Skeletal muscle VEGF gradients in peripheral arterial disease: simulations of rest and exercise

VEGF is a key promoter of angiogenesis and a major target of proangiogenic therapy for peripheral arterial disease (PAD). Greater understanding of VEGF angiogenic signaling and guidance by gradients for new capillaries will aid in developing new proangiogenic therapies and improving existing treatments. However, in vivo measurements of VEGF concentration gradients at the cell scale are currently impossible. We have developed a computational model to quantify VEGF distribution in extensor digitorum longus skeletal muscle using measurements of VEGF, VEGF receptor (VEGFR), and neuropilin-1 (NRP1) expression in an experimental model of rat PAD. VEGF is secreted by myocytes, diffuses through and interacts with extracellular matrix and basement membranes, and binds VEGFRs and NRP1 on endothelial cell surfaces of blood vessels. We simulate the effects of increased NRP1 expression and of therapeutic exercise training on VEGF gradients, receptor signaling, and angiogenesis. Our study predicts that angiogenic therapy for PAD may be achieved not only through VEGF upregulation but also through modulation of VEGFRs and NRP1. We predict that expression of 10(4) NRP1/cell can increase VEGF binding to receptors by 1.7-fold (vs. no NRP1); in nonexercise-trained muscle with PAD, angiogenesis is hindered due to limited VEGF upregulation, signaling, and gradients; in exercise-trained muscle, VEGF signaling is enhanced by upregulation of VEGFRs and NRP1, and VEGF signaling is strongest within the first week of exercise therapy; and hypoxia-induced VEGF release is important to direct angiogenesis towards unperfused tissue.     

The biology of VEGF and its receptors

Vascular endothelial growth factor (VEGF) is a key regulator of physiological angiogenesis during embryogenesis, skeletal growth and reproductive functions. VEGF has also been implicated in pathological angiogenesis associated with tumors, intraocular neovascular disorders and other conditions. The biological effects of VEGF are mediated by two receptor tyrosine kinases (RTKs), VEGFR-1 and VEGFR-2, which differ considerably in signaling properties. Non-signaling co-receptors also modulate VEGF RTK signaling. Currently, several VEGF inhibitors are undergoing clinical testing in several malignancies. VEGF inhibition is also being tested as a strategy for the prevention of angiogenesis, vascular leakage and visual loss in age-related macular degeneration.     

VEGF signaling inside vascular endothelial cells and beyond

Vascular endothelial growth factor-A (VEGF-A) has long been recognized as the key regulator of vascular development and function in health and disease. VEGF is a secreted polypeptide that binds to transmembrane tyrosine kinase VEGF receptors on the plasma membrane, inducing their dimerization, activation and assembly of a membrane-proximal signaling complex. Recent studies have revealed that many key events of VEGFR signaling occur inside the endothelial cell and are regulated by endosomal receptor trafficking. Plasma membrane VEGFR interacting molecules, including vascular guidance receptors Neuropilins and Ephrins also regulate VEGFR endocytosis and trafficking. VEGF signaling is increasingly recognized for its roles outside of the vascular system, notably during neural development, and blood vessels regulate epithelial branching morphogenesis. We review here recent advances in our understanding of VEGF signaling and its biological roles.     

VEGF binding to NRP1 is essential for VEGF stimulation of endothelial cell migration, complex formation between NRP1 and VEGFR2, and signaling via FAK Tyr407 phosphorylation

In endothelial cells, neuropilin-1 (NRP1) binds vascular endothelial growth factor (VEGF)-A and is thought to act as a coreceptor for kinase insert domain-containing receptor (KDR) by associating with KDR and enhancing VEGF signaling. Here we report mutations in the NRP1 b1 domain (Y297A and D320A), which result in complete loss of VEGF binding. Overexpression of Y297A and D320A NRP1 in human umbilical vein endothelial cells reduced high-affinity VEGF binding and migration toward a VEGF gradient, and markedly inhibited VEGF-induced angiogenesis in a coculture cell model. The Y297A NRP1 mutant also disrupted complexation between NRP1 and KDR and decreased VEGF-dependent phosphorylation of focal adhesion kinase at Tyr407, but had little effect on other signaling pathways. Y297A NRP1, however, heterodimerized with wild-type NRP1 and NRP2 indicating that nonbinding NRP1 mutants can act in a dominant-negative manner through formation of NRP1 dimers with reduced binding affinity for VEGF. These findings indicate that VEGF binding to NRP1 has specific effects on endothelial cell signaling and is important for endothelial cell migration and angiogenesis mediated via complex formation between NRP1 and KDR and increased signaling to focal adhesions. Identification of key residues essential for VEGF binding and biological functions provides the basis for a rational design of antagonists of VEGF binding to NRP1.     

Autocrine regulation of glioblastoma cell-cycle progression,viability and radioresistance through the VEGF-VEGFR2 (KDR) interplay

Vascular endothelial growth factor (VEGF) plays a crucial role in angiogenesis and progression of malignant brain tumors. Given the significance of tumor microenvironment in general, and the established role of paracrine VEGF signaling in glioblastoma (GBM) biology in particular, we explored the potential autocrine control of human astrocytoma behavior by VEGF. Using a range of cell and molecular biology approaches to study a panel of astrocytoma (grade III and IV/GBM)-derived cell lines and a series of clinical specimens from low- and high-grade astrocytomas, we show that co-expression of VEGF and VEGF receptors (VEGFRs) occurs commonly in astrocytoma cells. We found VEGF secretion and VEGF-induced biological effects (modulation of cell cycle progression and enhanced viability of glioblastoma cells) to function in an autocrine manner. Morevover, we demonstrated that the autocrine VEGF signaling is mediated via VEGFR2 (KDR), and involves co-activation of the c-Raf/MAPK, PI3K/Akt and PLC/PKC pathways. Blockade of VEGFR2 by the selective inhibitor (SU1498) abrogated the VEGF-mediated enhancement of astrocytoma cell growth and viability under unperturbed culture conditions. In addition, such interference with VEGF-VEGFR2 signaling potentiated the ionizing radiation-induced tumor cell death. In clinical specimens, both VEGFRs and VEGF were co-expressed in astroglial tumor cells, and higher VEGF expression correlated with tumor progression, thereby supporting the relevance of functional VEGF-VEGFR signaling in vivo. Overall, our results are consistent with a potential autocrine role of the VEGF-VEGFR2 (KDR) interplay as a factor contributing to malignant astrocytoma growth and radioresistance, thereby supporting the candidacy of this signaling cascade as a therapeutic target, possibly in combination with radiotherapy.     

A novel role of vascular endothelial cadherin in modulating c-Src activation and downstream signaling of vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is a potent mediator of angiogenesis and vascular permeability, in which c-Src tyrosine kinase plays an essential role. However, the mechanisms by which VEGF stimulates c-Src activation have remained unclear. Here, we demonstrate that vascular endothelial cadherin (VE-cadherin) plays a critical role in regulating c-Src activation in response to VEGF. In vascular endothelial cells, VE-cadherin was basally associated with c-Src and Csk (C-terminal Src kinase), a negative regulator of Src activation. VEGF stimulated Csk release from VE-cadherin by recruiting the protein tyrosine phosphatase SHP2 to VE-cadherin signaling complex, leading to an increase in c-Src activation. Silencing VE-cadherin with small interference RNA significantly reduced VEGF-stimulated c-Src activation. Disrupting the association of VE-cadherin and Csk through the reconstitution of Csk binding-defective mutant of VE-cadherin also diminished Src activation. Moreover, inhibiting SHP2 by small interference RNA and adenovirus-mediated expression of a catalytically inactive mutant of SHP2 attenuated c-Src activation by blocking the disassociation of Csk from VE-cadherin. Furthermore, VE-cadherin and SHP2 differentially regulates VEGF downstream signaling. The inhibition of c-Src, VE-cadherin, and SHP2 diminished VEGF-mediated activation of Akt and endothelial nitric-oxide synthase. In contrast, inhibiting VE-cadherin and SHP2 enhanced ERK1/2 activation in response to VEGF. These findings reveal a novel role for VE-cadherin in modulating c-Src activation in VEGF signaling, thus providing new insights into the importance of VE-cadherin in VEGF signaling and vascular function.     

The recombinant kringle domain of urokinase plasminogen activator inhibits VEGF165-induced angiogenesis of HUVECs by suppressing VEGFR2 dimerization and subsequent signal transduction

The recombinant kringle domain (UK1) of urokinase plasminogen activator was previously reported to exert antiangiogenic activity against Vascular Endothelial Growth Factor (VEGF)-induced angiogenesis in both in vitro and in vivo models. In this study, we explored the molecular signaling mechanisms involved in the antiangiogenic activity of UK1 by examining VEGF signaling proteins. VEGF165 stimulates the phosphorylation of VEGF signaling molecules, and pretreatment with UK1 blocked VEGF-induced signal transduction associated with proliferation, survival, and migration. UK1 also suppressed VEGF165-induced activation of MMP-2. Moreover, UK1 suppressed the phosphorylation and activation of VEGFR2 in VEGF-stimulated human umbilical cord vein endothelial cells (HUVECs) by blocking the dimerization of VEGFR2. Overall, our findings suggest that UK1 inhibits VEGF-induced proliferation, migration, and matrix metalloproteinase activity of HUVECs by suppressing VEGFR2 dimerization and subsequent angiogenic signals.     

AMPK activation inhibits the expression of HIF-1alpha induced by insulin and IGF-1

Insulin, insulin like growth factor (IGF)-1, and AMP-activated protein kinase (AMPK) signaling regulate independently angiogenesis through vascular endothelial growth factor (VEGF) expression. In the present study, we investigated a potential cross-talk between these signaling pathways on hypoxia-inducible factor (HIF)-1alpha and VEGF expression. Retinal epithelial ARPE-19 cells were treated with AICAR, an AMPK activator, alone or in combination with insulin and IGF-1. AICAR stimulated VEGF mRNA expression, but did not modify the insulin- and IGF-1-induced VEGF expression. We have investigated the effect of AICAR on insulin and IGF-1 signaling pathways. We observed that AICAR increased insulin- and IGF-1-induced phosphorylation of PKB, whereas phosphorylation of S6K-1 was decreased. Moreover, AICAR and metformin inhibited the ability of insulin and IGF-1 to induce HIF-1alpha expression. These results show that AICAR and insulin/IGF-1 regulate VEGF expression through different mechanisms.     

Signaling from across the way: transactivation of VEGF receptors by HSPGs

Heparan sulfate proteoglycans (HSPGs) influence the signaling of many growth factors, and recent work shows that they can promote VEGF signaling in trans from the surface of an adjacent cell. This mode of signaling also alters the activation state and internalization of VEGF receptors.     

Sphingosine kinase mediates vascular endothelial growth factor-induced activation of ras and mitogen-activated protein kinases

Vascular endothelial growth factor (VEGF) signaling is critical to the processes of angiogenesis and tumor growth. Here, evidence is presented for VEGF stimulation of sphingosine kinase (SPK) that affects not only endothelial cell signaling but also tumor cells expressing VEGF receptors. VEGF or phorbol 12-myristate 13-acetate treatment of the T24 bladder tumor cell line resulted in a time- and dose-dependent stimulation of SPK activity. In T24 cells, VEGF treatment reduced cellular sphingosine levels while raising that of sphingosine-1-phosphate. VEGF stimulation of T24 cells caused a slow and sustained accumulation of Ras-GTP and phosphorylated extracellular signal-regulated kinase (phospho-ERK) compared with that after EGF treatment. Small interfering RNA (siRNA) that targets SPK1, but not SPK2, blocks VEGF-induced accumulation of Ras-GTP and phospho-ERK in T24 cells. In contrast to EGF stimulation, VEGF stimulation of ERK1/2 phosphorylation was unaffected by dominant-negative Ras-N17. Raf kinase inhibition blocked both VEGF- and EGF-stimulated accumulation of phospho-ERK1/2. Inhibition of SPK by pharmacological inhibitors, a dominant-negative SPK mutant, or siRNA that targets SPK blocked VEGF, but not EGF, induction of phospho-ERK1/2. We conclude that VEGF induces DNA synthesis in a pathway which sequentially involves protein kinase C (PKC), SPK, Ras, Raf, and ERK1/2. These data highlight a novel mechanism by which SPK mediates signaling from PKC to Ras in a manner independent of Ras-guanine nucleotide exchange factor.     

Regulation of vascular endothelial growth factor signaling by miR-200b

Vascular endothelial growth factor (VEGF) signaling plays an important role in angiogenesis. In the VEGF signaling pathway, the key components are VEGF and its receptors, Flt-1 and KDR. In this study, we show that transfection of synthetic miR-200b reduced protein levels of VEGF, Flt-1, and KDR. In A549 cells, miR-200b targeted the predicted binding sites in the 3′-untranslated region (3′-UTR) of VEGF, Flt-1, and KDR as revealed by a luciferase reporter assay. When transfected with miR-200b, the ability of HUVECs to form a capillary tube on Matrigel and VEGF-induced phosphorylation of ERK1/2 were significantly reduced. Taken together, these results suggest that miR-200b negatively regulates VEGF signaling by targeting VEGF and its receptors and that miR-200b may have therapeutic potential as an angiogenesis inhibitor.     

Proteomic analysis of tyrosine phosphorylations in vascular endothelial growth factor- and reactive oxygen species-mediated signaling pathway

Vascular endothelial growth factor (VEGF) mediates angiogenic signaling by activating tyrosine kinase receptors. Endothelial cells treated with VEGF are known to increase reactive oxygen species (ROS) production and activate the MAPK pathway. To identify the target proteins of the VEGF receptor, we treated human umbilical vein endothelial cells (HUVECs) with VEGF or H2O2, and identified and semiquantified tyrosine-phosphorylated proteins, combining 2D-gel electrophoresis, Western analysis using antibody against phospho-tyrosine, and mass spectrometry. We detected 95 proteins that were differentially phosphorylated; some were specifically phosphorylated by VEGF but not by H2O2. 2D-gel electrophoresis revealed that heterogeneous populations of the same protein responded differently to H2O2 and VEGF. Bioinformatic studies examining the nature of the differential phosphorylation in various subpopulations of proteins should provide new insights into VEGF- and H2O2-induced signaling pathways.     

The role of VEGF and EGFR inhibition: implications for combining anti-VEGF and anti-EGFR agents

Multiple cellular pathways influence the growth and metastatic potential of tumors. This creates heterogeneity, redundancy, and the potential for tumors to bypass signaling pathway blockade, resulting in primary or acquired resistance. Combining therapies that inhibit different signaling pathways has the potential to be more effective than inhibition of a single pathway and to overcome tumor resistance. Vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR) inhibitors have become key therapies in several tumor types. Close relationships between these factors exist: VEGF signaling is up-regulated by EGFR expression and, conversely, VEGF up-regulation independent of EGFR signaling seems to contribute to resistance to EGFR inhibition. Therefore, inhibition of both pathways could improve antitumor efficacy and overcome resistance to EGFR inhibition. Preclinical studies have shown that VEGF and EGFR inhibitors can have additive effects and that combined inhibition is effective in EGFR inhibitor-resistant cell lines. Clinical trials have also produced promising data: combining the anti-VEGF monoclonal antibody bevacizumab with the anti-EGFR antibody cetuximab or the EGFR tyrosine kinase inhibitor erlotinib increases benefit compared with either of these anti-EGFR agents alone or combined with chemotherapy. The potential of this novel approach to anticancer therapy will be elucidated by large, ongoing clinical trials.     

VEGF 家族及其在肿瘤生长中作用的研究

血管内皮生长因子(Vascular Endothelial Growth Factor,VEGF)家族是一类多功能的细胞因子,在血管生成和淋巴管生成中具有直接和间接的调控作用,可促进内皮细胞增殖、促进血管生成以及增加血管的通透性。VEGF/VEGFR轴由多重配基和受体质量叠加交错组成,并且受体与配基结合具有专一性,在不同的细胞中具有不同的细胞类型表达和功能.启动VEGF信号通路,触发了一个网状的信号过程,从而促进血管内皮细胞生长、转移和存活。进来研究发现,VEGF的一个重要作用表现为可动员内皮祖细胞从骨髓向远处转移从而形成新生血管,因而有必要设计和发展针对这一途径的抑制因子。随着研究的深入,VEGF促进肿瘤血管生成的作用和与人类癌症的发病机制的关系是确定的,因此,抑制VEGF途径被确认为是一种重要的有效的抗癌模式     

Analysis of biological effects and signaling properties of Flt-1 (VEGFR-1) and KDR (VEGFR-2). A reassessment using novel receptor-specific vascular endothelial growth factor mutants

Endothelial cells express two related vascular endothelial growth factor (VEGF) receptor tyrosine kinases, KDR (kinase-insert domain containing receptor, or VEGFR-2) and Flt-1 (fms-like tyrosine kinase, or VEGFR-1). Although considerable experimental evidence links KDR activation to endothelial cell mitogenesis, there is still significant uncertainty concerning the role of individual VEGF receptors for other biological effects such as vascular permeability. VEGF mutants that bind to either KDR or Flt-1 with high selectivity were used to determine which of the two receptors serves to mediate different VEGF functions. In addition to mediating mitogenic signaling, selective KDR activation was sufficient for the activation of intracellular signaling pathways implicated in cell migration. KDR stimulation caused tyrosine phosphorylation of both phosphatidylinositol 3-kinase and phospholipase Cgamma in primary endothelial cells and stimulated cell migration. KDR-selective VEGF was also able to induce angiogenesis in the rat cornea to an extent indistinguishable from wild type VEGF. We also demonstrate that KDR, but not Flt-1, stimulation is responsible for the induction of vascular permeability by VEGF.     

An autocrine VEGF/VEGFR2 and p38 signaling loop confers resistance to 4-hydroxytamoxifen in MCF-7 breast cancer cells

Tamoxifen, a partial estrogen receptor antagonist, is part of the standard treatment of both primary and advanced breast cancers. However, significant proportions of breast cancers are either de novo resistant or develop tamoxifen resistance during the course of treatment through mechanisms which have been only partly characterized. We have previously found that high vascular endothelial growth factor (VEGF) or VEGF receptor 2 (VEGFR2) expression and concomitant high p38 mitogen-activated protein kinase activity within breast cancers predict a poor outcome for tamoxifen-treated patients. Here, we have molecularly dissected how VEGF/VEGFR2 and p38 are linked, and contribute to tamoxifen resistance within breast cancer using a MCF-7 BC cell model with different 4-hydroxytamoxifen (4-OHT) responsiveness. We report that MCF-7 breast cancer cell lines with tamoxifen resistance have increased secretion of VEGF and increased signaling through VEGFR2 compared with parental MCF-7 cells. 4-OHT treatment caused the ablation of VEGF secretion in parental MCF-7 cells, whereas in the tamoxifen-resistant subline, a VEGF/VEGFR2 signaling loop was still evident upon treatment. Increased basal levels of total and phosphorylated p38 were observed in tamoxifen-resistant cells. Pharmacologic inhibition of p38 reduced the proliferation of both tamoxifen-responsive and tamoxifen-resistant cells and showed an additive growth-inhibitory effect in combination with 4-OHT. A connection between VEGF/VEGFR2 and p38 signaling was identified by VEGF and VEGFR2 knockdown, which equally reduced both the total and the active forms of p38 in tamoxifen-resistant cells. Taken together, our results suggest that decreased sensitivity to 4-OHT is caused by a death-protecting VEGF/VEGFR2 and p38 growth factor loop in breast cancer cells. Inhibition of these signaling pathways may be beneficial to overcome tamoxifen resistance.