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Query: UNIPROT:P06889 (Mol)
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Cellular adaptation to hypoxia involves regulation of specific genes such as vascular endothelial growth factor (VEGF), erythropoietin (EPO) and hypoxia inducible factor (HIF)-1 . In this study, we have evaluated the protective effect of picroliv (a purified iridoid glycoside fraction from roots of Picrorhiza kurrooa with hepatoprotective, anti-inflammatory and antioxidant properties) against hypoxic injury by examining lactate dehydrogenase (LDH) release in Hep 3B and Glioma cells. The expression of hypoxia regulated genes, VEGF and HIF-1 was studied in human umbilical vein endothelial cells (HUVEC), Hep 3B and Glioma cells. Picroliv reduced the cellular damage caused by hypoxia as revealed by a significant reduction in LDH release compared to untreated control. The expression of VEGF and HIF-1 subunits (HIF-1alpha and HIF-1beta) was enhanced by treatment with picroliv during normoxia and hypoxia in HUVEC and Hep 3B cells and on reoxygenation the expression of these genes was significantly reduced as revealed by mRNA analysis using RT-PCR. Simultaneous treatment with picroliv during hypoxia inhibited VEGF and HIF-1 expression in Glioma cells whereas the expression was not reduced by picroliv treatment during reoxygenation as evidenced by both RT-PCR and Northern hybridization. VEGF expression as revealed by immunofluorescence studies correlates well with the regulations observed in the mRNA expression. We have also examined the kinase activity of tyrosine phosphorylated proteins and protein kinase C (PKC) in Glioma cells treated with picroliv during hypoxia/reoxygenation. A selective inhibition of protein tyrosine kinase activity leading to tyrosine dephosphorylation of several proteins including 80 kd protein, and a reduction in PKC was seen in cells treated with picroliv and hypoxia. These findings suggest that picroliv may act as a protective agent against hypoxia/reoxygenation induced injuries, and the underlying mechanism may involve a novel signal transduction pathway.
Mol Cell Biochem 1999 Apr
PMID:Picroliv -- a natural product protects cells and regulates the gene expression during hypoxia/reoxygenation. 1039 Nov 50

Studies with mammalian vascular cells have suggested growth inhibitory effects of estrogen on the vascular wall. To investigate the involvement of estrogen receptor-alpha (ER) in the control of endothelial cell proliferation, we have stably transfected human estrogen receptor-alpha cDNA into the endothelial cell line ECV304. The clone ECV-ER, thus obtained, over-expresses estrogen receptor to a level approximately 10-fold higher than the parent cell line. Effects of this over-expression were studied on the cell growth rate, and on the levels of secreted endothelin-1 and vascular endothelial growth factor (VEGF). Similar to the previously reported data in other cell types, we found the transfection of ER in ECV304 cells to be inhibitory to their growth. Our ER-over-expressing clone of ECV304 also showed an inhibition of secreted endothelin-1 and VEGF levels. Moreover, the growth inhibition of this ER-over-expressing clone was reversed by the addition of endothelin-1 or VEGF to the medium. In view of the growth-stimulatory effect of endothelin-1 and VEGF on vascular cells, our results indicate that estrogen receptor-alpha may bring about its growth inhibition partly by suppressing endothelin-1 and/or VEGF production in ECV304 cells.
Mol Cell Endocrinol 1999 Jun 25
PMID:Stable over-expression of estrogen receptor-alpha in ECV304 cells inhibits proliferation and levels of secreted endothelin-1 and vascular endothelial growth factor. 1043 18

Loss of function in the von Hippel-Lindau (VHL) tumor suppressor gene occurs in familial and most sporadic renal cell carcinomas (RCCs). VHL has been linked to the regulation of cell cycle cessation (G(0)) and to control of expression of various mRNAs such as for vascular endothelial growth factor. RCC cells express the Met receptor tyrosine kinase, and Met mediates invasion and branching morphogenesis in many cell types in response to hepatocyte growth factor/scatter factor (HGF/SF). We examined the HGF/SF responsiveness of RCC cells containing endogenous mutated (mut) forms of the VHL protein (VHL-negative RCC) with that of isogenic cells expressing exogenous wild-type (wt) VHL (VHL-positive RCC). We found that VHL-negative 786-0 and UOK-101 RCC cells were highly invasive through growth factor-reduced (GFR) Matrigel-coated filters and exhibited an extensive branching morphogenesis phenotype in response to HGF/SF in the three-dimensional (3D) GFR Matrigel cultures. In contrast, the phenotypes of A498 VHL-negative RCC cells were weaker, and isogenic RCC cells ectopically expressing wt VHL did not respond at all. We found that all VHL-negative RCC cells expressed reduced levels of tissue inhibitor of metalloproteinase 2 (TIMP-2) relative to the wt VHL-positive cells, implicating VHL in the regulation of this molecule. However, consistent with the more invasive phenotype of the 786-0 and UOK-101 VHL-negative RCC cells, the levels of TIMP-1 and TIMP-2 were reduced and levels of the matrix metalloproteinases 2 and 9 were elevated compared to the noninvasive VHL-positive RCC cells. Moreover, recombinant TIMPs completely blocked HGF/SF-mediated branching morphogenesis, while neutralizing antibodies to the TIMPs stimulated HGF/SF-mediated invasion in vitro. Thus, the loss of the VHL tumor suppressor gene is central to changes that control tissue invasiveness, and a more invasive phenotype requires additional genetic changes seen in some but not all RCC lines. These studies also demonstrate a synergy between the loss of VHL function and Met signaling.
Mol Cell Biol 1999 Sep
PMID:The von Hippel-Lindau tumor suppressor gene inhibits hepatocyte growth factor/scatter factor-induced invasion and branching morphogenesis in renal carcinoma cells. 1045 37

Osteogenic protein-1 (OP-1 or BMP-7) stimulates new bone formation in vivo and induces cell proliferation and differentiation of osteoblasts in vitro. In the present study, we examined effects of OP-1 on the expression of vascular endothelial growth factor (VEGF) in primary cultures of fetal rat calvaria (FRC) cells. OP-1 increased the steady-state level of VEGF mRNA by about 3-fold in an OP-1 concentration- and time-dependent manner. The increase in VEGF mRNA level depended on transcription and was sensitive to cell replication. The VEGF mRNA stability was unaffected. The mRNA levels for both types of VEGF receptors, Flk-1 and Flt-1 were low but detectable in FRC cells by RT-PCR and were not changed by OP-1. Inhibition of VEGF synthesis and function by antisense oligonucleotide and by suramin, respectively arrested the OP-1-induced alkaline phosphatase activity and mineralized bone nodule formation. Together with published studies of VEGF on vascular endothelial cells which are usually found in close proximity to osteoblastic cells in vivo, these results suggest that VEGF participates in the OP-1-induced osteogenesis by taking part in bone cell differentiation and by promoting angiogenesis at the site of bone formation.
Mol Cell Endocrinol 1999 Jul 20
PMID:Osteogenic protein-1 increases gene expression of vascular endothelial growth factor in primary cultures of fetal rat calvaria cells. 1045 59

Tumors associated with the VHL (von Hippel-Lindau) disease, such as hemangioblastomas and renal carcinomas and their sporadic counterparts, are cystic and well vascularized. Mutations of the VHL tumor-suppressor gene and elevated levels of vascular endothelial growth factor (VEGF) have been described in these tumors. The upregulation of VEGF has been shown in vitro as a consequence of alteration of the VHL gene. No comprehensive in vivo analysis has yet been carried out of the factors affecting tumor growth, vascularization, VEGF, and VHL expression. We performed immunohistochemistry and mRNA studies on primary sporadic renal carcinomas and matching normal renal tissue. We semiquantitatively analyzed 29 renal carcinomas (22 clear cell, 5 chromophilic, 2 chromophobic tumors) for VHL mRNA, and VEGF expression for morphology and tumor size. Immunohistochemistry was carried out for VEGF protein expression, vascularization, and macrophage infiltration. Vascularization of the chromophilic renal carcinomas was lower than that of the clear cell type of renal carcinoma. Low VEGF protein expression was seen in four of the five chromophilic renal carcinomas. We found two groups of clear cell renal cell carcinoma: one with reduced VHL mRNA and increased VEGF mRNA, and the other without significantly altered VHL or VEGF mRNAs. Tumor vascularization was correlated with VEGF protein and seemed to be independent of macrophage infiltration. Our in vivo findings support the inverse relationship between the regulation of VHL and that of VEGF. Our data also indicate that there may be an VHL-independent pathway for the induction of tumor vascularization.
J Mol Med (Berl) 1999 Jun
PMID:Inverse regulation of vascular endothelial growth factor and VHL tumor suppressor gene in sporadic renal cell carcinomas is correlated with vascular growth: an in vivo study on 29 tumors. 1047 65

Hypoxia-inducible factor (HIF)-2alpha is a recently identified hypoxia-inducible transcription factor abundantly expressed in vascular endothelial cells. As well as HIF-1alpha, HIF-2alpha forms a heterodimeric complex with the aryl hydrocarbon receptor nuclear translocator and upregulates hypoxia-inducible genes such as vascular endothelial growth factor. We found in this study that using green fluorescent protein (GFP) fusion constructs, the subcellular localization of HIF-2alpha was different from that of HIF-1alpha in bovine arterial endothelial cells (BAEC). HIF-1alpha was localized in the cytoplasm under normoxic cells and translocated from the cytoplasm into the nucleus in response to hypoxic induction. In contrast, HIF-2alpha was clearly localized in the nucleus of BAEC even under normoxic conditions. The regulation of HIF-2alpha might differ from that of HIF-1alpha in BAEC. We further showed that nuclear localization of HIF-2alpha was inhibited by either deletion or a single amino acid substitution within the C-terminal end of the protein. The amino acid sequence surrounding Lys737 and Arg738 functions as a nuclear localization signal of HIF-2alpha.
Mol Cell Biol Res Commun 1999 Aug
PMID:Nuclear localization of hypoxia-inducible factor-2alpha in bovine arterial endothelial cells. 1054 35

The Fab portion of a humanized antibody (Fab-12; IgG form known as rhuMAb VEGF) to vascular endothelial growth factor (VEGF) has been affinity-matured through complementarity-determining region (CDR) mutation, followed by affinity selection using monovalent phage display. After stringent binding selections at 37 degrees C, with dissociation (off-rate) selection periods of several days, high affinity variants were isolated from CDR-H1, H2, and H3 libraries. Mutations were combined to obtain cumulatively tighter-binding variants. The final variant identified here, Y0317, contained six mutations from the parental antibody. In vitro cell-based assays show that four mutations yielded an improvement of about 100-fold in potency for inhibition of VEGF-dependent cell proliferation by this variant, consistent with the equilibrium binding constant determined from kinetics experiments at 37 degrees C. Using X-ray crystallography, we determined a high-resolution structure of the complex between VEGF and the affinity-matured Fab fragment. The overall features of the binding interface seen previously with wild-type are preserved, and many contact residues are maintained in precise alignment in the superimposed structures. However, locally, we see evidence for improved contacts between antibody and antigen, and two mutations result in increased van der Waals contact and improved hydrogen bonding. Site-directed mutants confirm that the most favorable improvements as judged by examination of the complex structure, in fact, have the greatest impact on free energy of binding. In general, the final antibody has improved affinity for several VEGF variants as compared with the parental antibody; however, some contact residues on VEGF differ in their contribution to the energetics of Fab binding. The results show that small changes even in a large protein-protein binding interface can have significant effects on the energetics of interaction.
J Mol Biol 1999 Nov 05
PMID:Selection and analysis of an optimized anti-VEGF antibody: crystal structure of an affinity-matured Fab in complex with antigen. 1054 73

The trace element nutrient selenium (Se) has been shown to possess cancer-preventive activity in both animal models and humans, but the mechanisms by which this occurs remain to be elucidated. Because angiogenesis is obligatory for the genesis and growth of solid cancers, we investigated, in the study presented here, the hypothesis that Se may exert its cancer-preventive activity, at least in part, by inhibiting cancer-associated angiogenesis. The effects of chemopreventive levels of Se on the intra-tumoral microvessel density and the expression of vascular endothelial growth factor in 1-methyl-1-nitrosourea-induced rat mammary carcinomas and on the proliferation and survival and matrix metalloproteinase activity of human umbilical vein endothelial cells in vitro were examined. Increased Se intake as Se-enriched garlic, sodium selenite, or Se-methylselenocysteine led to a significant reduction of intra-tumoral microvessel density in mammary carcinomas, irrespective of the manner by which Se was provided: continuous exposure (7-wk feeding) with a chemoprevention protocol or acute bolus exposure (3 d) after carcinomas had established. Compared with the untreated controls, significantly lower levels of vascular endothelial growth factor expression were observed in a sizeable proportion of the Se-treated carcinomas. In contrast to the mammary carcinomas, the microvessel density of the uninvolved mammary glands was not altered by Se treatment. In cell culture, direct exposure of human umbilical vein endothelial cells to Se induced cell death predominantly through apoptosis, decreased the gelatinolytic activities of matrix metalloproteinase-2, or both. These results indicate a potential for Se metabolites to inhibit key attributes (proliferation, survival, and matrix degradation) of endothelial cells critical for angiogenic sprouting. Therefore, inhibition of angiogenesis associated with cancer may be a novel mechanism for the anticancer activity of Se in vivo, and multiple mechanisms are probably involved in mediating the anti-angiogenic activity.
Mol Carcinog 1999 Dec
PMID:Selenium-induced inhibition of angiogenesis in mammary cancer at chemopreventive levels of intake. 1056 99

The role of endothelial cell-specific growth factors in the vascularization of the primate peri-ovulatory follicle was examined. Experiments were designed firstly to detect expression of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) in granulosa cells and secondly, to determine whether gonadotrophins and/or steroids regulate their expression during the peri-ovulatory interval. Granulosa cells and follicular fluid were collected from rhesus macaques undergoing ovarian stimulation before (0 h), 12, or 36 h after a bolus of ovulatory human chorionic gonadotrophin (HCG), with or without steroid ablation and progestin replacement. VEGF, Ang-1 and Ang-2 mRNA were all detected prior to the ovulatory stimulus. Whereas follicular fluid VEGF concentrations increased 6-fold (P < 0.05) between 0 and 12 h, VEGF mRNA values were unchanged and were unaffected by steroid ablation. Ang-1 mRNA decreased from 0 to 12 h (P < 0.05), followed by a 30-fold increase (P < 0.05) at 36 h, while Ang-2 mRNA values were unchanged between 0, 12 and 36 h. Steroid ablation decreased (P < 0.05) Ang-1 mRNA at 36 h, and Ang-2 mRNA at 12 h, while only Ang-1 was restored by progestin replacement. These data suggest a dynamic expression of vascular-specific growth factors in a gonadotrophin-dependent, steroid-independent (VEGF) or steroid-dependent (Ang-1) manner in granulosa cells of peri-ovulatory follicles of primates.
Mol Hum Reprod 1999 Dec
PMID:Vascular endothelial growth factor (VEGF) and angiopoietin regulation by gonadotrophin and steroids in macaque granulosa cells during the peri-ovulatory interval. 1058 65

Src kinase activity was found to protect endothelial cells from apoptosis during vascular endothelial growth factor (VEGF)-, but not basic fibroblast growth factor (bFGF)-, mediated angiogenesis in chick embryos and mice. In fact, retroviral targeting of kinase-deleted Src to tumor-associated blood vessels suppressed angiogenesis and the growth of a VEGF-producing tumor. Although mice lacking individual Src family kinases (SFKs) showed normal angiogenesis, mice deficient in pp60c-src or pp62c-yes showed no VEGF-induced vascular permeability (VP), yet fyn-/- mice displayed normal VP. In contrast, inflammation-mediated VP appeared normal in Src-deficient mice. Therefore, VEGF-, but not bFGF-, mediated angiogenesis requires SFK activity in general, whereas the VP activity of VEGF specifically depends on the SFKs, Src, or Yes.
Mol Cell 1999 Dec
PMID:Selective requirement for Src kinases during VEGF-induced angiogenesis and vascular permeability. 1063 17


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