growth factor (VEGF) is strongly induced by oxidative stress in retinal pigment epithelial (RPE) cells, and VEGF-A is a survival factor for various cell types. This study was conducted to determine whether the autocrine VEGF signaling pathway in RPE cells is involved in the mechanism of adaptive response to oxidative stress. METHODS: ARPE-19 cells were treated with hydrogen peroxide, and cell death was measured by flow cytometry with annexin V-fluorescein isothiocyanate. Survival analysis was performed with pretreatment of VEGF-A-neutralizing antibodies, VEGF receptor tyrosine kinase inhibitor (SU5416), or VEGF-A receptor-neutralizing antibodies (anti-VEGF-R1 and anti-VEGF-R2). The expression of VEGF-A, -R1, -R2, and soluble VEGF-R1 was determined by semiquantitative RT-PCR or Western blot analysis. Phosphorylation of VEGF-R2 was detected with immunoprecipitation and immunoblot analysis. RESULTS: Hydrogen peroxide-induced cell death was promoted by pretreatment with VEGF-A and anti-VEGF-R2-neutralizing antibodies, but not with anti-VEGF-R1-neutralizing antibody. Phosphorylation of VEGF-R2 in RPE cells was induced by hydrogen peroxide, and pretreatment with anti-VEGF-A-neutralizing antibody inhibited phosphorylation. Phosphorylation of Akt under oxidative stress was abrogated by pretreatment with neutralizing antibodies against either VEGF-A or SU5416. CONCLUSIONS: Autocrine VEGF-A enhanced RPE cell survival under oxidative stress; the autocrine VEGF-A/VEGF-R2/PI3K/Akt pathway is involved. Neutralization of VEGF-A signaling, as in eyes with age-related macular degeneration, may influence RPE cell survival.
232: International journal of sports medicine, 2009 Nov, 30(11)
Nandrolone inhibits VEGF mRNA in rat muscle.
[Abstract]Vascular endothelial growth factor (VEGF) is a key compound for induction of angiogenesis in both physiological and pathological conditions. The aim of this study was to investigate the effect of androgenic-anabolic steroids (AAS) administration on VEGF mRNA expression in the rat soleus muscle after jumping training. Wistar rats were grouped into: sedentary (S); nandrolone decanoate-treated sedentary (AAS); trained without AAS (T) and trained and treated with AAS (AAST). Exercised groups performed a 7-weeks water-jumping program. Animals killed immediately after the last exercise bout showed significantly increased VEGF mRNA expression; however, the AAS treatment completely inhibited this effect. These results suggest that the AAS may be strongly prejudicial to muscle remodeling and performance at least partially due to an impaired angiogenesis.
233: Cancer biology & therapy, 2009 Dec 19, 8(23)
Combined anti-angiogenic therapy against VEGF and integrin alpha(V)beta(3) in an orthotopic model of ovarian cancer.
[Abstract]Purpose: We tested the efficacy of dual targeting of vascular endothelial growth factor (VEGF) and the alpha(V)beta(3) integrin in orthotopic mouse models of ovarian cancer. Results: In the SKOV3ip1 model, both single-agent bevacizumab and etaracizumab reduced tumor growth by 52-63% (p < 0.05), while combined therapy reduced growth by 63-74% compared to either agent alone (p < 0.05). Furthermore, bevacizumab/paclitaxel was superior to paclitaxel alone (weight reduction by 53%, p < 0.05), but etaracizumab/paclitaxel was not. Combining all three agents was more effective than either agent with paclitaxel (p < 0.05). Significantly, both bevacizumab and etaracizumab each sensitized the taxane-resistant SKOV3TRip2 cells to paclitaxel, reducing growth by 56-73% (p < 0.05). Both agents decreased proliferation and microvessel density, and increased apoptosis, alone and in combination with paclitaxel. In the HeyA8 model, there was significantly reduced growth with bevacizumab treatment, but not with etaracizumab, and combination therapy was not superior to bevacizumab alone. Experimental design: In vivo therapy experiments were conducted in chemo-sensitive (SKOV3ip1, HeyA8) and -resistant (SKOV3TRip2) ovarian cancer models. VEGF was targeted with bevacizumab and alpha(V)beta(3) with etaracizumab. Mice were treated with each agent alone, together, or in combination with paclitaxel for assessment of tumor growth. Tumor specimens were tested for proliferative index, microvessel density and apoptosis. Conclusions: Bevacizumab and etaracizumab are more effective in combination than individually in some ovarian cancer models, but not all. Both can sensitize taxane-resistant ovarian cancer cells to paclitaxel, though bevacizumab was superior to etaracizumab in this regard. Further study of this dual anti-angiogenic therapy is warranted.
234: The Journal of pathology, 2009 Dec, 219(4)
Impact of VEGF-dependent tumour micro-environment on EDB fibronectin expression by subcutaneous human tumour xenografts in nude mice.
[Abstract]Fibronectin (FN) is an extracellular matrix cell-adhesive glycoprotein. The alternative spliced isoform EDB-FN (extra domain B containing FN) is highly expressed in tumour blood vessels and stroma and represents a candidate for tumour targeting. To investigate the impact of different angiogenic micro-environments on EDB-FN expression, we used a tumour model in which human endometrial adenocarcinoma Tet-FGF2 cells overexpressing fibroblast growth factor-2 (FGF2) driven by the tetracycline-responsive promoter were further transfected with a VEGF antisense cDNA, generating AS-VEGF/Tet-FGF2 cells. In this model, the expression of FGF2 plus VEGF results in fast-growing, highly vascularized Tet-FGF2 tumours. Down-regulation of FGF2 production by tetracycline administration and/or of VEGF production by AS-VEGF transduction inhibited tumour growth and vascularization, with profound changes in tumour micro-environment. Quantitative RT-PCR analysis using human EDB-FN primers shows that subcutaneous grafting in immunodeficient mice is per se sufficient to cause a dramatic up-regulation of EDB-FN expression by these cells, as well as by human oesophageal cancer KYSE 30 cells and renal carcinoma Caki-1 cells. However, in vivo down-regulation of VEGF expression, as occurs in AS-VEGF/Tet-FGF2 tumours, and to a lesser extent of FGF2 expression, as occurs in tetracycline-treated Tet-FGF2 tumour-bearing animals, causes significant inhibition of EDB-FN production in tumour grafts, as shown by immunohistochemistry and quantitative RT-PCR analysis. Accordingly, treatment of Tet-FGF2 tumour-bearing animals with the neutralizing anti-murine VEGF receptor-2 antibody DC101, or of Caki-1 tumour-bearing animals with the anti-VEGF antibody bevacizumab, inhibited EDB-FN expression in tumour grafts. EDB-FN down-regulation was paralleled by a decrease in vascularity, thus confirming EDB-FN as a marker of tumour angiogenesis. These data demonstrate that the angiogenic micro-environment, and in particular the VEGF/VEGFR-2 system, plays a key role in modulating EDB-FN expression by tumour cells in vivo. This may have implications for the design of therapeutic strategies targeting EDB-FN in combination with anti-angiogenic and/or cytotoxic drugs.
235: Blood, 2009 Dec 24, 114(27)
Differential roles for ETS, CREB, and EGR binding sites in mediating VEGF receptor 1 expression in vivo.
[Abstract]Vascular endothelial growth factor receptor 1 (VEGFR1) is a marker for endothelial-specific gene expression. We previously reported that the human VEGFR1 promoter (between -748 and +284) contains information for expression in the intact endothelium of transgenic mice. The objective of this study was to dissect the cis-regulatory elements underlying VEGFR1 promoter activity in vitro and in vivo. In primary endothelial cells, binding sites for E74-like factor 1 (ELF-1; between -49 and -52), cyclic adenosine monophosphate response element binding (CREB; between -74 and -81), and early growth response factor 1/3 (EGR-1/3; between -16 to -25) were shown to play a positive role in gene transcription, whereas a putative E26 transformation-specificsequence (ETS) motif between -36 and -39 had a net negative effect on promoter activity. When targeted to the Hprt locus of mice, mutations of the ELF-1 binding site and the CRE element reduced promoter activity in the embryonic vasculature and resulted in a virtual loss of expression in adult endothelium. Postnatally, the EGR binding site mutant displayed significantly reduced promoter activity in a subset of vascular beds. In contrast, mutation of the -39 ETS site resulted in increased LacZ staining in multiple vascular beds. Together, these results provide new insights into the transcriptional regulatory mechanisms of VEGFR1.
236: Pediatric research, 2010 Jan, 67(1)
VEGF Polymorphisms Are Associated With Endocardial Cushion Defects: A Family-Based Case-Control Study.
[Abstract]Endocardial cushion defects (ECDs) of the cardiac outflow tract are among the most common congenital heart disease phenotypes. VEGF is essential for endocardial cushion formation and derangements in VEGF synthesis lead to ECD. Three functional single nucleotide polymorphisms (SNPs) in the VEGF gene -2578 C>A, -1154 G>A, and -634 G>C play a role in cardiogenesis. In a Dutch case-control family study of triads, 190 case and 317 control children with both parents, we investigated linkage and association between these VEGF SNPs and ECD. Allele frequencies for the three VEGF SNPs were comparable between ECD children and controls. However, VEGF alleles -2578 C and -1154 G were transmitted more frequently to children with ECD (p = 0.003 and p = 0.002), in particular perimembranous ventricular septal defects (p = 0.012 and p = 0.006). The -2578A/-1154A/-634G haplotype was associated with a reduced risk of ECD (OR 0.7; 95% CI, 0.6-1.0) and was significantly less transmitted to children with ECD (p = 0.002). In a Dutch population, we show that the VEGF 2578 C, -1154 G alleles, and the AAG haplotype are associated with ECD. Possible VEGF gene-environment interactions exposures are discussed. ABBREVIATIONS::
237: Domestic animal endocrinology, 2010 Apr, 38(3)
VEGF modulates the effects of gonadotropins in granulosa cells.
[Abstract]Follicle selection is associated with an increase in the expression of vascular endothelial growth factor (VEGF) and its receptors in granulosa cells, however, the roles of VEGF in regulating the function of these or other non-endothelial cells in the ovary have not been explored in detail. The current study used bovine cell cultures to investigate potential roles of VEGF in the regulation of granulosa cell function during follicle development. Granulosa cells were obtained from morphologically healthy follicles 4 to 8mm or 9 to 14mm in diameter (corresponding to diameters before and after the establishment of dominance, respectively, during a bovine follicular wave) and exposed to a range of VEGF concentrations (1 to 100ng/mL) encompassing concentrations found naturally in bovine dominant follicles. A concentration of VEGF of 1ng/mL induced significant proliferation of granulosa cells from 4- to 8-mm follicles (P=0.024) and increased the proliferative response of these cells to follicle-stimulating hormone (FSH; P=0.045); whereas higher doses of VEGF had no effect on proliferation (P=0.9). Treatment with VEGF induced an overall increase in mean extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation (P=0.02). In contrast, VEGF, alone or in combination with FSH, had no effect on expression of the steroidogenic enzyme, CYP11A1, by cells from 4- to 8-mm follicles (P=0.9). Granulosa cells from 9- to 14-mm follicles responded to 1ng/mL VEGF with an increase in expression of the ovulation-associated gene, PTGS2 (P=0.003) but higher VEGF doses had no effect (P=0.9). The PTGS2 response to 1ng/mL VEGF was similar to that induced by treatment with luteinizing hormone (LH). Interestingly, the stimulatory effects of LH on ERK1/2 phosphorylation (P=0.003) and PTGS2 expression (P<0.01) in granulosa cells from 9- to 14-mm follicles were abolished (P=0.2) by specific chemical inhibition of VEGF receptor 2 (VEGFR2). These results suggest novel and important roles of VEGF and its receptor, VEGFR2, in mediating and/or enhancing the effects of gonadotropins in granulosa cells.
238: Acta neurochirurgica. Supplement, 2010, 106(1)
Effects of VEGF administration or neutralization on the BBB of developing rat brain.
[Abstract]We investigated the effects of exogenous Vascular Endothelial Growth Factor VEGF combined with an enriched environment on BBB integrity after a minimal trauma induced during the first days of the critical visual period in rats, when peak levels of endogenous VEGF secretion are reached. VEGF was administered using osmotic mini-pumps placed in middle cortical layers of P18 Long-Evansrats. Tissue changes were evaluated using conventional histology. BBB integrity was shown by immunohistochemistry techniques for EBA and GluT-1. Mini-pump implantation produced a wider cavity in anti-VEGF infused rats. In VEGF-infused rats there was a damaged region around the cannula that was smaller in rats raised in an enriched environment (EE). The administration of VEGF induced a high concentration of plasma proteins in the neuropil around the point of cannula placement and a high inflammatory reaction. VEGF-infused rats raised in an EE showed a lower degree of extravasation and better tissue preservation. Anti-VEGF administration produced a lower protein expression profile and more widespread deterioration of tissue. Double immunofluorescence for EBA and GluT-1 showed that the administration of VEGF preserves the tissue, which remains present but not fully functional. In contrast, a combination of VEGF administration and an EE partially protects the functionally damaged tissue with a higher preservation of BBB integrity.
239: Biomaterials, 2010 Jan, 31(2)
Scaffolds with covalently immobilized VEGF and Angiopoietin-1 for vascularization of engineered tissues.
[Abstract]The aim of this study was to engineer a biomaterial capable of supporting vascularization in vitro and in vivo. We covalently immobilized vascular endothelial growth factor (VEGF) and Angiopoietin-1 (Ang1) onto three-dimensional porous collagen scaffolds using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) chemistry. Over both 3 and 7 days in vitro, seeded endothelial cells (ECs) had increased proliferation on scaffolds with immobilized VEGF and/or Ang1 compared to unmodified scaffolds and soluble growth factor controls. Notably, the group with co-immobilized VEGF and Ang1 showed significantly higher cell number (P=0.0079), higher overall lactate production rate (P=0.0044) and higher overall glucose consumption rate (P=0.0034) at Day 3, compared to its corresponding soluble control for which growth factors were added to culture medium. By Day 7, hematoxylin and eosin, live/dead, CD31, and von Willebrand factor staining all showed improved tube formation by ECs when cultivated on scaffolds with co-immobilized growth factors. Interestingly, scaffolds with co-immobilized VEGF and Ang1 showed increased EC infiltration in the chorioallantoic membrane (CAM) assay, compared to scaffolds with independently immobilized VEGF/Ang1. This study presents an alternative method for promoting the formation of vascular structures, via covalent immobilization of angiogenic growth factors that are more stable than soluble ones and have a localized effect.
240: Arteriosclerosis, thrombosis, and vascular biology, 2009 Dec, 29(12)
Shear Stress Increases Expression of the Arterial Endothelial Marker EphrinB2 in Murine ES Cells via the VEGF-Notch Signaling Pathways.
[Abstract]OBJECTIVE: Arterial-venous specification in the embryo has been assumed to depend on the influence of fluid mechanical forces, but its cellular and molecular mechanisms are still poorly understood. Our previous in vitro study revealed that fluid shear stress induces endothelial cell (EC) differentiation by murine embryonic stem (ES) cells. In the present study we investigated whether shear stress regulates the arterial-venous specification of ES-cell-derived ECs. METHODS AND RESULTS: When murine ES cell-derived VEGFR2(+) cells were exposed to shear stress, expression of the arterial EC marker protein ephrinB2 increased dose-dependently. The ephrinB2 mRNA levels also increased in response to shear stress, whereas the mRNA levels of the venous EC marker EphB4 decreased. Notch cleavage and translocation of the Notch intracellular domain (NICD) into the nucleus occurred as early as 30 minutes after the start of shear stress and increased with time. Gamma-Secretase inhibitors (DAPT and L685 458) and the recombinant extracellular domain of the Notch ligand DLL4 abolished the shear stress-induced NICD translocation, and that, in turn, blocked the shear stress-induced upregulation of ephrinB2 expression. In addition, the VEGF receptor kinase inhibitor SU1498 was found to suppress both the shear-stress-induced Notch cleavage and up-regulation of ephrinB2 expression. CONCLUSIONS: Exposure to shear stress induces an increase in expression of ephrinB2 in murine ES cells via VEGF-Notch signaling pathways.
241: Arteriosclerosis, thrombosis, and vascular biology, 2009 Dec, 29(12)
The ADMA/DDAH Pathway Regulates VEGF-Mediated Angiogenesis.
[Abstract]OBJECTIVE: Asymmetrical dimethylarginine (ADMA) is a nitric oxide synthase (NOS) inhibitor and cardiovascular risk factor associated with angiogenic disorders. Enzymes metabolising ADMA, dimethylarginine dimethylaminohydrolases (DDAH) promote angiogenesis, but the mechanisms are not clear. We hypothesized that ADMA/DDAH modifies endothelial responses to vascular endothelial growth factor (VEGF) by affecting activity of Rho GTPases, regulators of actin polymerization, and focal adhesion dynamics. METHODS AND RESULTS: The effects of ADMA on VEGF-induced endothelial cell motility, focal adhesion turnover, and angiogenesis were studied in human umbilical vein endothelial cells (HUVECs) and DDAH I heterozygous knockout mice. ADMA inhibited VEGF-induced chemotaxis in vitro and angiogenesis in vitro and in vivo in an NO-dependent way. ADMA effects were prevented by overexpression of DDAH but were not associated with decreased proliferation, increased apoptosis, or changes in VEGFR-2 activity or expression. ADMA inhibited endothelial cell polarization, protrusion formation, and decreased focal adhesion dynamics, resulting from Rac1 inhibition after decrease in phosphorylation of vasodilator stimulated phosphoprotein (VASP). Constitutively active Rac1, and to a lesser extent dominant negative RhoA, abrogated ADMA effects in vitro and in vivo. CONCLUSIONS: The ADMA/DDAH pathway regulates VEGF-induced angiogenesis in an NO- and Rac1-dependent manner.
242: The Journal of pathology, 2009 Dec, 219(4)
M¨¹ller cell-derived VEGF is a significant contributor to retinal neovascularization.
[Abstract]Vascular endothelial growth factor (VEGF-A) is a major pathogenic factor and a therapeutic target for age-related macular degeneration, diabetic retinopathy, and retinopathy of prematurity. Despite intensive effort in the field, the cellular mechanisms of VEGF action remain virtually uninvestigated. This situation makes it difficult to design cellular target-based therapeutics for these diseases. In light of the recent finding that VEGF is a potential neurotrophic factor, revealing the cellular mechanisms of VEGF action becomes necessary to preserve its beneficial effect and inhibit its pathological function in long-term anti-VEGF therapeutics for ocular vascular diseases. We therefore generated conditional VEGF knockout mice with an inducible Cre/lox system and determined the significance of M¨¹ller cell-derived VEGF in retinal development and maintenance and ischaemia-induced neovascularizartion and vascular leakage. Retinal development in the conditional VEGF knockout mice was analysed by examining retinal and choroidal vasculatures and retinal morphology and function. Ischaemia-induced retinal neovascularization and vascular leakage in the conditional VEGF knockout mice were analysed with fluorescein angiography, quantification of proliferative neovascular cells, immunohistochemistry, and immunoblotting using an oxygen-induced retinopathy model. Our results demonstrated that disruption of M¨¹ller cell-derived VEGF resulted in no apparent defects in retinal and choroidal vasculatures and retinal morphology and function, significant inhibition of the ischaemia-induced retinal neovascularization and vascular leakage, and attenuation of the ischaemia-induced breakdown of the blood-retina barrier. These results suggest that the retinal M¨¹ller cell-derived VEGF is a major contributor to ischaemia-induced retinal vascular leakage and pre-retinal and intra-retinal neovascularization. The observation that a significant, but not complete, reduction of VEGF in the retina does not cause detectable retinal degeneration suggests that appropriate doses of anti-VEGF agents may be important to the safe treatment of retinal vascular diseases.
243: Molecular and cellular endocrinology, 2010 Jan 15, 314(1)
Mechanism of VEGF expression by high glucose in proximal tubule epithelial cells.
[Abstract]Angiotensin II (Ang II) and vascular endothelial growth factor (VEGF) are important mediators of kidney injury in diabetes. VEGF expression is increased in proximal tubules of mice with type 1 diabetes. In mouse proximal tubular epithelial cells (MCT) cultured with 30 mM glucose (HG) for 24h, VEGF expression is increased at the protein and the mRNA level, suggesting a transcriptional mechanism. HG stimulation of VEGF synthesis is prevented by captopril, an inhibitor of angiotensin-converting enzyme, and, by losartan, a specific antagonist of angiotensin type 1 receptor (AT1), suggesting that VEGF synthesis is mediated by Ang II. Synthesis of angiotensinogen (AGT), a precursor of angiotensin II, is increased in MCTs cultured in HG. Although synthesis of renin and ACE is not affected by HG, their activity is increased in the conditioned medium. Concentrations of Ang I and Ang II are also increased in conditioned medium from HG-treated MCTs and captopril prevents increased Ang II, but not Ang I, synthesis. Finally, AT1 is activated in MCTs treated with HG, and its activation is prevented by captopril and losartan. The ERK pathway is activated by HG within minutes of stimulation and lasting for up to 24h. The initial phase of ERK activation is due to HG itself and leads to AGT upregulation and the sustained phase is mediated for the most part by Ang II-activated AT1 receptor and leads to increased VEGF synthesis. These data show that: (1) HG increases AGT synthesis and activation of renin and ACE by MCTs, leading to local production of Ang I and Ang II. (2) Ang II activates endogenous AT1 and stimulates synthesis of VEGF. (3) HG activation of ERK starts within minutes and lasts for up to 24h. Early ERK activation is involved in AGT upregulation and sustained ERK activation, mediated via AT1, is responsible for VEGF synthesis. In conclusion, our study shows that MCTs express an endogenous renin-angiotensin system that is activated by high glucose to stimulate the synthesis of VEGF, through activation of the ERK pathway.
244: Biochimica et biophysica acta, 2010 Mar, 1804(3)
Structure-function analysis of VEGF receptor activation and the role of coreceptors in angiogenic signaling.
[Abstract]Vascular endothelial growth factors (VEGFs) constitute a family of six polypeptides, VEGF-A, -B, -C, -D, -E and PlGF, that regulate blood and lymphatic vessel development. VEGFs specifically bind to three type V receptor tyrosine kinases (RTKs), VEGFR-1, -2 and -3, and to coreceptors such as neuropilins and heparan sulfate proteoglycans (HSPG). VEGFRs are activated upon ligand-induced dimerization mediated by the extracellular domain (ECD). A study using receptor constructs carrying artificial dimerization-promoting transmembrane domains (TMDs) showed that receptor dimerization is necessary, but not sufficient, for receptor activation and demonstrates that distinct orientation of receptor monomers is required to instigate transmembrane signaling. Angiogenic signaling by VEGF receptors also depends on cooperation with specific coreceptors such as neuropilins and HSPG. A number of VEGF isoforms differ in binding to coreceptors, and ligand-specific signal output is apparently the result of the specific coreceptor complex assembled by a particular VEGF isoform. Here we discuss the structural features of VEGF family ligands and their receptors in relation to their distinct signal output and angiogenic potential.
245: Expert opinion on biological therapy, 2009 Nov, 9(11)
VEGF as an inhibitor of tumor vessel maturation: implications for cancer therapy.
[Abstract]Tumor angiogenesis is an important component of cancer biology driven in part by the thesis that inhibition of tumor vessel growth would be expected to starve and thereby disrupt tumor growth. A significant portion of research on tumor angiogenesis has focused on VEGF and its blockade with the expectation that dramatic results would be demonstrated in cancer patients as previously documented in animal models. However, to date, anti-VEGF (bevacizumab, Avastin) therapy alone has demonstrated little if any antitumor activity or survival benefit in humans. Interestingly, bevacizumab in combination with chemotherapeutic agents appears to result in a modest survival benefit in patients with various malignancies. This has prompted the re-evaluation of the biological effects resulting from VEGF blockade. Recent reports indicate that inhibition of VEGF and its receptor can have dramatic and unexpected effects on mural and perivascular cells in the tumor microenvironment, leading to vascular smooth muscle cell/pericyte activation and vessel normalization/maturation. Specifically, when VEGF levels in tumors are high, recent studies suggest that this leads to reduced responsiveness of the mural cell to the related growth factor, platelet-derived growth factor. This raises the possibility that in addition to the demonstrated anti-angiogenic effect of VEGF neutralization, mural cell recruitment and thus vascular maturation might be among the most critical activities of anti-VEGF agents. This review seeks to explore how VEGF blockade impacts tumor vascular maturation and considers its implications for cancer therapy.
246: American journal of physiology. Regulatory, integrative and comparative physiology, 2009 Nov, 297(5)
Intramuscular VEGF repairs the failing heart: role of host-derived growth factors and mobilization of progenitor cells.
[Abstract]Skeletal muscle produces a myriad of mitogenic factors possessing cardiovascular regulatory effects that can be explored for cardiac repair. Given the reported findings that VEGF may modulate muscle regeneration, we investigated the therapeutic effects of chronic injections of low doses of human recombinant VEGF-A(165) (0.1-1 microg/kg) into the dystrophic hamstring muscle in a hereditary hamster model of heart failure and muscular dystrophy. In vitro, VEGF stimulated proliferation, migration, and growth factor production of cultured C2C12 skeletal myocytes. VEGF also induced production of HGF, IGF2, and VEGF by skeletal muscle. Analysis of skeletal muscle revealed an increase in myocyte nuclear [531 +/- 12 VEGF 1 microg/kg vs. 364 +/- 19 for saline (number/mm(2)) saline] and capillary [591 +/- 80 VEGF 1 microg/kg vs. 342 +/- 21 for saline (number/mm(2))] densities. Skeletal muscle analysis revealed an increase in Ki67(+) nuclei in the VEGF 1 microg/kg group compared with saline. In addition, VEGF mobilized c-kit(+), CD31(+), and CXCR4(+) progenitor cells. Mobilization of progenitor cells was consistent with higher SDF-1 concentrations found in hamstring, plasma, and heart in the VEGF group. Echocardiogram analysis demonstrated improvement in left ventricular ejection fraction (0.60 +/- 0.02 VEGF 1 microg/kg vs. 0.45 +/- 0.01 mm for saline) and an attenuation in ventricular dilation [5.59 +/- 0.12 VEGF 1 microg/kg vs. 6.03 +/- 0.09 for saline (mm)] 5 wk after initiating therapy. Hearts exhibited higher cardiomyocyte nuclear [845 +/- 22 VEGF 1 microg/kg vs. 519 +/- 40 for saline (number/mm(2))] and capillary [2,159 +/- 119 VEGF 1 microg/kg vs. 1,590 +/- 66 for saline (number/mm(2))] densities. Myocardial analysis revealed approximately 2.5 fold increase in Ki67+ cells and approximately 2.8-fold increase in c-kit(+) cells in the VEGF group, which provides evidence for cardiomyocyte regeneration and progenitor cell expansion. This study provides novel evidence of a salutary effect of VEGF in the cardiomyopathic hamster via induction of myogenic growth factor production by skeletal muscle and mobilization of progenitor cells, which resulted in attenuation of cardiomyopathy and repair of the heart.
247: Digestive diseases and sciences, 2010 Jul, 55(7)
Expression of VEGF, EGFR, and IL-6 in Gastric Adenomas and Adenocarcinomas by Endoscopic Submucosal Dissection.
[Abstract]BACKGROUND: The degree of intratumoral microvascular density is thought to affect tumor metastasis and prognosis in various human cancers, including gastric cancer. Despite recent medical advancements, gastric adenoma or adenocarcinoma remains a considerable therapeutic challenge. Endoscopic submucosal dissection (ESD) is a more recent approach that is now commonly used for radical resection of gastric adenoma and adenocarcinoma. AIM AND METHODS: The expression of vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR), and interleukin-6 (IL-6) are related to the prognosis of gastric adenocarcinoma. However, the expression of these factors in gastric adenoma/adenocarcinoma following ESD has not been clearly evaluated. Here, we report on our study of the expression of VEGF, EGFR, and IL-6 by immunohistochemical staining in extracted tissue from adenoma or adenocarcinoma of the stomach by ESD and subsequent evaluation of the correlation of VEGF, EGFR, and IL-6 with other clinicopathological parameters. The patient cohort consisted of 102 patients with adenoma or adenocarcinoma of the stomach. RESULTS: Immunohistochemical staining for VEGF and IL-6 was significantly higher in both high grade dysplasia and adenocarcinoma than in low grade dysplasia (P < 0.05). There was significant correlation between histological grade and intensity of immunohistochemical staining of VEGF (P = 0.039). Histological differentiation of adenocarcinoma was related to IL-6 expression (P = 0.028). The immunoreactivity of VEGF and IL-6 increased significantly in lesions >2 cm compared to lesions <2 cm (P < 0.05). CONCLUSION: The immunohistochemical expression of IL-6 and VEGF can be considered to be useful for clinical diagnosis and follow-up of adenoma or adenocarcinoma of the stomach.
248: American journal of physiology. Cell physiology, 2009 Dec, 297(6)
VEGF autoregulates its proliferative and migratory ERK1/2 and p38 cascades by enhancing the expression of DUSP1 and DUSP5 phosphatases in endothelial cells.
[Abstract]Vascular endothelial growth factor (VEGF) is a key angiogenic factor that regulates proliferation and migration of endothelial cells via phosphorylation of extracellular signal-regulated kinase-1/2 (ERK1/2) and p38, respectively. Here, we demonstrate that VEGF strongly induces the transcription of two dual-specificity phosphatase (DUSP) genes DUSP1 and DUSP5 in endothelial cells. Using fluorescence microscopy, fluorescence lifetime imaging (FLIM), and fluorescence cross-correlation spectroscopy (FCCS), we found that DUSP1/mitogen-activated protein kinases phosphatase-1 (MKP-1) was localized in both the nucleus and cytoplasm of endothelial cells, where it existed in complex with p38 (effective dissociation constant, K(D)(eff), values of 294 and 197 nM, respectively), whereas DUSP5 was localized in the nucleus of endothelial cells in complex with ERK1/2 (K(D)(eff) 345 nM). VEGF administration affected differentially the K(D)(eff) values of the DUSP1/p38 and DUSP5/ERK1/2 complexes. Gain-of-function and lack-of-function approaches revealed that DUSP1/MKP-1 dephosphorylates primarily VEGF-phosphorylated p38, thereby inhibiting endothelial cell migration, whereas DUSP5 dephosphorylates VEGF-phosphorylated ERK1/2 inhibiting proliferation of endothelial cells. Moreover, DUSP5 exhibited considerable nuclear anchoring activity on ERK1/2 in the nucleus, thereby diminishing ERK1/2 export to the cytoplasm decreasing its further availability for activation.
249: Chest, 2010 Jan, 137(1)
VEGF gene haplotypes are associated with sarcoidosis.
[Abstract]BACKGROUND: The cause of sarcoidosis is unclear. Evidence suggests that there is a genetic susceptibility toward the disease. In this study, we examined whether haplotypes of vascular endothelial growth factor (VEGF) and its receptors VEGFR-1 and VEGFR-2 are associated with the onset or the course of sarcoidosis. METHODS: Three hundred white patients with sarcoidosis and 381 matched controls were included. Sixty-three haplotype-tagging single nucleotide polymorphisms (SNPs) in the VEGF and VEGFR-1 and VEGFR-2 genes were selected from the HapMap Project phase 2. Mass spectrometry-based SNP genotyping was performed. RESULTS: Sarcoidosis, in general, was significantly associated with three SNPs in the VEGFR-1 gene: rs7337610 (P = .041), rs2296283 (P = .034), and rs12858139 (P = .027). In an acute course (defined as less than two episodes in a lifetime or a course lasting less than 2 years), an association of three SNPs in the VEGF gene was observed: rs833060 (P = .004), rs833068 (P = .008), and rs3025000 (P = .012). In the VEGFR-2 gene, one SNP was associated with an acute course of sarcoidosis (rs7667298, P = .023), whereas two SNPs were associated with a chronic course of sarcoidosis: rs7691507 (P = .029) and rs2125489 (P = .024). We then performed a haplotype analysis. After permutation-based correction, no significant haplotype association for the VEGF receptors was observed. However, we found two haplotypes associated with the onset of sarcoidosis in the VEGF gene. Even after correction for multiple testing, we obtained a P value of .0388. Moreover, patients with a chronic course of the disease showed a P value of .0103 for the same haplotype. CONCLUSIONS: There is strong evidence that VEGF and its receptors are involved in the onset of sarcoidosis and influence its course.
250: Experimental neurology, 2009 Dec, 220(2)
Inhibition of VEGF receptor 2 increased cell death of dentate hilar neurons after traumatic brain injury.
[Abstract]Post-traumatic epilepsy, partly due to the loss of hilar neurons of the hippocampus, is a frequent long-term consequence of traumatic brain injury (TBI). We and others found that the levels of vascular endothelial growth factor (VEGF) that can act as a neuroprotectant increase after TBI. Here we tested whether VEGF and its receptor VEGFR2 are involved in mediating the death or survival of hilar neurons after injury. We demonstrated that VEGFR2 is expressed by most, if not all, hilar neurons and that these neurons are dying in large numbers as indicated by Fluoro-Jade B histology after fluid percussion TBI. To directly test the involvement of VEGFR2 and VEGF in the injury-induced apoptotic death of hilar neurons, we delivered SU5416, an inhibitor to VEGFR2, or recombinant VEGF into the ipsilateral cerebral ventricle of injured animals. We found that blocking VEGFR2 by SU5416 significantly increased the number of apoptotic (TUNEL-positive) cells in the hilus. Infusion of VEGF, however, failed to reduce the number of TUNEL-positive cells. Our results suggest that VEGFR2 is involved in mediating death or survival of hilar neurons after injury but delivering additional exogenous VEGF does not provide further protection from TBI-induced death of hilar neurons.
251: Cancer science, 2009 Nov, 100(11)
Functional in vivo optical imaging of tumor angiogenesis, growth, and metastasis prevented by administration of anti-human VEGF antibody in xenograft model of human fibrosarcoma HT1080 cells.
[Abstract]Angiogenesis plays a crucial role in cancer progression and metastasis. Thus, blocking tumor angiogenesis is potentially a universal approach to prevent tumor establishment and metastasis. In this study, we used in vivo and ex vivo fluorescence imaging to show that an antihuman vascular endothelial growth factor (VEGF) antibody represses angiogenesis and the growth of primary tumors of human fibrosarcoma HT1080 cells in implanted nude mice. Interestingly, administering the antihuman VEGF antibody reduced the development of new blood vessels and normalized pre-existing tumor vasculature in HT1080 cell tumors. In addition, antihuman VEGF antibody treatment decreased lung metastasis from the primary tumor, whereas it failed to block lung metastasis in a lung colonization experiment in which tumor cells were injected into the tail vein. These results suggest that VEGF produced by primary HT1080 cell tumors has a crucial effect on lung metastasis. The present study indicates that the in vivo fluorescent microscopy system will be useful to investigate the biology of angiogenesis and test the effectiveness of angiogenesis inhibitors.
252: The Journal of pathology, 2009 Nov, 219(3)
VEGF-D deficiency in mice does not affect embryonic or postnatal lymphangiogenesis but reduces lymphatic metastasis.
[Abstract]Vascular endothelial growth factor-D (VEGF-D) is one of the two ligands of the VEGFR-3 receptor on lymphatic endothelial cells. Gene-silencing studies in mice and Xenopus tadpoles recently showed that the role of endogenous VEGF-D in lymphatic development is moderate. By contrast, exogenous VEGF-D is capable of stimulating lymphangiogenesis. Nonetheless, its endogenous role in pathological conditions remains largely unknown. Hence, we reassessed its role in disease, using Vegf-d(null) mice. Vegf-d(null) mice were generated that, under physiological conditions, displayed normal embryonic and postnatal lymphangiogenesis and lymphatic remodelling, efficient lymphatic functioning and normal health. Vegf-d(null) mice also reponded normally in models of skin wound healing and healing of infarcted myocardium, despite enhanced expression of VEGF-D in these models in wild-type mice. In contrast, Vegf-d(null) mice displayed reduced peritumoral lymphangiogenesis and lymph node metastasis in an orthotopic pancreatic tumour model. Together, our data indicate that endogenous VEGF-D in mice is dispensible for lymphangiogenesis during development, in postnatal and adult physiology and in several pathological conditions, but significantly contributes to lymphatic metastasis.
253: Investigative ophthalmology & visual science, 2010 Feb, 51(2)
Inhibition of vitreoretinal VEGF elevation and blood-retinal barrier breakdown in streptozotocin-induced diabetic rats by brimonidine.
[Abstract]PURPOSE: To determine whether long-term brimonidine (BRI) treatment prevents the hyperglycemia-induced increase in vitreoretinal vascular endothelial growth factor (VEGF) expression and breakdown of the blood-retinal barrier (BRB) in streptozotocin (STZ)-induced diabetic rats. METHODS: Brown Norway/Long-Evans rats were divided into three groups with similar distributions of blood glucose and body weight. Two groups received a single intravenous injection of STZ (65 mg/kg) and the remaining control group received vehicle. Drug treatment administered via miniosmotic pumps was initiated 1 or 6 weeks later. The STZ-induced diabetic rats were treated with BRI (1 mg/kg/d) or vehicle (VEH) and control nondiabetic rats were treated with VEH for 4 weeks. Vitreoretinal VEGF protein, vitreal glutamate, and BRB breakdown were then measured. RESULTS: At 5 weeks after STZ treatment, STZ-treated diabetic rats demonstrated significantly elevated vitreoretinal VEGF expression, vitreal glutamate concentrations, and BRB leakage compared with nondiabetic control rats. Chronic BRI treatment had no effect on vitreal glutamate concentrations in diabetic animals but significantly decreased vitreoretinal VEGF expression and BRB breakdown to levels similar to those observed in control rats. BRI also significantly reduced BRB breakdown in aged diabetic rats at 10 weeks after STZ treatment. CONCLUSIONS: BRI produced marked decreases in vitreoretinal VEGF and inhibition of BRB breakdown in diabetic rats. The mechanism for these effects may involve attenuation of retinal NMDA receptor activity by BRI. The results suggest that BRI would be useful for treatment of ocular diseases associated with BRB leakage, such as diabetic macular edema and retinopathy.
254: Atherosclerosis, 2010 Feb, 208(2)
Soluble VEGF receptor-2 is increased in sera of subjects with metabolic syndrome in association with insulin resistance.
[Abstract]OBJECTIVE: Metabolic syndrome (MetS) is associated with impaired angiogenesis. Vascular endothelial growth factor (VEGF) plays a key role in angiogenesis through binding to its specific receptor, VEGF receptor-2 (VEGFR-2), whereas the expression of VEGF and VEGFR-2 in the myocardium of insulin-resistant rats is down-regulated. Soluble VEGF receptor-1 (sVEGFR-1) and -2 (sVEGFR-2) have been reported to inhibit angiogenesis both in vitro and in vivo. However, the balance between circulating levels of VEGF and its soluble receptors, which may reflect and/or affect cardiovascular VEGF signaling, in subjects with MetS is unknown. METHODS AND RESULTS: We carried out a cross-sectional study including 272 consecutive, apparently healthy subjects who were not receiving any drugs. Plasma levels of VEGF and serum levels of its soluble receptors were determined using enzyme-linked immunosorbent assays. VEGF and sVEGFR-1 levels did not differ between subjects with and those without MetS. However, sVEGFR-2 levels were significantly increased in MetS compared with non-MetS subjects. Stepwise regression analysis revealed that HOMA-IR was the strongest independent determinant of the sVEGFR-2 level. Accordingly, the mean sVEGFR-2 levels increased in proportion to both the accumulation of components of MetS and quartile of HOMA-IR. Interestingly, multiple regression analyses revealed that independent determinants of VEGF were the body mass index and blood pressure, whereas, in contrast, those of sVEGFR-2 were HOMA-IR and high-sensitivity C-reactive protein. CONCLUSIONS: The correlation of sVEGFR-2 with insulin resistance supports the need for further investigations to define the clinical utility and predictive value of serum sVEGFR-2 levels in cardiovascular dysfunction in MetS.
255: Journal of experimental zoology. Part B, Molecular and developmental evolution, 2010 Mar 15, 314(2)
Evolution of viviparity and uterine angiogenesis: vascular endothelial growth factor (VEGF) in oviparous and viviparous skinks.
[Abstract]During pregnancy, uterine vasculature of live-bearing lizards proliferates to support embryonic growth and development. Vascular endothelial growth factor (VEGF) is the most potent of a suite of growth factors responsible for uterine vascularization in mammals. We have sequenced VEGF mRNA transcripts expressed in the uterus of oviparous and viviparous Australian skinks, and compared uterine VEGF expression in nonreproductive and late-reproductive Saiphos equalis, a fossorial viviparous skink. VEGF sequences differed between phylogenetic groups of skinks, rather than oviparous and viviparous skinks. Two transcripts were identified in the uterus of each species that had the same splice sites as human VEGF(165) and VEGF(189). A third transcript, found only in uterine and testis tissue from S. equalis, had the same splice sites as human VEGF(111). This is the first natural expression of VEGF(111), previously found only in human cultured cells subjected to environmental stress. All the three VEGF transcripts identified showed higher expression in uterus from late-reproductive S. equalis than nonreproductive females. The different angiogenic properties of VEGF transcripts provide a mechanism that may produce the variety of placental complexities observed in viviparous skinks. The presence of VEGF(111) in S. equalis may be an opportunity to investigate the function of this unique transcript in a whole animal system. J. Exp. Zool. (Mol. Dev. Evol.) 314B:148-156, 2010. (c) 2009 Wiley-Liss, Inc.
256: Biomedical chromatography : BMC, 2010 Apr, 24(4)
Quantification of henatinib maleate, a novel potent inhibitor of VEGF receptors, in rat plasma by LC-MS/MS.
[Abstract]Henatinib maleate (R,Z)-2-[(5-fluoro-1,2-dihydro-2-oxo-3H-indol-3-ylidene) methyl]-5-(2-hydroxy-3-morpholinopropyl)-3-methyl-5,6,7,8-tetrahydro-1H-pyrrolo[3,2-c] azepin-4-ketone maleate is a potent inhibitor of vascular endothelial growth factor receptors, and is currently under preclinical evaluation as an anticancer drug. A novel method for the quantification of henatinib maleate in rat plasma using high performance liquid chromatography-tandem mass spectrometry has been developed. The analyte (henatinib maleate) and internal standard (papaverine hydrochloride) were extracted from 50 muL of rat plasma by protein precipitation and separated on a C(18) column using a mixture of 25 mm ammonium acetate buffer : methanol : acetonitrile (35 : 50 : 15, v/v/v) as mobile phase with a run time of 4.5 min. The detection was performed by means of triple quadrupole mass spectrometer equipped with an ESI interface operating in the multiple-reaction monitoring mode. A linear response was observed over the concentration range 5.0-1000 ng/mL. The limit of quantification was 5.0 ng/mL. Both intra- and inter-day precision, defined as relative standard deviation, were within 9.7%. Accuracy, defined as relative error, was within +/- 3.1%. The developed method was successfully applied to preclinical pharmacokinetic studies of henatinib maleate in rat after a single oral administration of the drug. Copyright (c) 2009 John Wiley & Sons, Ltd.
257: Cancer letters, 2010 Feb 28, 288(2)
Cathepsin G-mediated enhanced TGF-beta signaling promotes angiogenesis via upregulation of VEGF and MCP-1.
[Abstract]Transforming growth factor (TGF)-beta signaling makes a significant contribution to the pathogenesis of breast cancer bone metastasis. In other tumor types, TGF-beta has been shown to promote tumor vascularity. Here, we report that inhibition of TGF-beta significantly reduces microvessel density in mammary tumor-induced bone lesions, mediated by decreased expression of both vascular endothelial growth factor (VEGF) and monocyte chemotactic protein (MCP)-1, both known angiogenic factors. Cathepsin G upregulation at the tumor-bone interface has been linked to increased TGF-beta signaling, and we also report that inhibition of Cathepsin G reduced tumor vascularity, as well as VEGF and MCP-1 expression.
258: The FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2009 Nov, 23(11)
Fatty acid binding protein 4 is a target of VEGF and a regulator of cell proliferation in endothelial cells.
[Abstract]Fatty acid binding protein 4 (FABP4) plays an important role in maintaining glucose and lipid homeostasis. FABP4 has been primarily regarded as an adipocyte- and macrophage-specific protein, but recent studies suggest that it may be more widely expressed. We found strong FABP4 expression in the endothelial cells (ECs) of capillaries and small veins in several mouse and human tissues, including the heart and kidney. FABP4 was also detected in the ECs of mature human placental vessels and infantile hemangiomas, the most common tumor of infancy and ECs. In most of these cases, FABP4 was detected in both the nucleus and cytoplasm. FABP4 mRNA and protein levels were significantly induced in cultured ECs by VEGF-A and bFGF treatment. The effect of VEGF-A on FABP4 expression was inhibited by chemical inhibition or short-hairpin (sh) RNA-mediated knockdown of VEGF-receptor-2 (R2), whereas the VEGFR1 agonists, placental growth factors 1 and 2, had no effect on FABP4 expression. Knockdown of FABP4 in ECs significantly reduced proliferation both under baseline conditions and in response to VEGF and bFGF. Thus, FABP4 emerged as a novel target of the VEGF/VEGFR2 pathway and a positive regulator of cell proliferation in ECs.
259: International journal of cancer. Journal international du cancer, 2009 Nov 1, 125(9)
Curcumin sensitizes human colorectal cancer to capecitabine by modulation of cyclin D1, COX-2, MMP-9, VEGF and CXCR4 expression in an orthotopic mouse model.
[Abstract]Because of the poor prognosis and the development of resistance against chemotherapeutic drugs, the current treatment for advanced metastatic colorectal cancer (CRC) is ineffective. Whether curcumin (a component of turmeric) can potentiate the effect of capecitabine against growth and metastasis of CRC was investigated. The effect of curcumin on proliferation of CRC cell lines was examined by mitochondrial dye-uptake assay, apoptosis by esterase staining, nuclear factor-kappaB (NF-kappaB) by electrophoretic mobility shift assay and gene expression by Western blot analysis. The effect of curcumin on the growth and metastasis of CRC was also examined in orthotopically implanted tumors in nude mice. In vitro, curcumin inhibited the proliferation of human CRC cell lines, potentiated capecitabine-induced apoptosis, inhibited NF-kappaB activation and suppressed NF-kappaB-regulated gene products. In nude mice, the combination of curcumin and capecitabine was found to be more effective than either agent alone in reducing tumor volume (p = 0.001 vs. control; p = 0.031 vs. capecitabine alone), Ki-67 proliferation index (p = 0.001 vs. control) and microvessel density marker CD31. The combination treatment was also highly effective in suppressing ascites and distant metastasis to the liver, intestines, lungs, rectum and spleen. This effect was accompanied by suppressed expression of activated NF-kappaB and NF-kappaB-regulated gene products (cyclin D1,c-myc, bcl-2, bcl-xL, cIAP-1, COX-2, ICAM-1, MMP-9, CXCR4 and VEGF). Overall, our results suggest that curcumin sensitizes CRC to the antitumor and antimetastatic effects of capecitabine by suppressing NF-kappaB cell signaling pathway.
260: Pediatric blood & cancer, 2009 Dec, 53(6)
VEGF expression as a prognostic marker in osteosarcoma.
[Abstract]BACKGROUND: The vascular endothelial growth factor (VEGF) pathway is the key regulator of angiogenesis. In osteosarcoma baseline VEGF is of proven prognostic value but prognostic potential of post-NACT VEGF expression is largely unexplored. PROCEDURE: Treatment naive patients with osteosarcoma were subjected to initial staging workup followed by three cycles of neoadjuvant chemotherapy (NACT) and surgery; resected tumors were assessed for histological necrosis by Huvos grading. Initial biopsy and resected tumor specimens post-NACT were examined for VEGF expression by immunohistochemistry. Positive VEGF expression was considered when intensive positive staining was observed in >10% of the tumor cells. VEGF expression at baseline was compared with grade of tumor; pre-NACT and post-NACT VEGF expression were compared with histological necrosis. Receiver operating characteristic curves were generated to assess best threshold and predictability. RESULTS: A total of 31 patients were recruited with median age of 17 years (range 5-66 years); male/female ratio was 25:6; 23 patients (74%) were non-metastatic. At baseline, there was 90% concordance between positive VEGF expression and higher histological grade (28/31); baseline VEGF expression did not correlate well with stage and histological necrosis. Twenty-one (67%) were poor and 10 (33%) were good histologic responders; post-NACT VEGF expression as well as VEGF change following NACT significantly correlated with histological necrosis. CONCLUSION: Positive VEGF expression in surviving tumor cells post-NACT in resected tumors appears to be an important negative prognostic factor in osteosarcoma which may help future therapies to be identified according to the angiogenic potential of the disease.
261: Archives of dermatological research, 2010 Apr, 302(3)
Gingko biloba extract reduces VEGF and CXCL-8/IL-8 levels in keratinocytes with cumulative effect with epigallocatechin-3-gallate.
[Abstract]In skin inflammation, vascular endothelial growth factor (VEGF) and CXCL-8/IL-8 play an important role and are produced by activated keratinocytes. Extracts from Ginkgo biloba leaves (GBE), widely used in phytotherapy, have been reported to exert antioxidant and anti-inflammatory properties in the skin. We therefore evaluated the effects of GBE on the release of VEGF and CXCL8/IL-8 by normal human keratinocytes (NHKs) activated by tumor necrosis factor alpha (TNFalpha). Moreover, as we previously showed that epigallocatechin-3-gallate (EGCG) reduces VEGF and CXCL8/IL-8 secretion in TNFalpha-activated NHKs, we also tested its effect in association with GBE. Our results showed that GBE exerted a potent inhibition on VEGF and CXCL8/IL-8 levels in activated cells. In association with EGCG, GBE down-regulated VEGF and CXCL8/IL-8 levels in a cumulative manner in TNFalpha-stimulated NHKs. These results suggest that GBE, alone or in association with EGCG may contribute to moderate inflammatory processes in skin diseases associated with angiogenesis.
262: The international journal of biochemistry & cell biology, 2009 Dec, 41(12)
Anti-HLA I antibodies induce VEGF production by endothelial cells, which increases proliferation and paracellular permeability.
[Abstract]Anti-human leukocyte antigen class I (HLA I) antibodies were shown to activate several protein kinases in endothelial cells (ECs), which induces proliferation and cell survival. An important phenomenon in antibody-mediated rejection is the occurrence of interstitial edema. We investigated the effect of anti-HLA I antibodies on endothelial proliferation and permeability, as one possible underlying mechanism of edema formation. HLA I antibodies increased the permeability of cultured ECs isolated from umbilical veins. Anti-HLA I antibodies induced the production of vascular endothelial growth factor (VEGF) by ECs, which activated VEGF receptor 2 (VEGFR2) in an autocrine manner. Activated VEGFR2 led to a c-Src-dependent phosphorylation of vascular endothelial (VE)-cadherin and its degradation. Aberrant VE-cadherin expression resulted in impaired adherens junctions, which might lead to increased endothelial permeability. This effect was only observed after cross-linking of HLA I molecules by intact antibodies. Furthermore, our results suggest that increased endothelial proliferation following anti-HLA I treatment occurs via autocrine VEGFR2 activation. Our data indicate the ability of anti-HLA I to induce VEGF production in ECs. Transactivation of VEGFR2 leads to increased EC proliferation and paracellular permeability. The autocrine effect of VEGF on endothelial permeability might be an explanation for the formation of interstitial edema after transplantation.
263: Schizophrenia research, 2009 Dec, 115(2-3)
Decreased VEGF mRNA expression in the dorsolateral prefrontal cortex of schizophrenia subjects.
[Abstract]BACKGROUND: The vascular endothelial growth factor (VEGF) pathway is the key regulator of angiogenesis. In osteosarcoma baseline VEGF is of proven prognostic value but prognostic potential of post-NACT VEGF expression is largely unexplored. PROCEDURE: Treatment naive patients with osteosarcoma were subjected to initial staging workup followed by three cycles of neoadjuvant chemotherapy (NACT) and surgery; resected tumors were assessed for histological necrosis by Huvos grading. Initial biopsy and resected tumor specimens post-NACT were examined for VEGF expression by immunohistochemistry. Positive VEGF expression was considered when intensive positive staining was observed in >10% of the tumor cells. VEGF expression at baseline was compared with grade of tumor; pre-NACT and post-NACT VEGF expression were compared with histological necrosis. Receiver operating characteristic curves were generated to assess best threshold and predictability. RESULTS: A total of 31 patients were recruited with median age of 17 years (range 5-66 years); male/female ratio was 25:6; 23 patients (74%) were non-metastatic. At baseline, there was 90% concordance between positive VEGF expression and higher histological grade (28/31); baseline VEGF expression did not correlate well with stage and histological necrosis. Twenty-one (67%) were poor and 10 (33%) were good histologic responders; post-NACT VEGF expression as well as VEGF change following NACT significantly correlated with histological necrosis. CONCLUSION: Positive VEGF expression in surviving tumor cells post-NACT in resected tumors appears to be an important negative prognostic factor in osteosarcoma which may help future therapies to be identified according to the angiogenic potential of the disease.
264: International journal of cancer. Journal international du cancer, 2010 Jan 1, 126(1)
Bisphosphonates suppress insulin-like growth factor 1-induced angiogenesis via the HIF-1alpha/VEGF signaling pathways in human breast cancer cells.
[Abstract]Adjunctive chemotherapy with bisphosphonates has been reported to delay bone metastasis and improve overall survival in breast cancer. Aside from its antiresorptive effect, bisphosphonates exhibit antitumor activities, in vitro and in vivo, via several mechanisms, including antiangiogenesis. In this study, we investigated the potential molecular mechanisms underlying the antiangiogenic effect of non-nitrogen-containing and nitrogen-containing bisphosphonates, clodronate and pamidronate, respectively, in insulin-like growth factor (IGF)-1 responsive human breast cancer cells. We tested whether bisphosphonates had any effects on hypoxia-inducible factor (HIF)-1alpha/vascular endothelial growth factor (VEGF) axis that plays a pivotal role in tumor angiogenesis, and our results showed that both pamidronate and clodronate significantly suppressed IGF-1-induced HIF-1alpha protein accumulation and VEGF expression in MCF-7 cells. Mechanistically, we found that either pamidronate or clodronate did not affect mRNA expression of HIF-1alpha, but they apparently promoted the degradation of IGF-1-induced HIF-1alpha protein. Meanwhile, we found that the presence of pamidronate and clodronate led to a dose-dependent decease in the newly-synthesized HIF-1alpha protein induced by IGF-1 in breast cancer cells after proteasomal inhibition, thus, indirectly reflecting the inhibition of protein synthesis. In addition, our results indicated that the inhibitory effects of bisphosphonates on the HIF-1alpha/VEGF axis are associated with the inhibition of the phosphoinositide 3-kinase/AKT/mammalian target of rapamycin signaling pathways. Consistently, we demonstrated that pamidronate and clodronate functionally abrogated both in vitro and in vivo tumor angiogenesis induced by IGF-1-stimulated MCF-7 cells. These findings have highlighted an important mechanism of the pharmacological action of bisphosphonates in the inhibition of tumor angiogenesis in breast cancer cells.
265: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association, 2009 Nov, 24(11)
Associations of VEGF and its receptors sVEGFR-1 and -2 with cardiovascular disease and survival in prevalent haemodialysis patients.
[Abstract]BACKGROUND: Vascular endothelial growth factor (VEGF) was recently shown to predict survival in prevalent haemodialysis patients. Soluble VEGF receptors (sVEGFR)-1 and -2 are circulating endogenous modulators of VEGF activity. We thus studied the relationship between sVEGFR-1 and -2 and survival in a cohort of prevalent haemodialysis (HD) patients. METHODS: Components of the VEGF system were measured (ELISAs) in 185 prevalent HD patients and levels related to clinical characteristics, biochemical markers and survival. The patients were followed up prospectively for a median 31 (20-37) months. RESULTS: While ischaemic heart disease was independently associated with a lower sVEGFR-2 (OR = 2.75, P = 0.02), sVEGFR-1 was positively associated with IL-6 (rho = 0.22, P = 0.003) and white blood cell count (rho = 0.22, P = 0.002). In survival analysis, the patients with a high sVEGFR-1 level had a higher all-cause mortality (Kaplan-Meier Chi-Square = 5.6, P = 0.02) and a higher adjusted mortality risk (Cox HR = 1.93, P = 0.009) than those with low levels. CONCLUSION: In the first clinical study of sVEGFR-1 and -2 in CKD, we found novel associations between the sVEGFRs and cardiac disease. This may be of clinical importance, as a high sVEGFR-1 was an independent risk factor for all-cause mortality.
266: Prostaglandins & other lipid mediators, 2009 Nov, 90(1-2)
Involvement of Rho-kinase in prostaglandin E(1)-stimulated VEGF synthesis through stress-activated protein kinase/c-Jun N-terminal kinase in osteoblast-like MC3T3-E1 cells.
[Abstract]We have previously shown that prostaglandin E(1) (PGE(1)) stimulates the synthesis of vascular endothelial growth factor (VEGF) through p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) but not p44/p42 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the involvement of Rho-kinase in the PGE(1)-stimulated VEGF synthesis in these cells. PGE(1) induced within 3min the phosphorylation of myosin phosphatase targeting subunit (MYPT-1), a substrate of Rho-kinase. Y27632 and fasudil, specific inhibitors of Rho-kinase, which attenuated the MYPT-1 phosphorylation, significantly suppressed the PGE(1)-stimulated VEGF synthesis. Y27632 and fasudil markedly reduced the PGE(1)-induced phosphorylation of SAPK/JNK without affecting the phosphorylation levels of p38 MAP kinase or p44/p42 MAP kinase. These results strongly suggest that Rho-kinase functions at a point upstream of SAPK/JNK and regulates PGE(1)-stimulated VEGF synthesis in osteoblasts.
267: Archives of gynecology and obstetrics, 2010 Mar, 281(3)
Regulated expression of the Renin-Angiotensin-System in human granulosa lutein cells: Angiotensin II increases VEGF expression but its synthesis is reduced by hCG.
[Abstract]INTRODUCTION: The Renin-Angiotensin-System (RAS) has been suspected not only to control vascular tone but also to regulate angiogenesis. Angiotensin II has been shown to influence angiogenic factors such as vascular endothelial growth factor (VEGF). The Corpus luteum undergoes intense VEGF-dependent angiogenesis, regulated by luteinising hormone (LH) and human chorionic gonadotrophin (hCG). We therefore hypothesised, that locally produced Angiotensin II could act as a physiological co-regulator with hCG in luteal VEGF expression. MATERIALS AND METHODS: We investigated the expression of RAS components and their regulation by hCG in human granulosa lutein cells using RT-PCR, immunocytochemistry and Western blotting. In addition, we studied the effect of Angiotensin II on basal and hCG-stimulated VEGF expression using TaqMan-analysis, ELISA, and Western blot analysis. RESULTS: Human granulosa lutein cells express angiotensinogen and angiotensin converting enzyme (ACE) and synthesise Angiotensin. In addition, they express both Angiotensin receptors. Angiotensin II stimulated VEGF mRNA (p < 0.05) and protein expression (p < 0.05). However, hCG decreased angiotensinogen (p < 0.05) and Angiotensin II (p < 0.05). Both, the addition of Angiotensin II and its inhibition using Candesartan did not change the magnitude of hCG-increased VEGF expression. CONCLUSION: These findings suggest a role for locally synthesised Angiotensin II in the regulation of luteal VEGF expression. However, Angiotensin II does not appear to have a major contribution in the presence of hCG when the RAS pathway is down-regulated.
268: Leukemia research, 2009 Dec, 33(12)
Strong correlation between VEGF and MCL-1 mRNA expression levels in B-cell chronic lymphocytic leukemia.
[Abstract]Expression of the anti-apoptotic myeloid cell leukemia-1 (MCL-1) gene is a novel prognostic factor in B-cell chronic lymphocytic leukemia (B-CLL). Vascular and endothelial growth factor (VEGF) and interleukin-6 (IL-6) are able to upregulate MCL-1 via autocrine signaling loops. In 88 B-CLL patients, we found a strong correlation of MCL-1 gene expression with VEGF (P<10(-7)) but not with IL-6 mRNA levels. VEGF but not IL-6 expression influenced patient prognosis. VEGF may be a positive autocrine in vivo regulator of MCL-1 in B-CLL. Inhibition of VEGF and its signaling may prove to be useful in the treatment of B-CLL patients.
269: Journal of biomedical materials research. Part A, 2010 Apr, 93(1)
VEGF-E enhances endothelialization and inhibits thrombus formation on polymeric surfaces.
[Abstract]Thrombotic complications of long-term blood-contacting devices can be avoided by formation of an endothelial cell layer on the blood-contacting surface. The endothelial cells form a bioactive boundary between the synthetic surface and blood, regulating haemostasis and inflammation. Biofunctionalization of synthetic blood-contacting surfaces is necessary to accommodate growth of endothelial cells. Vascular endothelial growth factor E (VEGF-E) or collagen I may stimulate endothelialization of a polymeric surface coating of a prototype small diameter vascular prosthesis. VEGF-E was produced in Escherichia coli and could be easily purified in large quantities. Recombinant VEGF-E or purified collagen I was allowed to adsorb onto the polymeric surfaces and enhanced formation of an endothelial cell layer. Adsorption of VEGF-E was increased by the inclusion of the anti-coagulant drug heparin in the polymeric coating. Collagen I adsorption induced rapid thrombin generation and increased platelet adhesion on surfaces with or without heparin. VEGF-E inhibited thrombus formation, and did not interfere with the anti-thrombogenic effect of heparin. Additionally, VEGF-E did not affect platelet adhesion. Adsorption of VEGF-E, especially on heparin containing surfaces, provides an economical strategy to improve endothelialization of cardiovascular implants without disturbing blood-compatibility. (c) 2009 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.
270: Cancer letters, 2009 Nov 28, 285(2)
Curcumin inhibits the migration and invasion of human A549 lung cancer cells through the inhibition of matrix metalloproteinase-2 and -9 and Vascular Endothelial Growth Factor (VEGF).
[Abstract]It is well known that matrix metalloproteinases (MMPs) act an important role in the invasion, metastasis and angiogenesis of cancer cells. Agents suppressed the MMPs could inhibited the cancer cells migration and invasion. Numerous evidences had shown that curcumin (the active constituent of the dietary spice turmeric) has potential for the prevention and therapy of cancer. Curcumin can inhibit the formation of tumors in animal models of carcinogenesis and act on a variety of molecular targets involved in cancer development. There is however, no available information to address the effects of curcumin on migration and invasion of human lung cancer cells. The anti-tumor invasion and migration effects of lung cancer cells induced by curcumin were examined. Here, we report that curcumin suppressed the migration and invasion of human non-small cell lung cancer cells (A549) in vitro. Our findings suggest that curcumin has anti-metastatic potential by decreasing invasiveness of cancer cells. Moreover, this action was involved in the MEKK3, p-ERK signaling pathways resulting in inhibition of MMP-2 and -9 in human lung cancer A549 cells. Overall, the above data shows that the anticancer effect of curcumin is also exist for the inhibition of migration and invasion in lung cancer cells.
271: The Journal of nutritional biochemistry, 2010 Jul, 21(7)
Ferulic acid augments angiogenesis via VEGF, PDGF and HIF-1 alpha.
[Abstract]Therapeutic angiogenesis is critical to wound healing and ischemic diseases such as myocardial infarction and stroke. For development of therapeutic agents, a search for new angiogenic agents is the key. Ferulic acid, a phytochemical found in many fruits and vegetables, exhibits a broad range of therapeutic effects on human diseases, including diabetes and cancer. This study investigated the augmenting effect of ferulic acid on angiogenesis through functional modulation of endothelial cells. Through endothelial cell migration and tube formation assays, ferulic acid (10(-6)-10(-4) M) was found to induce significant angiogenesis in human umbilical vein endothelial cells (HUVECs) in vitro without cytotoxicity. With chorioallantoic membrane assay, ferulic acid (10(-6)-10(-5) M) was also found to promote neovascularization in vivo. Using Western blot analysis and quantitative real-time polymerase chain reaction, we found that ferulic acid increased vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) expression in HUVECs. Furthermore, the amounts of hypoxic-induced factor (HIF) 1 alpha mRNA and protein, the major regulator of VEGF and PDGF, also showed up-regulation by ferulic acid. Electrophoretic migration shift assay showed that the binding activity of HIF-1 alpha was also enhanced with ferulic acid treatment of HUVECs. Moreover, inhibitors of extracellular-signal-regulated kinase 1/2 and phosphoinositide-3 kinase (PI3K) abolished the binding activity of HIF-1 alpha and the subsequent activation of VEGF and PDGF production by ferulic acid. Thus, both mitogen-activated protein kinase and PI3K pathways were involved in the angiogenic effects of ferulic acid. Taken together, ferulic acid serves as an angiogenic agent to augment angiogenesis both in vitro and in vivo. This effect might be observed through the modulation of VEGF, PDGF and HIF-1 alpha.
272: Molecular and cellular biochemistry, 2009 Nov, 331(1-2)
PTEN regulate angiogenesis through PI3K/Akt/VEGF signaling pathway in human pancreatic cancer cells.
[Abstract]Phosphoinositide 3-kinase (PI3K) pathway exerts its effects through Akt, its downstream target molecule, and thereby regulates various cell functions including cell proliferation, cell transformation, apoptosis, tumor growth, and angiogenesis. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has been implicated in regulating cell survival signaling through the PI3K/Akt pathway. However, the mechanism by PI3K/PTEN signaling regulates angiogenesis and tumor growth in vivo remains to be elucidated. Vascular endothelial growth factor (VEGF) plays a pivotal role in tumor angiogenesis. The effect of PTEN on VEGF-mediated signal in pancreatic cancer is unknown. This study aimed to determine the effect of PTEN on both the expression of VEGF and angiogenesis. Toward that end, we used the siRNA knockdown method to specifically define the role of PTEN in the expression of VEGF and angiogenesis. We found that siRNA-mediated inhibition of PTEN gene expression in pancreatic cancer cells increase their VEGF secretion, up-modulated the proliferation, and migration of co-cultured vascular endothelial cell and enhanced tubule formation by HUVEC. In addition, PTEN modulated VEGF-mediated signaling and affected tumor angiogenesis through PI3K/Akt/VEGF/eNOS pathway.
273: Journal of biomedical materials research. Part A, 2010 Mar 15, 92(4)
Targeted molecular imaging of VEGF receptors overexpressed in ischemic microvasculature using chitosan-DC101 conjugates.
[Abstract]Expression of vascular endothelial growth factor receptors (VEGFRs) increases in ischemic muscles, and thus, VEGFR could potentially be used as marker to detect ischemia. Here, we investigated whether (99m)Tc or Cy5.5-labeled chitosan-DC101 conjugates could identify VEGFR-2 overexpressed in ischemia. To this end, chitosan was conjugated with the DC101 antibody and Cy5.5, FITC, or the HYNIC chelator for (99m)Tc-labeling. Targeting of the conjugate was evaluated in vitro and in vivo through cell-binding studies and gamma/optical imaging, respectively. A hindlimb ischemic mouse model was surgically created by femoral artery occlusion. The chitosan-DC101 conjugates exhibited VEGFR-selective cell binding properties as determined by both confocal microscopy and flow cytometry. At postoperative times of 2, 12, and 24 h, (99m)Tc or Cy5.5-labeled chitosan-DC101 conjugates were intravenously injected into the mice, and gamma/optical imaging studies were conducted at 1 or 3 h. Both the gamma and optical imaging results indicated a significantly higher uptake in ischemic muscles when compared with the contralateral nonischemic muscle. Further, semiquantitative analysis of scintigraphic imaging data revealed that the ischemic to contralateral limb ratio was 4.5 +/- 0.25 at 24 h postoperation. Western blotting analysis confirmed VEGFR expression in the ischemic muscle. In conclusion, we believe that (99m)Tc or Cy5.5-labeled chitosan-DC101 conjugates have the potential to be useful as VEGFR-2-targeted imaging agents for monitoring ischemia. (c) 2009 Wiley Periodicals, Inc. J Biomed Mater Res 2010.
274: The Journal of surgical research, 2010 Apr, 159(2)
Significance of Portal Venous VEGF During Liver Regeneration After Hepatectomy.
[Abstract]BACKGROUND: Although some studies have hypothesized portal venous blood is important for liver regeneration, no studies have established organs whose venous effluent flow into the portal vein secrete liver regenerating factors into the portal vein during liver regeneration. The aim of this study was to elucidate up-regulation of vascular endothelial growth factor (VEGF) in the portal vein, and expressions of hepatic regenerating factors in organs whose venous effluent flows into the portal vein during liver regeneration. MATERIALS AND METHODS: VEGF protein in systemic and portal venous blood, as well as expression of VEGF, hypoxia-inducible factor-1alfa (HIF-1alpha), hepatocyte growth factor (HGF), and HGF activator (HGFA) mRNA were evaluated in the regenerating liver, spleen, and intestine following 70% partial hepatectomy (PHx) in rats. RESULTS: The portal VEGF protein level was significantly higher than the systemic level post-PHx (portal/systemic at 72, 120, and 168 h post-PHx: 17.2/13.0, 20.2/12.8, and 24.0/14.7 pg/mL; P = 0.003, P = 0.022 and P = 0.032, respectively). VEGF mRNA expressions were significantly higher in the liver (P = 0.000027: 168 h), spleen (P = 0.000059: 72 h) and intestine (P = 0.01: 24-72 h) post-PHx compared with pre-PHx. HIF-1alpha, HGF, and HGFA mRNA expressions in the liver, intestine, and spleen were also significantly higher post-PHx compared to pre-PHx. CONCLUSIONS: Portal VEGF was significantly higher than systemic VEGF, and expressions of VEGF, HIF-1alpha, HGF, and HGFA mRNA in the liver, spleen and intestine were also up-regulated during liver regeneration. These results suggest that hepatic regenerating factors derived from the spleen or intestine may contribute liver regeneration.
275: Fertility and sterility, 2010 Apr, 93(6)
Thiazolidinediones decrease vascular endothelial growth factor (VEGF) production by human luteinized granulosa cells in vitro.
[Abstract]OBJECTIVE: To determine the effect of thiazolidenedione derivatives (TZDs) on vascular endothelial growth factor (VEGF) production by human luteinized granulosa cells and the morphologic development of murine embryos. DESIGN: Prospective, experimental, in vitro and in vivo study. SETTING: Research laboratory. PATIENT(S): Follicular aspirates from 10 women undergoing oocyte retrieval. INTERVENTION(S): Isolated human granulosa cells were treated with a dimethyl sulfoxide (DMSO) control or ciglitazone, in the presence and absence of an hCG stimulus. Embryos extracted from superovulated B6C3F1 female mice were cultured in the presence of DMSO or pioglitazone. MAIN OUTCOME MEASURE(S): Vascular endothelial growth factor concentrations at 24 and 48 hours. Morphologic development of murine embryos at 96 hours. RESULT(S): Following an hCG stimulus, treatment with 20 microM or 40 microM ciglitazone decreased VEGF production in a statistically significant manner at both time intervals. Blastocyst development at 96 hours did not significantly differ between untreated zygotes and those treated with pioglitazone. CONCLUSION(S): Ciglitazone significantly decreased VEGF production by human granulosa cells in an in vitro model. Pioglitazone did not adversely impact the development of cultured murine embryos. Although mechanistic evidence is not provided, the pivotal role of VEGF in ovarian hyperstimulation syndrome prompts investigation of TZDs as a novel treatment for this condition.
276: Human gene therapy, 2009 Mar 2,
Genetic Delivery of Bevacizumab to Suppress VEGF-induced High-permeability Pulmonary Edema.
[Abstract]High-permeability pulmonary edema causing acute respiratory distress syndrome is associated with high mortality. Using a model of intratracheal adenovirus (Ad)-mediated overexpression of human vascular endothelial growth factor (VEGF) A165 in the mouse lung to induce alveolar permeability and consequent pulmonary edema, we hypothesized that systemic administration of a 2nd Ad vector expressing an anti-VEGF antibody (AdVEGFAb) would protect the lung from pulmonary edema. Pulmonary edema was induced in mice by intratracheal administration of AdVEGFA165. To evaluate anti-VEGF antibody therapy, the mice were treated intravenously with AdVEGFAb, an Ad vector encoding the light and heavy chains of an anti-human VEGF antibody with the bevacizumab (Avastin(R)) antigen-binding site. Lung VEGFA165 and phosphorylated VEGF receptor (VEGFR) 2 levels, histology, lung wet/dry weight ratios and bronchoalveolar lavage fluid (BALF) levels of total protein were assessed. Administration of AdVEGFAb to mice decreased AdVEGFA165-induced levels of human VEGFA165 and phosphorylated VEGFR 2 in the lung. Histological analysis of AdVEGFAb-treated mice demonstrated a reduction of edema fluid in the lung tissue that correlated with a reduction of lung wet/dry ratios and BALF total protein levels. Importantly, administration of AdVEGFAb at 48 hr after induction of pulmonary edema with AdVEGFA165 was effective in suppressing pulmonary edema. Administration of an Ad vector encoding an anti-VEGF antibody that is the equivalent of bevacizumab effectively suppresses VEGFA165-induced high-permeability pulmonary edema, suggesting that anti-VEGF antibody therapy may represent a novel therapy for high-permeability pulmonary edema.
277: Acta biochimica Polonica, 2009 Feb 27,
Chimeric protein ABRaA-VEGF(121) is cytotoxic towards VEGFR-2-expressing PAE cells and inhibits B16-F10 melanoma growth.
[Abstract]It has been known that VEGF(121) isoform can serve as a carrier of therapeutic agents targeting tumor endothelial cells. We designed and constructed synthetic cDNA that encodes a chimeric protein comprising abrin-a (ABRaA) toxin A-chain and human VEGF(121). Expression of the ABRaA-VEGF(121) chimeric protein was carried out in E. coli strain BL21(DE3). ABRaA-VEGF(121) preparations were isolated from inclusion bodies, solubilized and purified by affinity and ion-exchanged chromatography (Ni-agarose and Q-Sepharose). Finaly, bacterial endotoxin was removed from the recombinant protein. Under non-reducing conditions, the recombinant protein migrates in polyacrylamide gel as two bands (~84 kDa homodimer and ~42 kDa monomer). ABRaA-VEGF(121) is strongly cytotoxic towards PAE cells expressing VEGFR-2, as opposed to VEGFR-1 expressing or parental PAE cells. The latter are about 400 times less sensitive to the action of this fusion protein. The biological activity of the ABRaA domain forming part of the chimeric protein was assessed in vitro: ABRaA-VEGF(121) inhibited protein biosynthesis in cell-free translation system. Preincubation of ABRaA-VEGF(121) with antibody neutralizing the biological activity of human VEGF abolishe the cytotoxic effect of the chimeric protein in PAE/KDR cells. Experiments in vivo demonstrated that ABRaA-VEGF(121) inhibits growth of B16-F10 murine melanoma tumors.
278: Molecular cancer, 2009 Feb 28, 8(1)
Overexpression of RRM2 decreases thrombspondin-1 and increases VEGF production in human cancer cells: implication of RRM2 in angiogenesis.
[Abstract]ABSTRACT: BACKGROUND: In addition to its essential role in ribonucleotide reduction, ribonucleotide reductase (RNR) small subunit, RRM2, has been known to play a critical role in determining tumor malignancy. Overexpression of RRM2 significantly enhances the invasive and metastatic potential of tumor. Angiogenesis is critical to tumor malignancy; it plays an essential role in tumor growth and metastasis. It is important to investigate whether the angiogenic potential of tumor is affected by RRM2. RESULTS: We examined the expression of antiangiogenic thrombospondin-1 (TSP-1) and proangiogenic vascular endothelial growth factor (VEGF) in two RRM2-overexpressing KB cells: KB-M2-D and KB-HURs. We found that TSP-1 was significantly decreased in both KB-M2-D and KB-HURs cells compared to the parental KB and mock transfected KB-V. Simultaneously, RRM2-overexpressing KB cells showed increased production of VEGF mRNA and protein. In contrast, attenuating RRM2 expression via siRNA resulted in a significant increased TSP-1 expression in both KB and LNCaP cells; while the expression of VEGF by the two cells was significantly decreased under both normoxia and hypoxia. In comparison with KB-V, overexpression of RRM2 had no significant effect on proliferation in vitro, but it dramatically accelerated in vivo subcutaneous growth of KB-M2-D. KB-M2-D possessed more angiogenic potential than KB-V, as sho