Vascular Endothelial Growth Factor Receptor 2 Protein Levels Research Articles

Regulation of VEGFR2 and AKT Signaling by Musashi-2 in Lung Cancer.

Lung cancer is the most frequently diagnosed cancer type and the leading cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC) represents most of the diagnoses of lung cancer. Vascular endothelial growth factor receptor-2 (VEGFR2) is a member of the VEGF family of receptor tyrosine kinase proteins, which are expressed on both endothelial and tumor cells, are one of the key proteins contributing to cancer development, and are involved in drug resistance. We previously showed that Musashi-2 (MSI2) RNA-binding protein is associated with NSCLC progression by regulating several signaling pathways relevant to NSCLC. In this study, we performed Reverse Protein Phase Array (RPPA) analysis of murine lung cancer, which suggests that VEGFR2 protein is strongly positively regulated by MSI2. Next, we validated VEGFR2 protein regulation by MSI2 in several human lung adenocarcinoma cell line models. Additionally, we found that MSI2 affected AKT signaling via negative PTEN mRNA translation regulation. In silico prediction analysis suggested that both VEGFR2 and PTEN mRNAs have predicted binding sites for MSI2. We next performed RNA immunoprecipitation coupled with quantitative PCR, which confirmed that MSI2 directly binds to VEGFR2 and PTEN mRNAs, suggesting a direct regulation mechanism. Finally, MSI2 expression positively correlated with VEGFR2 and VEGF-A protein levels in human lung adenocarcinoma samples. We conclude that the MSI2/VEGFR2 axis contributes to lung adenocarcinoma progression and is worth further investigations and therapeutic targeting.

Placental growth factor stabilizes VEGF receptor-2 protein in retinal pigment epithelial cells by downregulating glycogen synthase kinase 3 activity

Placental growth factor (PlGF) belongs to the vascular endothelial growth factor (VEGF) family of proteins that participate in angiogenesis and vasculogenesis. Anti-VEGF therapy has become the standard treatment for ocular angiogenic disorders in ophthalmological practice. However, there is emerging evidence that anti-VEGF treatment may increase the risk of atrophy of the retinal pigment epithelium (RPE), which is important for the homeostasis of retinal tissue. Whereas the cytoprotective role of VEGF family molecules, particularly that of VEGF A (VEGFA) through its receptor VEGF receptor-2 (VEGFR-2), has been recognized, the physiological role of PlGF in the retina is still unknown. In this study, we explored the role of PlGF in the RPE using PlGF-knockdown RPE cells generated by retrovirus-based PlGF-shRNA transduction. We show that VEGFA reduced apoptosis induced by serum starvation in RPE cells, whereas the antiapoptotic effect of VEGFA was abrogated by VEGFR-2 knockdown. Furthermore, PlGF knockdown increased serum starvation–induced cell apoptosis and unexpectedly reduced the protein level of VEGFR-2 in the RPE. The antiapoptotic effect of VEGFA was also diminished in PlGF-knockdown RPE cells. In addition, we found that glycogen synthase kinase 3 activity was involved in proteasomal degradation of VEGFR-2 in RPE cells and inactivated by PlGF via AKT phosphorylation. Overall, the present data demonstrate that PlGF is crucial for RPE cell viability and that PlGF supports VEGFA/VEGFR-2 signaling by stabilizing the VEGFR-2 protein levels through glycogen synthase kinase 3 inactivation.

Abstract 874: MSI2 regulates VEGFR2 signaling and tumor progression in NSCLC

Abstract Lung cancer holds second place in incidence and first place in mortality among all cancers, both in men and women, with metastasis contributing to the vast majority of deaths. Recently, expression of vascular endothelial growth factor receptor-2 (VEGFR2) on both endothelial and tumor cells was demonstrated as one of the key mechanisms contributing to growth of non-small cell lung cancer (NSCLC), the most common subtype of lung cancer. VEGFR2 dependent signaling is critical both for tumor autocrine functions related to NSCLC migration, and in paracrine control of tumor angiogenesis through action on tumor-proximal endothelial cells, suggesting a potential mechanism for growth control. Several drugs targeting VEGFR2 signaling pathway demonstrated their clinical efficacy in NSCLC setting, including VEGFR2 and VEGF-A inhibitors, VEGF-A/PIGF trap. Recently our group demonstrated that RNA-binding protein Musashi-2 (MSI2) is driving progression and metastasis of NSCLC. MSI2 regulates mRNA translation of multiple targets, its expression is upregulated in the metastasis-competent murine and human lung cancer cell lines and is progressively elevated in lung cancer samples. The goal of this study is to define the role of MSI2 in sustaining VEGFR2 signaling during NSCLC progression. Using reverse RNA immunoprecipitation (RIP) qPCR analysis, we demonstrate that MSI2 directly binds VEGFR2 mRNA. Reverse protein phase array (RPPA) of several NSCLC cell line models with expression or knockdown of MSI2 and subsequent direct validation showed that MSI2 strongly and positively regulates expression of VEGFR2. Subsequent mechanistical analysis revealed that MSI2 knockdown leads to decreased VEGFR2 protein and mRNA levels expression in several NSCLC cell lines in vitro, resulting in increased apoptosis via inhibition of VEGFR2 signaling pathway. To better characterize MSI2 biology, we’ve crossed the B6/129S4-Msi2tm1.1Cjl/J (M2) mice with B6/129S/Sv-Krastm3Tyj/J;Trp53tm1Brn/J (KP) mice and generated novel B6/129S/Sv-KP;Msi2-/- (KPM2) mouse model. Induction of tumorigenesis in KP mice using Cre adenovirus leads to development of lung adenocarcinoma. Induction of lung-specific Cre resulted in tumorigenesis in both KP and KPM2 mice. Interestingly, we observed significant decrease in both total lung tumor number and total lung tumor burden in KPM2 compared to KP mice. Next, we established three novel KP and KPM2 derived cell lines from mouse lung tumors and found that KPM2 cell lines demonstrate reduced proliferation capacity and VEGFR2 mRNA level relative to KP cell lines. Additional studies to understand an impact of MSI2 deletion in KP cell lines are ongoing. Taken together, MSI2 is a promising target for NSCLC treatment, as this protein regulates VEGFR2 signaling and apoptosis in cell models and contributes to KP tumorigenesis in vivo. Citation Format: Igor Bychkov, Alexander Deneka, Iuliia Topchu, Petr Makhov, Alexander Kudinov, Anna Nikonova, John Karanicolas, Erica Golemis, Christopher Lengner, Hossein Borghaei, Jyoti Patel, Yanis Boumber. MSI2 regulates VEGFR2 signaling and tumor progression in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 874.

Effects of silencing epididymal vascular endothelial growth factor (VEGF) expression on hyaluronidase (HYD) activity in arsenic poisoning rats through downregulating VEGF receptor 2 (VEGFR2)

ABSTRACT RNA interference (RNAi) was used to investigate the role of epididymal vascular endothelial growth factor (VEGF) gene expression on sperm hyaluronidase (HYD) in a rat model of arsenic poisoning and to identify a new gene therapy target for male infertility caused by arsenic poisoning. The Rat model of chronic arsenic poisoning was established. And we found that positive expression of VEGF and VEGF receptor 2 (VEGFR2) was observed by Immunohistochemical staining in the epididymal tissues of arsenic-exposed rats. Subsequently, VEGF-shRNA-1, VEGF-shRNA-2 and VEGF shRNA-3 expression vectors containing epididymal VEGF-shRNA lentivirus were constructed and injected into the bilateral epididymis of each group of rats (Control group, NC-shRNA negative infection group, VEGF-shRNA-1 group, VEGF-shRNA-2 group, VEGF-shRNA-3 group) (n = 10 per group). Compared with the negative infection group and the normal control group, the expression of VEGF and VEGFR2 mRNA and protein levels were significantly decreased following epididymal infection. In addition, the HYD activity was all significantly lower than that in the normal control group and the negative infection group. Taken together, epididymal VEGF gene silencing may inhibit the activity of sperm HYD through downregulating VEGFR2.

Carom, a novel protein induced by Homocysteine suppresses endothelial cell migration through inhibiting Vascular endothelial growth factor receptor 2 (VEGFR2) endocytosis

Hyperhomocysteinemia (HHcy), a syndrome displayed by high concentration of homocysteine (Hcy) in blood has been demonstrated as a significant risk factor for cardiovascular disease (CVD). We have shown that Hcy leaded to endothelial cells (EC) injuries through inhibition of proliferation and migration. Previously we have demonstrated a Hcy specifically induced gene, Carom from cultured EC and shown its inhibition of EC migration by wound healing assay. Here, through further biochemical studies, we explore mechanistic insights behind these regulations. Carom protein belongs to Bin‐Amphiphysin‐Rvs (BAR) superfamily and specifically it is a FBAR protein since it displays Fes/CIP homology (FCH) domain on N‐terminus. Using biotin homologue to label plasma membrane proteins, we find Carom overexpression with adenovirus transduction increases Vascular endothelial growth factor receptor 2 (VEGFR2) internalization in human vein umbilical endothelial cells (HUVEC) after VEGFA stimulation. The same phenomena also exist in Hcy pretreated ECs. This process is mainly dependent on clathrin mediated endocytosis, since both Monensin and dynamin GTPase inhibitor MitMAB eliminate Carom's effects on VEGFR2 endocytosis. The VEGFR2 trafficking is tightly correlated with VEGFR2 protein levels. Carom adenovirus transduction leads to significant reduction of total cellular VEGFR2 levels. Mass spectrometric analysis of Carom interacting proteins in ECs show some partner proteins are involved in subcellular organelle trafficking and cytoskeleton organization, suggesting co‐ordinations between Carom and these partners in regulating receptor endocytosis, sorting and its function. Regulation of VEGFR2 trafficking and the ensuing signaling pathways are one of the major causes accounted for Carom's inhibitory effects on EC migration.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Increased serum levels of soluble CD146 and vascular endothelial growth factor receptor 2 in patients with exudative age-related macular degeneration.

To investigate serum levels of soluble CD146 (sCD146) and vascular endothelial growth factor receptor 2 (VEGFR2) in patients with age-related macular degeneration (AMD). Eighty-eight patients with exudative AMD and 45 sex- and age-matched healthy controls were enrolled in this study conducted in China. Serum samples was obtained from the patients with exudative AMD and from the controls. Serum sCD146 and VEGFR2 protein levels were measured using an enzyme-linked immunosorbent assay. We found that serum sCD146 and VEGFR2 protein levels were significantly higher in the patients with exudative AMD group than in the controls (t=3.859, P<0.001 and t=3.829, P<0.001, respectively). Serum sCD146 levels were significantly higher in patients with classic choroidal neovascularization (CNV) than in those with occult CNV (t=9.899, P<0.001). There was a significant difference in the trend for exudative AMD in the highest versus lowest quartile of circulating sCD146 levels (χ 2=10.29, P=0.001). The receiver operating characteristic curve analysis showed that the area under the curve was 0.696 for sCD146 (95%CI: 0.601-0.791) with an optimum diagnostic cut-off value of 157.16 ng/mL, a sensitivity of 55.7%, and a specificity of 82.2%. The serum sCD146 level increases and may be a biomarker for exudative AMD.

VEGFR2 inhibition by RNA interference affects cell proliferation, migration, invasion, and response to radiation in Calu-1 cells.

To investigate the role of the vascular endothelial growth factor receptor 2 (VEGFR2) in the proliferation, migration, invasion, and radiation-induced apoptosis of the non-small cell lung cancer (NSCLC) cell line Calu-1. VEGFR2 gene was silenced by RNA interference in Calu-1 cells, and the expression of VEGFR2 was measured by qRT-PCR and Western blot analysis. The cells were divided into control, VEGF-treated, VEGFR2 knockdown, and VEGFR2 knockdown and VEGF-treated groups. A CCK8 assay and Transwell assay were performed to assess cell proliferation, migration, and invasion, respectively, after VEGFR2 knockdown. Western blot assays were used to detect signaling proteins downstream of VEGFR2. Cells in the groups listed above were also subjected to radiation treatment, followed by apoptosis analysis. (1) RNA interference of VEGFR2 in Calu-1 cells reduced VEGFR2 mRNA (P < 0.01) and protein levels (P < 0.01). (2) VEGFR2 knockdown inhibited proliferation (P < 0.05), migration (P < 0.05), and invasion (P < 0.05) in Calu-1 cells. (3) VEGFR2 knockdown blocked the phosphorylation of protein kinase B (Akt, also known as PKB), extracellular regulated kinase (ERK) 1/2, and p38 mitogen-activated protein kinase (p38 MAPK) to various extent (P < 0.05), but did not change their total protein expression. (4) Knockdown of VEGFR2 suppressed HIF-1α protein synthesis (P < 0.05), and exacerbated apoptosis induced by radiation (P < 0.05). VEGFR2 gene knockdown significantly suppressed a number of cellular activities in Calu-1 cells and increased radiation-induced cell death.

Abstract 1388A: Assessment of the anti-angiogenic effect of VEGFR2 siRNA in HUVEC using the Lonza 4D-Nucleofecto system

Abstract Angiogenesis is a hallmark of most cancers, and is thus an attractive target for the treatment of cancer. One of the easiest screening and target validation strategies for anti-angiogenic target identification involves knocking down targets in Human Umbilical Vein Endothelial Cells (HUVEC) and assessing subsequent effects on tube formation in Corning's Matrigel product. The usage of small interfering RNA (siRNA) is one of the strategies to knock-down RNA, and thereby protein expression within cells. siRNA can be delivered within cells using either chemical transfection or electroporation-based strategies such as the one offered by the Lonza Nucleofector™ Technology. In the current study we used the Lonza 4D-Nucleofector™ X-Unit to transfect single-donor Clonetics™ HUVEC cultured in EGM™-2 Endothelial Cell Growth Media. Cells were transfected with siRNA directed against Vascular Endothelial Growth Factor Receptor 2 (VEGFR2). Transfection conditions were fine-tuned using pmaxGFP™ vector, and it was shown that the Nucleofection™ process had no deleterious effect on the tube formation potential of HUVEC on Corning's Matrigel product. Efficient knock-down of VEGFR2 protein levels was demonstrated after the transfection of VEGFR2-siRNA. Best knock-down efficiency was observed 24 hours after transfection. VEGFR2-siRNA transfected cells demonstrated significant inhibition of tube formation on Corning's Growth Factor Reduced Matrigel product in comparison to control samples. Since the efficient knock-down of the VEGFR2 protein in Clonetics™ HUVEC using the Nucleofector™ Technology can be demonstrated at time points as early as 24 hours after transfection, this is an efficient approach for target identification, validation and screening of siRNA based anti-angiogenesis therapeutics for cancer treatment. Citation Format: Srinivasan Kokatam, Kanchan Tiwari, Jenny Schroeder, Andrea Toell, Lubna Hussain, Preeti Kapoor. Assessment of the anti-angiogenic effect of VEGFR2 siRNA in HUVEC using the Lonza 4D-Nucleofecto system. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1388A. doi:10.1158/1538-7445.AM2015-1388A

Receptors for advanced glycation end products (RAGE) is associated with microvessel density and is a prognostic biomarker for clear cell renal cell carcinoma.

The receptor for advanced glycation end products (RAGE) is involved in a variety of biological processes, including tumorigenisis. Previous studies have demonstrated that RAGE regulates the neo-angiogenesis related downstream molecule-vascular endothelial growth factor receptor 2 (VEGFR-2). Here, we investigated the potential relationship between RAGE, VEGFR-2 and angiogenesis in 80 renal cell carcinoma (RCC) patients. Real-time quantitative PCR and ELISA analysis were used to explore the RAGE and VEGFR-2 gene expression levels and the protein of VEGFR-2 expression. Meanwhile, angiogenesis was detected by the semi-quantification of endothelial cell marker CD34 combined with caldesmon, which was detected by microvessel density (MVD) technique and immunohistochemistry. Tumors were classified as low or high RAGE-expressing using the median as the cut-off. Immunofluorescence staining for RAGE protein was performed as well. Additionally, the median gene expression levels of VEGFR-2 in the tumors were significantly lower expressing low levels of RAGE expression, 0.34 (95% CI, 0.28-0.39) compared to the expressing high levels of RAGE expression, 0.45 (95% CI, 0.29-0.61), (P=0.03). The median MVD was significantly lower in the tumors expressing low levels of RAGE, 6.5 (95% CI, 6.21-7.43), compared to the expressing high levels, 7.9 (95% CI, 6.25-8.93), (P<0.01). Further, a positive association was certified with VEGFR-2 protein levels, P=0.07. Besides, RCC with high levels of RAGE expression are associated with high VEGFR-2 mRNA/protein levels and a higher density of microvessels; conversely, Kaplan-Meier survival analysis suggests that a significant correlation of elevated RAGE expression with decreased overall survival and metastasis-free survival. Our results establish that RAGE was identified as a potential prognostic biomarker for disease prognosis of RCC.

Elevated microRNA-185 is associated with high vascular endothelial growth factor receptor 2 expression levels and high microvessel density in clear cell renal cell carcinoma.

MicroRNAs (miRNAs) are involved in a number of biological processes, including tumor biology. Previous studies have demonstrated that miRNA-185 regulates signaling downstream of vascular endothelial growth factor receptor 2 (VEGFR-2) and, consequently, angiogenesis. The aim of this study was to analyze the potential relationship between miRNA-185, VEGFR-2, and angiogenesis in samples from renal cell carcinoma (RCC) patients. Tumor tissue was obtained from 82 patients. The miRNA-185 and VEGFR-2 gene expression levels were analyzed by PCR, and the protein concentrations of VEGFR-2 were detected by ELISA. Angiogenesis, visualized by the endothelial cell marker CD34 combined with caldesmon, was assessed by immunohistochemistry and the microvessel density (MVD) technique. In situ hybridization was performed for miRNA-185. Tumors were classified as low or high miRNA-185-expressing using the median as the cutoff. The median gene expression levels of VEGFR-2 were significantly lower in the tumors expressing low levels of miRNA-185, 0.31 (95% CI, 0.25-0.37), compared to those expressing high levels of miRNA-185, 0.47 (95% CI, 0.27-0.59), p = 0.02. A positive association was certified with VEGFR-2 protein levels, p = 0.06. The median MVD was significantly lower in the tumors expressing low levels of miRNA-185, 6.8 (95% CI, 6.33-7.67), compared to those expressing high levels, 8.0 (95% CI, 6.33-9.00), p < 0.01. miRNA-185 was detected in endothelial cells by in situ hybridization detection. The results suggest that high levels of miRNA-185 in RCC are associated with high VEGFR-2 mRNA and protein levels and a higher density of microvessels. However, further investigation should be performed to analyze the prognostic value of miRNA-185 in RCC.

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.

Attenuation of VEGFR-2 Expression by sFlt-1 and Low Oxygen in Human Placenta

Vascular endothelial growth factor receptor 2 (VEGFR-2), the primary receptor for VEGF, is crucial for normal endothelial function. sFlt-1, a truncated and soluble form of VEGFR-1 which binds and inhibits VEGF, is increased in preeclampsia and is positively regulated by low oxygen. Here, we investigated the effects of sFlt-1 and hypoxia on VEGFR-2 expression and signaling in the human placenta. VEGFR-2 transcript and protein levels were significantly decreased in preeclamptic placentae compared to controls (1.82 and 1.85 fold, respectively). An inverse correlation was observed for VEGFR-2 and sFlt-1 levels in both singleton and twin placentae from patients with preeclampsia. Immunofluorescence analyses revealed co-localization of VEGFR-2 and sFlt-1 in placental vasculature and co-immunoprecipitation analyses confirmed VEGFR-2 and sFlt-1 interaction only in preeclamptic placentae compared to age-matched controls. VEGFR-2 transcript and protein levels from explants cultured in 3% O2 were significantly decreased compared to those incubated at 20% O2 (5.9 and 12.47 fold, respectively). Also, VEGFR-2 transcript levels were significantly decreased in early first trimester placentae (low oxygen environment) compared to late first trimester placentae (2.05 fold). We next explored whether sFlt-1 directly affects VEGFR-2 expression. Treatment of first trimester placental explants with sFlt-1 resulted in significantly decreased levels of VEGFR-2 (2.03 fold) and downstream signaling proteins phospho-ERK (1.60 fold) and phospho-Akt (1.64 fold). Our findings show a novel hypoxia-induced and PE-related down-regulation of VEGFR-2 in the human placenta. sFlt-1, which is known to be increased in hypoxic conditions and PE, directly attenuates VEGFR-2 expression and signaling. A direct interaction between sFlt-1 and VEGFR-2 may represent an important mechanism in VEGFR-2 regulation, inhibition of VEGFR-2-mediated processes in placentation and a novel platform to examine the onset of preeclampsia.

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&amp;lt;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

Vascular endothelial growth factor receptor 2 gene (KDR) polymorphisms and expression levels in depressive disorder

Recent research findings suggest that vascular endothelial growth factor (VEGF) participates in the development of depressive disorder. VEGF is involved in neurogenesis and neuroprotection processes, mediated by vascular endothelial growth factor receptor 2 (VEGFR2). VEGFR2 also plays a role in angiogenesis, a process related to neurogenesis and other biological processes.We examined VEGFR2 (KDR) gene polymorphism, mRNA expression levels, as well as VEGFR2 protein levels in 268 patients diagnosed with a recurrent depressive disorder (rDD) using the ICD-10 criteria, and in 200 healthy controls. Genotyping and gene expression level analysis was performed using polymerase chain reaction (PCR)—based methods. An Enzyme—Linked Immunosorbent Assay (ELISA) was used for measurement of KDR protein levels.Our study found that distribution of KDR polymorphism +1416T/A differs significantly in patients with rDD when compared to healthy subjects, while A allele and AA genotype are risk factors for rDD. KDR mRNA and protein expression are higher in patients with rDD. We also observed a significant association between the −271A/G variant and gene and protein levels.Our study is the first to demonstrate that the KDR gene may serve as a novel genetic marker that could participate in the etiology of rDD. This new pathway may play a role in the inflammatory pathophysiology of depression.

Angiogenesis impairment in diabetes: role of methylglyoxal-induced receptor for advanced glycation endproducts, autophagy and vascular endothelial growth factor receptor 2.

Diabetes impairs physiological angiogenesis by molecular mechanisms that are not fully understood. Methylglyoxal (MGO), a metabolite of glycolysis, is increased in patients with diabetes. This study defined the role of MGO in angiogenesis impairment and tested the mechanism in diabetic animals. Endothelial cells and mouse aortas were subjected to Western blot analysis of vascular endothelial growth factor receptor 2 (VEGFR2) protein levels and angiogenesis evaluation by endothelial cell tube formation/migration and aortic ring assays. Incubation with MGO reduced VEGFR2 protein, but not mRNA, levels in a time and dose dependent manner. Genetic knockdown of the receptor for advanced glycation endproducts (RAGE) attenuated the reduction of VEGFR2. Overexpression of Glyoxalase 1, the enzyme that detoxifies MGO, reduced the MGO-protein adducts and prevented VEGFR2 reduction. The VEGFR2 reduction was associated with impaired angiogenesis. Suppression of autophagy either by inhibitors or siRNA, but not of the proteasome and caspase, normalized both the VEGFR2 protein levels and angiogenesis. Conversely, induction of autophagy either by rapamycin or overexpression of LC3 and Beclin-1 reduced VEGFR2 and angiogenesis. MGO increased endothelial LC3B and Beclin-1, markers of autophagy, which were accompanied by an increase of both autophagic flux (LC3 punctae) and co-immunoprecipitation of VEGFR2 with LC3. Pharmacological or genetic suppression of peroxynitrite (ONOO−) generation not only blocked the autophagy but also reversed the reduction of VEGFR2 and angiogenesis. Like MGO-treated aortas from normglycemic C57BL/6J mice, aortas from diabetic db/db and Akita mice presented reductions of angiogenesis or VEGFR2. Administration of either autophagy inhibitor ex vivo or superoxide scavenger in vivo abolished the reductions. Taken together, MGO reduces endothelial angiogenesis through RAGE-mediated, ONOO–dependent and autophagy-induced VEGFR2 degradation, which may represent a new mechanism for diabetic angiogenesis impairment.

Elevated microRNA-126 is associated with high vascular endothelial growth factor receptor 2 expression levels and high microvessel density in colorectal cancer

MicroRNAs (miRNAs) are involved in a number of biological processes, including tumour biology. Pre-clinical studies have shown that miRNA-126 regulates signalling downstream of vascular endothelial growth factor receptor 2 (VEGFR-2) and, consequently, angiogenesis. The aim of this study was to analyse the possible relationship between miRNA-126, VEGFR-2 and angiogenesis in tumour tissue from patients with colorectal cancer (CRC). Tumour tissue was obtained from 81 patients. The miRNA-126 and VEGFR-2 gene expression levels were analysed by PCR and the protein concentrations of VEGFR-2 were analysed by ELISA. Angiogenesis, visualised by the endothelial cell marker CD105 combined with caldesmon, was assessed by immunohistochemistry and the microvessel density (MVD) technique. In situ hybridisation was performed for miRNA-126. Tumours were classified as low or high miRNA-126-expressing using the median as the cut-off. The median gene expression levels of VEGFR-2 were significantly lower in the tumours expressing low levels of miRNA-126, 0.30 (95% CI, 0.24-0.36), compared to those expressing high levels of miRNA-126, 0.48 (95% CI, 0.28-0.60), p=0.02. A positive association was observed with VEGFR-2 protein concentrations, p=0.06. The median MVD was significantly lower in the tumours expressing low levels of miRNA-126, 5.8 (95% CI, 5.33-6.67), compared to those expressing high levels, 8.0 (95% CI, 6.33-9.00), p<0.01. miRNA-126 was detected in endothelial cells by in situ hybridisation analysis. These results suggest that high levels of miRNA-126 in CRC are associated with high VEGFR-2 mRNA and protein levels and a higher density of newly formed microvessels. However, further studies should be conducted to analyse the clinical value of miRNA-126 in CRC.

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 ...

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.

VE-cadherin increases the half-life of VEGF receptor 2

VE-cadherin plays a critical role in cell–cell interactions by forming adherens junctions in endothelial cells. VE-cadherin has increasingly been implicated in the cell signaling cascades initiated by the activation of growth factor receptors. Vascular endothelial growth factor receptor 2 (VEGFR-2) is present in regions of cell–cell contact and coimmunoprecipitates with VE-cadherin. In this study, we report that stable overexpression of VE-cadherin in two different endothelial cells induced an increase in VEGFR-2 protein levels. The increase in VEGFR-2 was also induced by overexpression of other classical cadherins such as E-cadherin or N-cadherin. Removing the extracellular domain of VE-cadherin abolished this effect, and a truncated form of VE-cadherin lacking the intracellular domain decreased VEGFR-2 instead of increasing it. VE-cadherin-induced changes in VEGFR-2 levels were paralleled by a corresponding shift in the VEGF-dependent activation of MAPK signaling, which demonstrated the functional relevance of varying the VEGFR-2 levels. Since VE-cadherin upregulated endogenous VEGFR-2 or exogenously expressed VEGFR-2, we hypothesized that the mechanism may be posttranslational. Indeed, the half-life of VEGFR-2 was 70 min in control cells whereas in cells overexpressing VE-cadherin the half-life was extended to 146 min. These results support the existence of a novel layer of functional regulation of VEGFR-2 by VE-cadherin.