Vascular Endothelial Growth Factor Receptor-2 Protein Expression Research Articles

Suppression of alpha-tocopherol ether-linked acetic acid in VEGF-induced angiogenesis and the possible mechanisms in human umbilical vein endothelial cells

Alpha-tocopherol ether-linked acetic acid (α-TEA) has been reported to exhibit both anti-tumor and anti-metastatic activities in cell culture and animal studies. However, it is unclear whether α-TEA possesses anti-angiogenic effects. In this study, we investigated the effect of α-TEA on vascular endothelial growth factor (VEGF)-induced angiogenesis and matrix metalloproteinase (MMP) expression both in vitro and ex vivo. We found that the α-TEA inhibited tube formation, invasion, and migration in human umbilical vein endothelial cells (HUVECs) and that such actions were accompanied by reduced expression of MMP-2. α-TEA also inhibited ex vivo angiogenesis, as indicated by chicken egg chorioallantoic membrane assay. We further showed that α-TEA attenuated protein expression of VEGF receptor-2 (VEGFR-2)-mediated p38 mitogen-activated protein kinase (p38), phosphorylated p38, and focal adhesion kinase (FAK). Moreover, α-TEA (30μM) significantly up-regulated protein expression of tissue inhibitors of MMP (TIMP)-2 (by 138%) and the metastasis suppressor gene nm23-H1 (by 54%). These results demonstrate that the anti-angiogenic effect of α-TEA both in vitro and ex vivo and its possible mechanistic action appears to involve the inhibition of MMP-2 level through VEGFR-2-mediated FAK and p38 signaling pathways and through up-regulation of TIMP-2 and nm23-H1 expression.

Azithromycin Attenuates Pulmonary Inflammation and Emphysema in Smoking-Induced COPD Model in Rats

The role of inflammation and immunity in COPD treatment is increasingly being recognized. The relationship between anti-inflammation/immunoregulation and emphysema in COPD lungs remains to be elucidated. The aim of this study was to investigate the effects of azithromycin (Azm) on the development of emphysema in smoking-induced COPD in rats. Sprague-Dawley rats (n = 50) were randomly assigned to normal, COPD, saline-treated, Azm-treated, and levofloxacin-treated (Lev) groups. The effects of treatment were assessed by measuring the levels of vascular endothelial growth factor (VEGF) by enzyme-linked immunosorbent assay and measuring the numbers of neutrophil and macrophage in bronchoalveolar lavage fluid, vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR2) protein expression by western blotting. Lung function measurements and histopathological evaluations (mean linear intercept and destructive index) were performed. FEV0.3/FVC and peak expiratory flow were lower in the COPD group than in the normal group. Mean linear intercept and destructive index were lower in the Azm-treated group than in the COPD, saline-treated, and Lev-treated groups. The numbers of neutrophil and macrophage in bronchoalveolar lavage fluid were lower in the Azm-treated group than in the COPD, saline-treated, and Lev-treated groups. As confirmed by western blotting, the levels of VEGF in lung homogenates were higher in the Azm-treated group than in the COPD, saline-treated, and Lev-treated groups. VEGFR2 protein expression was higher in the Azm-treated group than in the COPD, saline-treated, and Lev-treated groups. Azm attenuates pulmonary emphysema by partly reversing the decrease in the numbers of inflammatory cells (neutrophil and macrophage) and VEGF secretion and VEGFR2 protein expression in smoking-induced COPD in rats.

Inhibitory effects of 5-lipoxygenase on oxygen-induced retinal neovascularization in mice

Objective To investigate the inhibitory effects of 5-lipoxygenase (5-LOX) on oxygeninduced retinal neovascularization in mice and to explore its possible mechanisms.Methods 7-day-old C57BL/6J mice were randomly divided into normal group,oxygen induced retinopathy (OIR) model group,large-dose group,small-dose group and control group with 12 mice in each group.The mice with their mothers were kept in (75±2)％ of oxygen environment for 5 days and then returned to normoxia for 5 days to establish the OIR model except for normal group.From postnatal day 12 to 17,the large-dose group and small-dose group received intravitreous injection of 5-LOX at dose of 100 mg/kg and 50 mg/kg respectively,while the control group received the same volume of 1 ％ dimethyl sulfoxide.The mice in the OIR group received no treatment.The number of endothelium cell nuclei breaking through the inner limiting membrane (ILM) was counted on hematoxylin and eosin-stained retinal section.The mRNA expression of 5 LOX,vascular endothelial growth factor (VEGF) a,VEGF receptor 2 (VEGFR-2) on retinal tissue were detected by reverse transcription polymerase chain reaction (RT-PCR).The protein expression of 5 LOX,VEGF a,VEGFR-2 and phosphorylation extracellular signal-regulated kinase (P-ERK) 1/2 on retinal tissue were detected by Western blot.Results The number of vascular cell nuclei breaking through the ILM in the large-dose group and small dose group decreased significantly compared with the OIR group and control group (F=73.390,P＜0.05).The mRNA expression and protein expression of 5-LOX,VEGFa,VEGFR 2 on retinal tissue were decreased significantly in the large-dose group and small dose group as compared with the OIR group and control group (F =92.668,P＜ 0.05).The difference of VEGFR-2 protein expression between large-dose group and small dose group was not significant (F=2.118,P＞0.05).The differences of 5-LOX,VEGF a,P-ERK 1/2 protein expression between large-dose group and small-dose group were significant (F=86.490,165.128,139.424; P＜0.05).Conclusion Hypoxia may induce 5 LOX expression in the retina.Retinal neovascularization was significantly inhibited by selective inhibition of 5 LOX. Key words: Retinal neovascularization/prevention & control; Arachidonate 5-lipoxygenase; Extracellular signal-regulated MAP kinases; Animal experimentation

Glycaemic control improves perfusion recovery and VEGFR2 protein expression in diabetic mice following experimental PAD

Diabetes mellitus (DM) is associated with poor clinical outcomes in humans with peripheral arterial disease (PAD) and in pre-clinical models of PAD, but the effects of glycaemic control are poorly understood. We investigated the effect of glycaemic control on experimental PAD in mice with Type 1 DM and explored the effects of hyperglycaemia on vascular endothelial growth factor receptor 2 (VEGFR2) expression in ischaemia. Hind limb ischaemia was induced in non-diabetic, untreated Type 1 DM, and treated Type 1 DM mice. We assessed perfusion recovery, capillary density, VEGFR2 levels, and VEGFR2 ubiquitination in ischaemic hind limbs. We found that untreated Type 1 DM mice showed impaired perfusion recovery, lower hind limb capillary density 5 weeks post-ischaemia, and lower VEGFR2 protein in Day 3 post-ischaemic hind limbs when compared with non-DM controls. Treated Type 1 DM mice had perfusion recovery, capillary density, and VEGFR2 protein levels comparable with that of non-diabetic mice at the same time points. Treatment with anti-VEGFR2 antibody negated that the improved perfusion recovery displayed by treated Type 1 DM mice. In ischaemic Type 1 DM hind limbs and endothelial cells exposed to simulated ischaemia, high glucose impaired VEGFR2 expression and was associated with increased VEGFR2 ubiquitination. Inhibition of the ubiquitin-proteasome complex restored normal endothelial VEGFR2 expression in simulated ischaemia. Hyperglycaemia in Type 1 DM impairs VEGFR2 protein expression in ischaemic hind limbs, likely due to increased ubiquitination and degradation by the proteasome complex. Glycaemic control allows normal levels of VEGFR2 in ischaemia and improved perfusion recovery.

3D study of capillary network derived from human cord blood mesenchymal stem cells and differentiated into endothelial cell with VEGFR2 protein expression

Background and Aim: New blood forming vessels are produced by differentiation of mesodermal precursor cells to angioblasts that b ecome endothelial cells (ECs) which in turn give rise to primitive capillary netw ork. Human cord blood (HCB) contains large subsets of mononuclear cells (MNCs) that can be differentiated into endothelial-like cells in vitro . Methods: Human mononuclear progenitor cells were purified f rom fresh umbilical cord blood by the expression of CD34 and FLK-1 antigens expressed in both angioblasts and hematopoetic stem cells. The H CB derived mesenchymal stem cells (MSCs) can differentiate into adipocyte, osteocyte, chondrocyte and ECs. Results: In this study, the differentiation of human cord b lood mesenchymal stem cells (hCBMSCs) into endothelial-like cells was ind uced in presence of vascular endothelial growth factor (VEGF) and insulin-like g rowth factor (IGF-1). The differentiated ECs were then examined for their abi lity to express VEGF receptor- 2 (VEGFR2) and von Willebrand factor (vWF). These cells were adopted to grow, proliferate and develop into a capillary network in a semisolid gel matrix in vitro . Conclusion: The capillary network formation in each well of 24 -well plate was found to be 80% in presence of VEGF (40 ng/ml) and IGF-1 (20 ng/ml) of culture media, suggesting that the capillary network format ion is associated with endothelial-like cells derived from hCBMSCs by expression of their markers .

Abstract 5254: Cedarinib inhibits VEGF-stimulated proliferation in NSCLC cell lines.

Abstract Background: Vascular endothelial growth factor (VEGF) is a key stimulator of physiological and pathological angiogenesis. VEGF signals primarily through VEGF receptor 2 (VEGFR2), a receptor tyrosine kinase whose expression is generally considered to be restricted to endothelial cells. Recently, tumor cell VEGFR2 expression has been reported in several tumor settings, including non-small cell lung cancer (NSCLC). The purpose of this study was to determine the role of VEGFR2 in NSCLC cells, and to investigate the impact of inhibiting VEGFR2 signaling. Methods: NSCLC tissue sections (n=52) were stained for VEGFR2 and CD31 expression by immunohistochemistry (IHC). Total and phosphorylated VEGFR2, AKT and MAPK protein levels in human NSCLC cell lines (n=8) were determined by Western blotting. VEGFR2 mRNA levels were measured by RT-PCR. For cell proliferation assays, cells were seeded (4x10ˆ3 per well) and 24 h later treated with VEGF (0-100 ng/mL) +/- cediranib (a potent and selective inhibitor of VEGF signaling). Alternatively, cells were treated with VEGFR2 siRNA 24 h post plating (4x10ˆ3 per well) and then treated with VEGF (0-100 ng/mL). Cell growth was quantified 5 d after VEGF treatment using crystal violet staining. For cytotoxicity assays, cells (2x10ˆ4 per well) were plated and 24 h later treated with VEGF, chemotherapy agents or cedarinib and incubated for 48 h. For radiation, cells were exposed to 137Cs (10 Gy, 1.958 Gy/min) 4 h after VEGF treatment. 48 h after VEGF treatment Hoechst 33258 (25 μM, 30 min) was added and apoptotic cells quantified by fluorescence (INCELL Analyzer 1000). Results: IHC analysis of tissue sections demonstrated VEGFR2 expression in both adenocarcinoma and squamous cell carcinoma, and in endothelial cells. Western blot analysis showed expression of VEGFR2 protein in 3/8 NSCLC cell lines that correlated with VEGFR2 mRNA expression levels. VEGF-dependent VEGFR2 activation (increased pVEGFR2 and pMAPK but not pAKT) was apparent in NSCLC cells, and was associated with increased proliferation (1.4 - 1.9 fold, p<0.05) in 2/3 of the VEGFR2-expressing NSCLC cell lines. Cedarinib inhibited both VEGF-dependent increases in cell proliferation and VEGFR2 downstream signaling. Knock down of VEGFR2 protein expression using siRNA also reduced VEGF-stimulated NSCLC cell proliferation. Inhibition of VEGFR2 tyrosine kinase activity using cediranib was more effective than inhibition of AKT (MK2206) or MEK (AZD6244) for overcoming VEGFR2-driven cell proliferation. VEGF treatment did not affect cell survival following treatment with radiation, cisplatin, docetaxel or gemcitabine. Conclusion: Our data suggest that a subset of NSCLC patients may express functional tumor cell VEGFR2 which can act to promote VEGF-dependent tumor cell growth. In this patient subset, therapies targeting VEGFR2 signaling, such as cedarinib, may have the potential to inhibit both tumor cell proliferation and angiogenesis. Citation Format: Aoife M. Devery, Rekha Wadeka, Sivan M. Bokobza, Anika M. Weber, Yanyan Jiang, Anderson J. Ryan. Cedarinib inhibits VEGF-stimulated proliferation in NSCLC cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5254. doi:10.1158/1538-7445.AM2013-5254

Sorafenib treatment improves hepatopulmonary syndrome in rats with biliary cirrhosis

HPS (hepatopulmonary syndrome) is characterized by oxygen desaturation in patients with chronic liver disease. The initiation of HPS comes from abnormal pulmonary vasodilatation and/or angiogenesis. In the present study, we evaluated anti-angiogenesis therapy using sorafenib in experimental HPS animals. HPS was induced by CBDL (common bile duct ligation) in rats. A 2-week 10mg·(kg of body weight)-1·day-1 treatment regimen of sorafenib or distilled water (control) was initiated 2weeks after the surgical procedure. Haemodynamics, liver biochemistry, plasma VEGF (vascular endothelial growth factor) measurements and blood gas analysis of the CBDL rats were performed. The livers of the CBDL rats were dissected for histopathology examination, and the lungs were examined by immunohistochemical staining, real-time PCR and Western blot analysis. In another two parallel groups, intrapulmonary shunts were determined. The AaPO2 (alveolar-arterial O2 gradient) and plasma VEGF levels were reduced after sorafenib treatment [AaPO2, 7.2±3.4 mmHg in sorafenib-treated rats compared with 15.3±4.2 mmHg in controls (P=0.004); VEGF, 45.3±2.7 pg/ml in sorafenib-treated rats compared with 54.4±7.7 pg/ml in controls (P=0.021)]. Sorafenib attenuated pulmonary VEGF mRNA and VEGF, VEGFR-2 (VEGF receptor 2), phospho-VEGFR-2 and Akt protein expression. In addition, sorafenib significantly attenuated intrapulmonary angiogenesis and decreased the degree of intrapulmonary shunting by 33.7% (11.2±5.7% in sorafenib-treated rats compared with 16.9±5.9% in controls; P=0.003). Our findings suggest that sorafenib attenuates intrapulmonary shunting and decreases the AaPO2 in CBDL rats, implicating the improvement of HPS in this experimental animal model. The beneficial effect may be attributed to the reduction in intrapulmonary angiogenesis through inhibition of the VEGF/VEGFR-2/Akt pathway.

Vascular endothelial growth factor receptor 2 inhibition in-vivo affects tumor vasculature in a tumor type-dependent way and downregulates vascular endothelial growth factor receptor 2 protein without a prominent role for miR-296

The precise molecular effects that antiangiogenic drugs exert on tumor vasculature remain to be poorly understood. We therefore set out to investigate the molecular and architectural changes that occur in the vasculature of two different tumor types that both respond to vascular endothelial growth factor receptor 2 (VEGFR2) inhibitor therapy. Mice bearing Lewis lung carcinoma (LLC) or B16.F10 melanoma were treated with vandetanib (ZD6474), a VEGFR2/epidermal growth factor receptor (EGFR)/REarranged during Transfection (RET) kinase inhibitor, resulting in a significant 80% reduction in tumor outgrowth. Although in LLC the vascular density was not affected by vandetanib treatment, it was significantly decreased in B16.F10. In LLC, vandetanib treatment induced a shift in vascular gene expression toward stabilization, as demonstrated by upregulation of Tie2 and N-cadherin and downregulation of Ang2 and integrin β3. In contrast, only eNOS and P-selectin responded to vandetanib treatment in B16.F10 vasculature. Strikingly, vandetanib reduced protein expression of VEGFR2 in both models, whereas mRNA remained unaffected. Analysis of miR-296 expression allowed us to exclude a role for the recently proposed microRNA-296 in VEGFR2 posttranslational control in LLC and B16.F10 in vivo. Our data demonstrate that VEGFR2/EGFR inhibition through vandetanib slows down both LLC and B16.F10 tumor growth. Yet, the underlying molecular changes in the vasculature that orchestrate the antitumor effect differ between tumor types. Importantly, in both models, vandetanib treatment induced loss of its pharmacological target, which was not directly related to miR-296 expression. Validation of our observations in tumor biopsies from VEGFR2 inhibitor-treated patients will be essential to unravel the effects of VEGFR2 inhibitor therapy on tumor vasculature in relation to therapeutic efficacy.

Abstract 4251: Inhibition of membrane bound and soluble VEGF receptor-2 activity enhances the proliferation of the BON carcinoid cell line

Abstract There is a growing interest in using angiogenesis inhibitors to treat neuroendocrine tumors. Previously, we detected the expression of vascular endothelial growth factor receptor-2 (VEGFR-2) and its soluble isoform (sVEGFR-2) in the epithelial component of carcinoid tumors and in the BON carcinoid cell line. Recent studies have demonstrated that VEGF inhibitors transiently inhibit tumor growth, followed by increased tumor invasion and metastasis. Also, we have reported that shRNA reduction of VEGFR-2 increases BON cell proliferation and invasion. Thus, the purpose of our current study was to: i) assess the effect that the VEGF signaling inhibitors bevacizumab, an antibody against VEGF-A, or sunitinib, a small molecule inhibitor of VEGFR-2, have on BON cell proliferation and, ii) evaluate the effect of knockdown and overexpression of sVEGFR-2 on BON cell proliferation. Methods. i) BON cells were cultured in media supplemented with bevacizumab (50 ng/ml), sunitinib (50 ng/ml), or vehicle control for two weeks. Media was replaced every other day, and cell lysates for western blot and RNA analysis were collected after one and two weeks. After two weeks, cell proliferation was measured using a cell counting kit-8 assay. ii) Knockdown or upregulation of sVEGFR-2 was achieved by transfecting BON cells with shRNA to sVEGFR-2 or a cDNA plasmid expressing the sVegfr2 gene, respectively. Proliferation following sVEGFR-2 knockdown or upregulation was determined and compared to cells transfected with the appropriate controls. Results. i) BON cell proliferation was significantly increased in cells treated with bevacizumab or sunitinib compared to vehicle control. Western blot analysis demonstrated an increase in phospho-ERK and phospho-Akt following treatment with bevacizumab. Interestingly, there were no observed changes in activation of ERK or Akt following treatment with sunitinib. However, sunitinib treatment increased protein and mRNA expression of VEGFR-2, as assessed by western blot and qRT-PCR, respectively. ii) BON cells with reduced expression of sVEGFR-2 demonstrated increased proliferation at 24 h, 48 h, 72 h, 96 h, and 120 h following plating. Conversely, BON cells transfected with plasmid cDNA encoding sVEGFR-2 displayed reduced proliferation at the aforementioned time points compared to cells transfected with empty plasmid. Conclusions. We have demonstrated that the VEGF inhibitors bevacizumab and sunitinib can enhance the proliferation of BON cells in vitro and that sVEGFR-2 inhibits BON cell proliferation, possibly by sequestering VEGF ligands. While VEGF inhibitors have been shown to effectively inhibit angiogenesis, they may also enhance the aggressiveness of surviving tumor cells, suggesting that a multimodal treatment plan is warranted in carcinoid patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4251. doi:10.1158/1538-7445.AM2011-4251

Anti-TNF-α Antibody (Infliximab) Therapy Supports the Recovery of eNOS and VEGFR2 Protein Expression in Endothelial Cells

The aim of this study is to investigate the effect of sera obtained from patients of Crohn's disease treated by anti-TNF-alpha antibody (Infliximab) on the expression of endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor receptor-2 (VEGFR2) protein in human umbilical vein endothelial cells (HUVEC) cultured in vitro. HUVEC was cultured in the presence of sera derived from patients before and after treatment, or from healthy individuals. Effects of sera on the expression of eNOS and VEGFR2 were monitored by determination of mRNA and protein levels using real time quantitative PCR and Western blot analysis, respectively. The serum of Crohn's patients contained elevated levels of TNF-alpha (34±1.80 pg/mL), which resulted in a decrease in the protein level of eNOS in HUVEC with a simultaneous induction of VEGFR2. Infliximab treatment normalized the expression level of these proteins by decreasing TNF-alpha level, particularly in those cases when clinical healing was also recorded, and it also conferred restitution of the level of angiogenic cytokines. Results suggest that altered angiogenesis possibly contributes to the initiation and perpetuation of inflammatory processes in inflammatory bowel disease (IBD). Endothelial dysfunction, a selective feature of Crohn's disease is beneficially affected by intravascular TNF-alpha neutralization.

PPARδ agonists suppress angiogenesis in a VEGFR2-dependent manner

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that have a pleiotropic impact on the regulation of differentiation, cell growth, and the metabolism of lipids and glucose. PPARδ agonists display a variety of effects on pro- and anti-tumor processes, and seem to have pro-angiogenic activity at very low concentrations. We analyzed the influence of higher concentrations of PPARδ agonists on angiogenesis and its underlying mechanisms. We found that treatment with PPARδ agonists inhibited the formation of capillary-like structures and endothelial cell migration. Since signaling via the vascular endothelial growth factor receptor-2 (VEGFR2) pathway is critical for angiogenic responses during chronic inflammation and tumor development, we explored whether PPARδ agonist inhibition acted by diminishing VEGFR2 expression. PPARδ agonists inhibited endothelial VEGFR2 protein expression in a time- and concentration-dependent manner. In contrast, neither tie-2, neuropilin-1 nor VEGFR1 expression was significantly affected by PPARδ agonist treatment. We also demonstrated that PPARδ agonists significantly suppressed accumulation of VEGFR2 mRNA. Consistent with these results, promoter luciferase assays showed that the inhibitory effects of PPAR agonists occur through suppression of VEGFR2 promoter activity. Hence, VEGFR-2 expression may be a critical molecular target of PPAR δ agonists, which may be responsible for their anti-angiogenic effects. These results may help to define the optimal therapeutic doses of PPARδ agonists in prospective therapeutic applications.

Effect of mitomycin C on concentrations of vascular endothelial growth factor and its receptors in bladder cancer cells and in bladders of rats intravesically instilled with mitomycin C

• To examine, using in vitro and in vivo models, the largely unexamined effect of mitomycin C (MMC), an effective intravesical treatment for superficial bladder cancer and carcinoma in situ, on expression of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor-2 (VEGFR-2), which mediates many of the angiogenic properties of VEGF. • To measure, as a positive control, concentrations of the inhibitor of apoptosis, survivin, as an assessment of MMC effectiveness. • To measure MMC-induced changes in proliferation in the presence and absence of VEGF-A small interfering RNA (siRNA). • After treatment with increasing MMC concentrations (5-200 µg/mL), we measured proliferation, as well as VEGF, survivin, VEGF receptor-1 (VEGFR-1) and VEGFR-2 concentrations in RT-4 and T-24 bladder cancer cells. • The effect of pre-treatment of VEGF siRNA and survivin siRNA on MMC-induced decreases in proliferation was measured. • Urinary VEGF concentrations and bladder and kidney concentrations of VEGF-A, VEGFR-1, VEGFR-2 and interleukin-6 (IL-6) mRNA were measured in rats intravesically instilled with saline or MMC (200 µg/mL). • Although MMC treatment inhibited cell proliferation and decreased survivin mRNA expression in T-24 and RT4 cells, MMC (12-50 µg/mL) increased VEGF-A mRNA and VEGFR-2 mRNA and protein expression. • Pre-treatment with VEGF-A siRNA or survivin siRNA before MMC treatment reduced proliferation more than MMC alone. • MMC-induced reductions in proliferation were reduced additively by pre-treatment with survivin siRNA, but were potentiated by pre-treatment with VEGF-A siRNA. • VEGFR-2 mRNA and protein concentrations and urinary VEGF concentrations were increased in bladders of rats instilled with MMC. • Intravesically instilled MMC increases urinary VEGF and bladder VEGFR-2 protein and mRNA in rats. • MMC increases VEGF mRNA and VEGFR-2 protein and mRNA concentrations in bladder cancer cells. Therefore, we speculate that MMC could increase the angiogenic potential of both cancer and normal cells. • In cancer cells this effect is largest at lower MMC concentrations. • Combining MMC with agents that reduce EGF concentrations could be of value in treatment of transitional cell carcinoma of the bladder (TCC).

Effects of Radix Ginseng and Radix Notoginseng formula on secretion of vascular endothelial growth factor and expression of vascular endothelial growth factor receptor-2 in human umbilical vein endothelial cells

To investigate the effects of Radix Ginseng and Radix Notoginseng formula on secretion of vascular endothelial growth factor (VEGF) and expression of vascular endothelial growth factor receptor-2 (VEGFR-2) in human umbilical vein endothelial cells (HUVECs) in vitro. HUVECs were cultured in vitro. Bovine basic fibroblast growth factor (bFGF) at concentration of 320 U/mL and Radix Ginseng and Radix Notoginseng formula at concentrations of 0.1, 0.2 and 0.4 mg/mL were used to culture with HUVECs. And HUVECs in blank control group were cultured with culture solution only. After 24-hour culture, the content of VEGF in supernatant was detected by enzyme-linked immunosorbent assay and the expression of VEGFR-2 was detected by immunocytochemical staining and Western-blotting. Radix Ginseng and Radix Notoginseng formula at 0.4 mg/mL, the same as bFGF, increased VEGF content in the HUVEC supernatant and the number of VEGFR-2-positive HUVECs. Expression of VEGFR-2 protein in high-dose Radix Ginseng and Radix Notoginseng formula group was up-regulated as compared with the blank control group. Radix Ginseng and Radix Notoginseng formula can promote HUVEC proliferation and secretion of VEGF, as well as the expression of VEGFR-2 protein, which may be one of the mechanisms of Radix Ginseng and Radix Notoginseng formula in promoting angiogenesis.

Accumulation of VEGFR2 in zoledronic acid-treated endothelial cells

Nitrogen-containing bisphosphonates (BIS) are potent inhibitors of bone resorption and are used in the treatment of a number of medical conditions, including multiple myeloma, breast cancer and osteoporosis. Recent experimental evidence demonstrates that BIS also affect endothelial cell functions and angiogenesis; however, the molecular mechanism(s) are unclear. Vascular endothelial growth factor (VEGF) is a potent pro-angiogenic signal for endothelial cells. BIS inhibit VEGF responses in endothelial cells. The VEGF receptor-2 (VEGFR2) is the main signaling receptor for VEGF in endothelial cells. We hypothesized that altered VEGFR2 expression in BIS-treated endothelial cells may account for these attenuated responses to VEGF. The affect of the BIS zoledronic acid (ZOL) was investigated in human umbilical vein endothelial cells using confocal microscopy, Western blotting, real-time PCR and flow cytometry. VEGFR2 accumulated within the ZOL-treated endothelial cells (p=0.0002), though not on the cell surface (p>0.05). ZOL did not induce VEGFR2-specific mRNA (p>0.05). ZOL inhibited endothelial cell chemotaxis towards VEGF (p=0.001). VEGF stimulation significantly reduced the amount of VEGFR2 in the endothelial cells (p=0.01). This response to VEGF was reduced by ZOL (p>0.05). The effects of ZOL on endothelial cell migration, VEGFR2 protein expression and response to VEGF were attenuated by geranylgeranyl pyrophosphate. Two- and one-way ANOVAs with Tukey or Dunnett's multiple comparison adjustments were used. The data suggest that ZOL induces aberrant VEGFR2 accumulation. This is not likely due to the induction of mRNA transcription, but rather to the disruption of the mevalonate pathway.

1153 EFFECT OF MITOMYCIN C ON LEVELS OF VASCULAR ENDOTHELIAL GROWTH FACTOR AND ITS RECEPTORS IN BLADDER CANCER CELLS AND IN BLADDERS OF RATS INTRAVESICALLY INSTILLED WITH MITOMYCIN C

You have accessJournal of UrologyBladder Cancer: Basic Research III1 Apr 20101153 EFFECT OF MITOMYCIN C ON LEVELS OF VASCULAR ENDOTHELIAL GROWTH FACTOR AND ITS RECEPTORS IN BLADDER CANCER CELLS AND IN BLADDERS OF RATS INTRAVESICALLY INSTILLED WITH MITOMYCIN C Avanti Verma, Marcia A. Wheeler, Justin DeGrado, Hristos Z. Kaimakliotis, Adam B. Hittelman, and Robert M. Weiss Avanti VermaAvanti Verma More articles by this author , Marcia A. WheelerMarcia A. Wheeler More articles by this author , Justin DeGradoJustin DeGrado More articles by this author , Hristos Z. KaimakliotisHristos Z. Kaimakliotis More articles by this author , Adam B. HittelmanAdam B. Hittelman More articles by this author , and Robert M. WeissRobert M. Weiss More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.652AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The chemotherapeutics, mitomycin C (MMC) and Bacillus Calmette-Guérin (BCG) are effective intravesical treatments for superficial bladder cancer and carcinoma in situ. Intravesical instillation of BCG does not change vascular endothelial growth factor (VEGF) immunoreactivity in bladders from TCC patients and upregulates VEGF receptors in rats. The impact of MMC on expression of the soluble form of VEGF (VEGF-A) and on VEGF receptor-2 (VEGFR-2) which mediates many angiogenic properties of VEGF, has been largely unexamined. We plan to examine the effect of MMC on proliferation and on levels of VEGF-A, VEGF receptor-1 (VEGFR-1) and VEGFR-2 in in vitro and in vivo models. METHODS One day after MMC treatment (6-100 μg/ml), we measured proliferation, and VEGF-A, VEGFR-1 and VEGFR-2 levels in T-24 and RT4 bladder cancer cells. The effect of pretreatment of VEGF siRNA on MMC induced decreases in proliferation was measured. Rats were intravesically instilled with saline or MMC (200 μg/rat). Urinary VEGF levels, and bladder levels of VEGFR-2 protein relative to actin and VEGF, VEGFR-1, and VEGFR-2 mRNA relative to GAPDH mRNA were measured. RESULTS Although MMC treatment inhibited cell proliferation, it did not decrease VEGF protein expression in T-24 and RT4 cells. In fact, in T-24 cells, MMC (6-50 μg/ml) increased VEGF mRNA expression normalized to GAPDH 3-13 fold and increased VEGFR-2 mRNA and protein expression, 2-12 fold and 2-5 fold, respectively. Levels of VEGFR-1 were unchanged. In T-24 cells, MMC (25 μg/ml) reduced proliferation 40 ± 5%, while increasing VEGF mRNA levels 3.4 fold. However, pretreatment of MMC treated cells with VEGF siRNA blocked VEGF mRNA production and potentiated MMC induced reductions in proliferation by an additional 27 ± 4%. Similar results were seen with RT-4 cells. In rats intravesically instilled with MMC, urinary VEGF/creatinine was increased 80% and VEGFR-2 mRNA and protein levels were increased 80% and 150%, respectively, compared to levels in saline instilled rats (n = 4-6). CONCLUSIONS MMC treatment increases levels of VEGF mRNA and VEGFR-2 mRNA and protein in bladder cancer cells. VEGF siRNA potentiates mitomycin inhibitory effects on proliferation in bladder cancer cells. Intravesical Instillation of MMC increases urinary VEGF and bladder VEGFR-2 levels. We speculate that MMC increases the angiogenic potential of both cancer and normal cells. Combining MMC with agents that reduce VEGF levels may be of value in treatment of transitional cell carcinoma of the bladder. New Haven, CT© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e446-e447 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Avanti Verma More articles by this author Marcia A. Wheeler More articles by this author Justin DeGrado More articles by this author Hristos Z. Kaimakliotis More articles by this author Adam B. Hittelman More articles by this author Robert M. Weiss More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The expression of angiogenic growth factors and their receptors in ovarian follicles throughout the estrous cycle in the ewe

Healthy follicles are highly vascularized whereas those undergoing atresia have poor vascularity, suggesting a relationship between follicular vascularization and follicular function. Vascularization is regulated by angiogenic factors, and among them vascular endothelial growth factor (VEGF) and angiopoietin-Tie (Ang-Tie) systems are of central importance. The objectives of this study were to determine if VEGF, VEGF receptor-2 (VEGFR-2), and components of the Ang-Tie system are expressed in ovarian follicles at both the protein and mRNA levels and to explore if their expression is related to the stage of the estrous cycle in the ewe. Ovaries from cyclic ewes were collected during the luteal phase (n = 5) or before (n = 5), during (n = 4), and after (n = 4) the preovulatory luteinizing hormone (LH) surge. After fixation, ovaries were wax-embedded, serially sectioned, and analyzed for both protein and mRNA expression of VEGF, VEGFR-2, angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), Tie-1 (mRNA only), and Tie-2. mRNA was studied by in situ hybridization using digoxigenin-11-UTP–labeled ovine riboprobes. A similar pattern of expression was observed for mRNA and protein for all of the factors. Both mRNA and protein expression of VEGF, VEGFR-2, Ang-1, Ang-2, Tie-1 (mRNA only), and Tie-2 in the granulosa and theca cells of follicles ≥2 mm in diameter was significantly different among the stages of the estrous cycle, with the highest expression detected at the post-LH surge stage. Theca cells expressed significantly greater levels of the six angiogenic factors compared with granulosa cells at all stages of the estrous cycle. Expression levels in granulosa and theca cells were comparable between small (2.0 to 2.5 mm) and medium (2.5 to 4.0 mm) follicles, but large follicles (>4.0 mm) expressed higher mRNA and protein levels (all P < 0.05) for all factors at all stages of the estrous cycle. These data show (i) that VEGF, VEGFR-2, and the Ang-Tie system are present in both granulosa and theca cells of the ovarian follicle, (ii) that thecal cells consistently express greater levels of all of these factors compared with granulosa cells, and (iii) that their levels of expression are related to the stage of the estrous cycle and to follicle size.

Amplification and overexpression of KIT, PDGFRA, and VEGFR2 in medulloblastomas and primitive neuroectodermal tumors

Medulloblastomas (MB) and primitive neuroectodermal tumors (PNET) are the most common malignant brain tumors in children. These two tumor types are histologically similar, but have different genetic backgrounds and clinical outcomes. Other brain tumors, such as gliomas, frequently have coamplification and overexpression of receptor tyrosine kinases KIT, platelet-derived growth factor receptor alpha (PDGFRA), and vascular endothelial growth factor receptor 2 (VEGFR2). We investigated protein expression and gene copy numbers of KIT, PDGFRA, and VEGFR2 in 41 MB and 11 PNET samples by immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH). KIT and PDGFRA expression was detected in both MBs and PNETs, whereas VEGFR2 expression was weak in these tumors. KIT, PDGFRA, and VEGFR2 amplifications were all present in 4% of MBs/PNETs, and KIT amplification was associated with concurrent PDGFRA and VEGFR2 amplifications (P <or= 0.001). Most strikingly, increased gene copy number of PDGFRA was associated with poor overall survival (P = 0.027). We suggest that coamplification of PDGFRA or VEGFR2 with KIT may be clinically useful novel molecular markers in MBs and PNETs.

Inhibition of Rac1 GTPase downregulates vascular endothelial growth factor receptor‐2 expression by suppressing Sp1‐dependent DNA binding in human endothelial cells

RhoA, Rac1 and CDC42 are small GTP-binding proteins of the Rho family that play a crucial role in regulation of the actin-based cytoskeleton. In addition to cell growth regulation, they are implicated in transcriptional activation, oncogenic transformation and angiogenesis. The small Rho-GTPases have been linked to vascular endothelial growth factor (VEGF)-induced signalling pathways, but their role has not yet been elucidated. As signalling via the VEGF receptor-2 (VEGFR2) pathway is critical for angiogenic responses in cancer, wound repair and ischaemic and inflammatory diseases, we investigated whether the small Rho-GTPase Rac1 influences VEGFR2 expression in human endothelial cells. In this study, we show that a dominant negative Rac1 expression vector led to a pronounced decrease in VEGFR2 mRNA and protein expression. To identify minimal promoter requirements and potential applications of the small Rho-GTPases, we used VEGFR2 promoter-reporter gene constructs containing various deletions. The inhibitory effects of dominant negative Rac1 on the transcriptional activity of the VEGFR2 promoter localized to an element between -77 and -60 that contains an Sp1 transcription factor binding site. Electrophoretic mobility shift assays demonstrated that constitutive Sp1-dependent DNA binding decreased with Rac1 inhibition. Hence, repression of the small Rho GTPase Rac1 seems to be an additional critical molecular mechanism in the regulation of VEGFR2 expression.

Oral N‐acetylcysteine attenuates pulmonary emphysema and alveolar septal cell apoptosis in smoking‐induced COPD in rats

The role of apoptosis in lung destruction in emphysema/COPD is increasingly being recognized. The relationship between anti-oxidants and alveolar septal cell apoptosis in COPD lungs remains to be elucidated. The aim of this study was to investigate the effects of the anti-oxidant, N-acetylcysteine (NAC), on the development of emphysema and alveolar septal cell apoptosis in smoking-induced COPD in rats. Sprague-Dawley rats (n = 48) were randomly assigned to normal, COPD, sham and NAC groups. The effects of treatment were assessed by measuring the levels of vascular endothelial growth factor (VEGF) in BAL fluid by ELISA, VEGF and VEGF receptor-2 (VEGFR2) protein expression by western blotting, and the apoptotic index (AI) of alveolar septal cells by terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay. Histopathological evaluations (mean linear intercept (MLI), destructive index (DI)) and lung function measurements were performed. FEV(0.3)/FVC and PEF were lower in the COPD group than in the normal group. MLI and DI were lower in the NAC-treated group than in the COPD or sham-treated groups. As confirmed by western blotting, the levels of VEGF in BAL fluid were higher in the NAC-treated group than in the COPD group. VEGFR2 protein expression was higher in the NAC-treated group than in the COPD group. The AI was significantly lower in the NAC-treated group than in the COPD group. There was an inverse correlation between levels of VEGF in BAL fluid and the AI of alveolar septal cells. NAC attenuates lung damage, pulmonary emphysema and alveolar septal cell apoptosis by partly reversing the decrease in VEGF secretion and VEGFR2 protein expression in smoking-induced COPD in rats.

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.