7422

VEGFA

OBJECTIVE: To examine the relationship between effect of vascular endothelial growth factor (VEGF) on epithelial-myofibroblast transition (EMT) of HK-2 cells and changes in expressions of bone morphogenetic protein-7 (BMP-7) and inhibitor of DNA binding/differentiation (Id) 2, Id3. METHODS: The cultured HK-2 cells were co-treated with transforming growth factor-beta1 (TGF-beta1) (5 ng/ml) and VEGF165 (0.1, 1, 10, 100 ng/ml), or with TGF-beta1 (5 ng/ml) and VEGF receptor-1 neutralized antibody (10 microg/ ml), and were also co-treated with TGF-beta1 (5 ng/ml) and VEGF165 (100 ng/ml) with or without activin receptor-like kinase 6 (Alk6)/Fc Chimera (2 microg/ml, to neutralize endogenous BMP-7) for 48 hours. mRNA and protein expressions of alpha-smooth muscle actin (alpha-SMA), E-cadherin, BMP-7, Id2 and Id3 of HK-2 cells were assessed with double-stain immunocytochemistry, real-time PCR and Western blot respectively. RESULTS: Compared with normal controls, alpha-SMA expression significantly increased, while E-cadherin, BMP-7, Id2, and Id3 mRNA and protein expressions markedly decreased in HK-2 cells treated with TGF-beta1 (5 ng/ml) (P < 0.05). VEGF165 interrupted TGF-beta1 induced alpha-SMA expression in a dose-dependent manner and upregulated BMP-7, Id2 mRNA and protein expressions of the cells (P < 0.05). alpha-SMA expression increased, while E-cadherin, BMP-7, and Id2 expressions decreased further in HK-2 cells co-treated with TGF-beta1 and VEGFR1 antibody compared with normal controls (P < 0.05). When endogenous BMP-7 was neutralized with Alk6/Fc Chimera in the cells co-treated with TGF-beta1 and VEGF165, alpha-SMA expression upregulated (P < 0.05), while Id2 was not changed. CONCLUSIONS: VEGF165 may partially inhibit TGF-beta1-induced EMT of HK-2 cells in vitro. This effect is related to the upregulated expressions of BMP-7 and Id2. Id2 may be upregulated directly by VEGF165, but not related to BMP-7.

epithelial-mesenchymal transition (EMT) is an important process that contributes to renal fibrogenesis. TGF-beta1 and EGF stimulate EMT. Recent studies suggested that parathyroid hormone-related protein (PTHrP) promotes fibrogenesis in the damaged kidney, apparently dependent on its interaction with vascular endothelial growth factor (VEGF), but whether it also interacts with TGF-beta and EGF to modulate EMT is unknown. Here, PTHrP(1-36) increased TGF-beta1 in cultured tubuloepithelial cells and TGF-beta blockade inhibited PTHrP-induced EMT-related changes, including upregulation of alpha-smooth muscle actin and integrin-linked kinase, nuclear translocation of Snail, and downregulation of E-cadherin and zonula occludens-1. PTHrP(1-36) also induced EGF receptor (EGFR) activation; inhibition of protein kinase C and metalloproteases abrogated this activation. Inhibition of EGFR activation abolished these EMT-related changes, the activation of ERK1/2, and upregulation of TGF-beta1 and VEGF by PTHrP(1-36). Moreover, inhibition of ERK1/2 blocked EMT induced by either PTHrP(1-36), TGF-beta1, EGF, or VEGF. In vivo, obstruction of mouse kidneys led to changes consistent with EMT and upregulation of TGF-beta1 mRNA, p-EGFR protein, and PTHrP. Taken together, these data suggest that PTHrP, TGF-beta, EGF, and VEGF might cooperate through activation of ERK1/2 to induce EMT in renal tubuloepithelial cells.

Inhibition of VEGF signaling leads to a proinvasive phenotype in mouse models of glioblastoma multiforme (GBM) and in a subset of GBM patients treated with bevacizumab. Here, we demonstrate that vascular endothelial growth factor (VEGF) directly and negatively regulates tumor cell invasion through enhanced recruitment of the protein tyrosine phosphatase 1B (PTP1B) to a MET/VEGFR2 heterocomplex, thereby suppressing HGF-dependent MET phosphorylation and tumor cell migration. Consequently, VEGF blockade restores and increases MET activity in GBM cells in a hypoxia-independent manner, while inducing a program reminiscent of epithelial-to-mesenchymal transition highlighted by a T-cadherin to N-cadherin switch and enhanced mesenchymal features. Inhibition of MET in GBM mouse models blocks mesenchymal transition and invasion provoked by VEGF ablation, resulting in substantial survival benefit.CI - Copyright (c) 2012 Elsevier Inc. All rights reserved.

AIM: The aim of the present study was to investigate the molecular mechanism of fibroblast growth factor (FGF)-9 in prostate cancer cells. MATERIALS AND METHODS: expression of vascular endothelial growth factor (VEGF)s and cadherins in LNCaP cells by incubation with FGF9 was assessed by western blot analysis. Tissues obtained during a radical prostatectomy in 88 patients were immunohistochemically-stained using anti-FGF9, anti-cadherin, and anti-VEGF antibodies. RESULTS: expression of N-cadherin and VEGF-A were induced in LNCaP cells incubated in FGF9-containing medium. The biochemical relapse-free survival rate in cases with FGF9, N-cadherin and VEGF-A-positive cells was significantly lower than the rate in cases where positive cells were not detectable. The prevalence of both VEGF-A- and N-cadherin-positive cells in the sample with FGF9-positive cells were significantly higher in comparison to FGF9-negative cases. CONCLUSION: FGF9 can be associated with epithelial-to-mesenchymal transition and invasion by inducing VEGF-A expression in prostate cancer cells.

We previously found that CCL20 induced primarily cultured healthy breast cell proliferation and migration. The objective of this study was to investigate the hypothesis that CCL20 modulated the epithelial-mesenchymal transition (EMT) of primarily cultured healthy breast epithelial cells and the angiogenesis in areas adjacent to the tumor. Key results showed that CCL20 (a) down-regulated E-cadherin and ZO-1; (b) up-regulated N-cadherin, vimentin, and Snail expressions; (c) increased mRNA and secretion of VEGF and (d) increased angiogenic micro vessel sprouting. Thus, the signal transduction pathways evoked by CCL20 were investigated. We showed that NF-kB p65 down-regulation (by small interfering RNA, siRNA) reversed CCL20-induced Snail and blocked the up-regulation of vimentin and N-cadherin mRNAs. Furthermore, PI3K/AKT inhibition (by LY294002) completely blocked CCL20-induced Snail and NF-kB activation. Inhibition of JNK1/2 (by SP60125) or PKC-alpha (by siRNA) or src (by PP1) blocked NF-kB activation and Snail expression suggesting that these kinases are all upstream of NF-kB/Snail. Inhibition of mTOR (by rapamycin) abolished the effects of CCL20 on N-cadherin and vimentin protein synthesis. Furthermore, siRNA of PKC-delta inhibited the phosphorylation of CCL20-induced mTOR and S6, increased vimentin and N-cadherin expressions and, finally, blocked the CCL20 induced-EMT. CCL20 increased mRNA and secretion of VEGF by healthy breast cells by using PKC-alpha, src, Akt, NF-kB, and Snail signalling. In summary, tumor cells signal to the surrounding healthy cells through CCL20 inducing the modulation of the expression of molecules involved in EMT and promoting angiogenesis directly and indirectly through the secretion of VEGF, a major contributor to angiogenesis. (c) 2015 Wiley Periodicals, Inc.CI - (c) 2015 Wiley Periodicals, Inc.

PURPOSE: The purpose of this study was to identify potential therapeutic strategies to slow down or prevent the expression of early-onset epithelial to mesenchymal transition (EMT) marker proteins (fibronectin and alpha smooth muscle actin, alpha-SMA) without sacrificing the synthesis and accumulation of the prosurvival protein vascular endothelial growth factor (VEGF) in cultured virally transformed human lens epithelial (HLE) cells. METHODS: HLE-B3 cells, maintained in a continuous hypoxic environment (1% oxygen), were treated with SB216763, a specific inhibitor of glycogen synthase kinase-3beta (GSK-3beta) catalytic activity. Western blot analysis was employed to detect the cytoplasmic and nuclear levels of beta-catenin, as well as the total lysate content of fibronectin and alpha-SMA. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of VEGF in cell culture medium. A hypoxia-inducible factor-1alpha (HIF-1alpha) translation inhibitor and an HIF-2alpha translation inhibitor were independently employed to evaluate the effect of hypoxia inducible factor inhibition on EMT marker protein and VEGF expression. XAV932 was used to assess the suppression of nuclear beta-catenin and its downstream effect on EMT marker proteins and VEGF expression. RESULTS: SB216763-treated HLE-B3 cells caused marked inhibition of GSK-3beta activity prompting a significant increase in the translocation of cytoplasmic beta-catenin to the nucleus. The enhancement of nuclear beta-catenin looked as if it positively correlated with a significant increase in the basal expression of VEGF as well as increased expression of fibronectin and alpha-SMA. In conjunction with SB216763, coadministration of an HIF-1alpha translation inhibitor, but not an HIF-2alpha translation inhibitor, markedly suppressed the expression of fibronectin and alpha-SMA without affecting VEGF levels. Treatment with XAV932 significantly reduced the level of nuclear beta-catenin, but the levels of neither the EMT marker proteins nor VEGF were changed. CONCLUSIONS: Recently, we reported that nuclear beta-catenin, but not HIF-2alpha, regulates the expression of fibronectin and alpha-SMA in atmospheric oxygen. In marked contrast, data from the hypoxic condition clearly establish that nuclear beta-catenin plays little apparent role in the expression of EMT marker proteins. Instead, the loss of HIF-1alpha (but not HIF-2alpha) decreases the expression of the EMT marker proteins without sacrificing the levels of the prosurvival protein VEGF. These findings support the development of a potentially relevant therapeutic strategy to undermine the progression of normal cells to the mesenchymal phenotype in the naturally hypoxic lens without subverting cell viability.

Autocrine vascular endothelial growth factor (VEGF) can regulate the survival and progression of cancers through its various receptors. But the mechanisms and mediators for these functions are largely uncovered, especially in breast cancer. We examined the potential roles and mechanisms of VEGF/neuropilin-1 (NRP-1) axis in regulating the tumorigenesis and metastasis of breast cancer and found the expression of VEGF and NRP-1 correlated with aggressiveness of breast cancer. Knockdown of VEGF or NRP-1 inhibited the proliferation, migration and invasion, but enhanced the apoptosis of MDA-MB-231 cells. In contrast, induction of NRP-1 over-expression promoted the proliferation, migration and invasion of MCF-7 cells. VEGF or NRP-1 silencing attenuated the epithelial-mesenchymal transition (EMT) process and the activation of NF-kappaBp65, but enhanced GSK-3beta expression in MDA-MB-231 cells while NRP-1 over-expression reversed the effects in MCF-7 cells. Treatment with hVEGF165 did not change the inhibition in NRP-1 silencing MDA-MB-231 cells, but enhanced the aggressiveness of NRP-1 over-expressing MCF-7 cells. In addition, VEGF-silencing inhibited the growth and metastasis of implanted MDA-MB-231 tumors in vivo. Our novel data suggest that the positive regulation of the VEGF/NRP-1 axis on the tumorigenesis and metastasis of breast cancer may be associated with enhancing the EMT process and the NF-kappaB and beta-catenin signaling. Hence, the VEGF/NRP-1 axis may be a valuable target for design of therapies for intervention of breast cancer.CI - Copyright (c) 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Cardiac malformations constitute the most common birth defects, of which heart septal and valve defects are the most frequent forms diagnosed in infancy. These cardiac structures arise from the endocardial cushions through dynamic interactions between cells and the extracellular matrix (cardiac jelly). Targeted deletion of the hyaluronan synthase-2 (Has2) gene in mice results in an absence of hyaluronan (HA), cardiac jelly, and endocardial cushions, a loss of vascular integrity, and death at embryonic day 9.5. Despite the requirements for Has2 and its product, HA, in the developing heart, little is known about the normal processing and removal of HA during development. Cell culture studies show that HA obtains new bioactivity after depolymerization into small oligosaccharides. We previously showed reduction in Has2 expression and diminished presence of HA at later stages of heart development as tissue remodeling formed the leaflets of the cardiac valves. Here we show that small oligosaccharide forms of HA (o-HA) act antagonistically to developmental epithelial-to-mesenchymal transformation (EMT), which is required to generate the progenitor cells that populate the endocardial cushions. We further show that o-HA induces vascular endothelial growth factor (VEGF), which acts as a negative regulator of EMT. This is the first report illustrating a functional link between oligosaccharide HA and VEGF. Collectively, our data indicate that following endocardial cell EMT, native HA is likely processed to o-HA, which stimulates VEGF activity to attenuate cardiac developmental EMT.