Vascular Endothelial Growth Factor Receptor-2 Protein Expression Research Articles

Requirement of Fut8 for the expression of vascular endothelial growth factor receptor-2: a new mechanism for the emphysema-like changes observed in Fut8-deficient mice

alpha1,6-Fucosylation plays key roles in many biological functions, as evidenced by the study of alpha1,6-fucosyltransferase (Fut8) knockout (Fut8(-/-)) mice. Phenotypically, Fut8(-/-) mice exhibit emphysema-like changes in the lung, and severe growth retardation. Fut8(-/-) cells also show marked dysregulation of the TGF-beta1 receptor, EGF receptor, integrin activation and intracellular signalling, all of which can be rescued by reintroduction of Fut8. The results of the present study demonstrated that vascular endothelial growth factor receptor-2 (VEGFR-2) expression was significantly suppressed in Fut8(-/-) mice, suggesting that Fut8 was required for VEGFR-2 expression. The expression of VEGFR-2 mRNA and protein was consistently down-regulated by knockdown of the Fut8 gene with small interference RNA in A549 cells, as well as in TGP49 cells, suggesting that suppression occurs at the level of transcription. In contrast, the expression level of ceramide, an inducer of cell apoptosis, was increased in the lungs of Fut8(-/-) mice. The terminal transferase dUTP nick end-labelling (TUNEL) assay was used to identify apoptotic cells. The number of TUNEL-positive septal epithelia and endothelia cells was significantly increased in the alveolar septa of lungs from Fut8(-/-) mice when in comparison with lungs from wild-type mice. It is well known that, in emphysema, ceramide expression can be greatly enhanced by blockade of the VEGFR-2. Thus, suppression of VEGFR-2 expression may provide a novel explanation for the emphysema-like changes in Fut8(-/-) mice.

The role of VEGFR-2 genetic variation in breast cancer progression.

Abstract Abstract #904 Background: Substantial laboratory and clinical data have demonstrated the critical role of angiogenesis in breast tumor progression. A significant correlation between vascular endothelial growth factor receptor-2 (VEGFR-2) expression and cell proliferation has been described in invasive breast carcinomas, suggesting that VEGF stimulates mammary cell growth through VEGFR-2. We sought to examine whether variability in VEGFR-2 expression and activation in tumors might be due to individual genetic variations, which may also play a role in response to anti-angiogenic therapy. To our knowledge, no study has correlated genetic variation in VEGFR-2 to expression and activity in primary breast tumors.
 Methods: DNA from 42 primary breast tumors was extracted from fresh frozen tissue. The core promoter, 5'-untranslated region (UTR), 3'-UTR, exons and intron-exon boundary regions of VEGFR-2 were sequenced for all tumors. Tissue microarrays were constructed, and tumor and paired normal breast tissue were stained with anti-VEGFR-2 antibody (Calbiochem). Microvessel density (MVD) was determined by immunohistochemical staining using a primary antibody against platelet endothelial cell adhesion molecule (anti-CD34, Novocastra). Semiquantitative analysis was performed independently by two pathologists. VEGFR-2 expression was correlated with genotype and MVD using the Mann-Whitney test. VEGFR-2 expression in normal and tumor tissue was compared using the Wilcoxon signed-rank test.
 Results: Two-thirds of tumors were from self-reported African Americans (AA), and the majority were ER positive. Twenty-three different single nucleotide polymorphisms (SNPs) were identified; ten were previously reported in dbSNP. Three of these SNPs were common (minor allele frequency >10%): one was located in the core promoter region and the other two were located in exons 7 and 11 (both non-synonymous SNPs). Using PolyPhen prediction software (http://genetics.bwh.harvard.edu/pph/), the two non-synonymous SNPs were predicted to affect protein function. Of the 23 different SNPs identified, 11 were only seen in tumors from AA and 3 were only observed in tumors from Caucasians. Thirty-six of the 42 tumors (86%) had at least one SNP. VEGFR-2 expression in tumor was significantly higher than in paired normal tissue (p=0.0002). VEGFR-2 expression was significantly lower in tumors with the AA genotype of the 4032 A/G core promoter SNP as compared to those with the AG and GG genotypes combined (p=0.02). VEGFR-2 expression was significantly associated with MVD in tumor tissues (p=0.04).
 Discussion: Our preliminary study suggests an association between genetic variations in the VEGFR-2 gene and protein expression in tumor tissue. Future work will examine the spectrum of these genetic variations in diverse populations and their potential role in predicting response to anti-angiogenic therapy.
 This study was funded by the University of Chicago Breast SPORE NCI P50 CA125183. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 904.

Differential Regulation of VEGF Signaling by PKC-α and PKC-ε in Endothelial Cells

Vascular endothelial growth factor (VEGF) stimulates proangiogenic signal transduction and cell function in part through activation of protein kinase C (PKC). Our aim was to examine how individual isoforms of PKC affect VEGF action. Transfection of bovine aortic endothelial cells with small interfering RNA (siRNA) targeting either PKC-alpha, delta, or epsilon caused a reduction in the cognate PKC protein by 76% to 89% without changing expression of nontargeted isoforms. Downregulation of PKC-epsilon abrogated VEGF-stimulated phosphorylation of Akt at Ser473 and eNOS at Ser1179 and decreased VEGF-stimulated NO synthase activity in intact cells. In contrast, PKC-alpha knockdown increased Akt and eNOS phosphorylation, whereas PKCdelta knockdown had no significant effect. PKC-epsilon knockdown also decreased VEGF-stimulated Erk1/2 phosphorylation and abolished VEGF-stimulated DNA synthesis. Consistent with an effect on several pathways of VEGF signaling, VEGF receptor-2 (VEGFR2) tyrosine phosphorylation and expression of VEGFR2 protein and mRNA was decreased by 81, 90, and 84%, respectively, during knockdown of PKC-epsilon, but increased during PKC-alpha knockdown. By regulating VEGFR2 expression and activation, PKC-epsilon expression is critical for activation of Akt and eNOS by VEGF and contributes to VEGF-stimulated Erk activation, whereas PKC-alpha has opposite effects.

Z-Guggulsterone, a constituent of Ayurvedic medicinal plant Commiphora mukul, inhibits angiogenesis in vitro and in vivo

Our previous studies have shown that z-guggulsterone, a constituent of Indian Ayurvedic medicinal plant Commiphora mukul, inhibits the growth of human prostate cancer cells by causing apoptosis. We now report a novel response to z-guggulsterone involving the inhibition of angiogenesis in vitro and in vivo. The z-guggulsterone treatment inhibited capillary-like tube formation (in vitro neovascularization) by human umbilical vein endothelial cells (HUVEC) and migration by HUVEC and DU145 human prostate cancer cells in a concentration- and time-dependent manner. The z- and E-isomers of guggulsterone seemed equipotent as inhibitors of HUVEC tube formation. The z-guggulsterone-mediated inhibition of angiogenesis in vitro correlated with the suppression of secretion of proangiogenic growth factors [e.g., vascular endothelial growth factor (VEGF) and granulocyte colony-stimulating factor], down-regulation of VEGF receptor 2 (VEGF-R2) protein level, and inactivation of Akt. The z-guggulsterone-mediated suppression of DU145 cell migration was increased by knockdown of VEGF-R2 protein level. Ectopic expression of constitutively active Akt in DU145 cells conferred protection against z-guggulsterone-mediated inhibition of cell migration. Oral gavage of 1 mg z-guggulsterone/d (five times/wk) to male nude mice inhibited in vivo angiogenesis in DU145-Matrigel plug assay as evidenced by a statistically significant decrease in tumor burden, microvessel area (staining for angiogenic markers factor VIII and CD31), and VEGF-R2 protein expression. In conclusion, the present study reveals that z-guggulsterone inhibits angiogenesis by suppressing the VEGF-VEGF-R2-Akt signaling axis. Together, our results provide compelling rationale for further preclinical and clinical investigation of z-guggulsterone for its efficacy against prostate cancer.

Myoferlin Regulates Vascular Endothelial Growth Factor Receptor-2 Stability and Function

Myoferlin and dysferlin are members of the ferlin family of membrane proteins. Recent studies have shown that mutation or genetic disruption of myoferlin or dysferlin promotes muscular dystrophy-related phenotypes in mice, which are the result of impaired plasma membrane integrity. However, no biological functions have been ascribed to myoferlin in non-muscle tissues. Herein, using a proteomic analysis of endothelial cell (EC) caveolae/lipid raft microdomains we identified myoferlin in these domains and show that myoferlin is highly expressed in ECs and vascular tissues. The loss of myoferlin results in lack of proliferation, migration, and nitric oxide (NO) release in response to vascular endothelial growth factor (VEGF). Western blotting and surface biotinylation experiments show that loss of myoferlin reduces the expression level and autophosphorylation of VEGF receptor-2 (VEGFR-2) in native ECs. In a reconstituted cell system, transfection of myoferlin increases VEGFR-2 membrane expression and autophosphorylation in response to VEGF. In vivo, VEGFR-2 levels and VEGF-induced permeability are impaired in myoferlin-deficient mice. Mechanistically, myoferlin forms a complex with dynamin-2 and VEGFR-2, which prevents CBL-dependent VEGFR-2 polyubiquitination and proteasomal degradation. These data are the first to report novel biological activities for myoferlin and reveal the role of membrane integrity to VEGF signaling.

Inhibition of Cell Proliferation and in Vitro Markers of Angiogenesis by Indole-3-carbinol, a Major Indole Metabolite Present in Cruciferous Vegetables

A variety of studies have suggested a cancer protective role of cruciferous vegetables. In the present study, we investigated the effect of indole-3-carbinol (I3C), a major indole metabolite in cruciferous vegetables, on cell proliferation and in vitro markers of angiogenesis in phorbol myristate acetate (PMA)-stimulated endothelial EA hy926 cells. The results showed that I3C inhibited the growth of EA hy926 cells in a concentration-dependent manner. The capillary-like tube formation by PMA-activated endothelial cells was significantly suppressed by I3C, and such inhibition was associated with decreased vascular endothelial growth factor (VEGF) and increased interleukin-8 (IL-8) secretion, but not with the expression of VEGF receptor-2 protein. Additionally, gelatin zymography analysis indicated that I3C suppressed activities of matrix metalloproteinases-2 (MMP-2) and MMP-9 stimulated by PMA. These results suggest that the dietary I3C may be useful in the treatment of human cancers and angiogenic diseases.

Vitamins C and E downregulate vascular VEGF and VEGFR-2 expression in apolipoprotein-E-deficient mice

Anti-angiogenic therapy reduces both plaque growth and intimal neovascularization in apolipoprotein-E-deficient mice (apoE−/−). Vascular endothelial growth factor (VEGF) has been suggested as playing a role in the development of atherosclerosis. We examined the hypothesis that VEGF and VEGF receptor-2 (VEGFR-2) expression is upregulated in apoE−/− and, since it could be driven by oxidative stress, tested whether dietary supplementation with vitamins C and E could downregulate it. Two-month-old apoE−/− received vitamin C combined with α- or β-tocopherol for 4 weeks. Aortic VEGF and VEGFR-2 expression were measured by RT-qPCR and western blot. ApoE−/− showed significantly higher expression of aortic VEGF and VEGFR-2 mRNA ( P<0.001) and protein ( P<0.001) than wild-type mice, as well as increased plasma VEGF ( P<0.001). Vitamin C and α-tocopherol significantly reduced aortic VEGF and VEGFR-2 expression in apoE−/− ( P<0.001), circulating VEGF ( P<0.01) and plasma lipid peroxidation ( P<0.01). apoE−/− receiving vitamin C and β-tocopherol showed diminished lipid peroxidation and VEGFR-2, but only partial reduction of VEGF expression. These data demonstrate that augmented VEGF and VEGFR-2 expression in apoE−/− vasculature can be downregulated by vitamins C and E, at least partially through oxidative stress reduction. This novel mechanism could contribute to explaining the beneficial effects of antioxidant vitamins in experimental atherosclerosis.