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.     

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.     

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.     

Walking the walk: migration and other common themes in blood and vascular development

In this issue of Cell, a study by N. Cho and coworkers provides insight into the role of vascular endothelial growth factor (VEGF) signaling in Drosophila hematopoiesis. Their work suggests that an ancestral function of VEGF was to guide blood cell migration and highlights the conservation of at least one aspect of VEGF signaling during evolution.     

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.     

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.     

Targeting neuropilin-1 to inhibit VEGF signaling in cancer: Comparison of therapeutic approaches

Angiogenesis (neovascularization) plays a crucial role in a variety of physiological and pathological conditions including cancer, cardiovascular disease, and wound healing. Vascular endothelial growth factor (VEGF) is a critical regulator of angiogenesis. Multiple VEGF receptors are expressed on endothelial cells, including signaling receptor tyrosine kinases (VEGFR1 and VEGFR2) and the nonsignaling co-receptor Neuropilin-1. Neuropilin-1 binds only the isoform of VEGF responsible for pathological angiogenesis (VEGF₁₆₅), and is thus a potential target for inhibiting VEGF signaling. Using the first molecularly detailed computational model of VEGF and its receptors, we have shown previously that the VEGFR–Neuropilin interactions explain the observed differential effects of VEGF isoforms on VEGF signaling in vitro, and demonstrated potent VEGF inhibition by an antibody to Neuropilin-1 that does not block ligand binding but blocks subsequent receptor coupling. In the present study, we extend that computational model to simulation of in vivo VEGF transport and binding, and predict the in vivo efficacy of several Neuropilin-targeted therapies in inhibiting VEGF signaling: (a) blocking Neuropilin-1 expression; (b) blocking VEGF binding to Neuropilin-1; (c) blocking Neuropilin–VEGFR coupling. The model predicts that blockade of Neuropilin–VEGFR coupling is significantly more effective than other approaches in decreasing VEGF–VEGFR2 signaling. In addition, tumor types with different receptor expression levels respond differently to each of these treatments. In designing human therapeutics, the mechanism of attacking the target plays a significant role in the outcome: of the strategies tested here, drugs with similar properties to the Neuropilin-1 antibody are predicted to be most effective. The tumor type and the microenvironment of the target tissue are also significant in determining therapeutic efficacy of each of the treatments studied.     

TGF-beta1 induces rearrangement of FLK-1-VE-cadherin-beta-catenin complex at the adherens junction through VEGF-mediated signaling

VEGF and TGF-beta1 induce angiogenesis but have opposing effects on vascular endothelial cells: VEGF promotes survival; TGF-beta1 induces apoptosis. We have previously shown that TGF-beta1 induces endothelial cell apoptosis via up-regulation of VEGF expression and activation of signaling through VEGF receptor-2 (flk-1). In context with TGF-beta1, VEGF signaling is transiently converted from a survival into an apoptotic one. VEGF promotes cell survival in part via activation of PI3K/Akt by a mechanism dependent on the formation of a multi-protein complex that includes flk-1 and the adherens junction proteins VE-cadherin and beta-catenin. Here we report that TGF-beta1 induces rearrangement of the adherens junction complex by separating flk-1 from VE-cadherin and increasing beta-catenin association with both flk-1 and VE-cadherin. This rearrangement is caused neither by changes in adherens junction mRNA or protein expression nor by post-translational modification, and requires VEGF signaling through flk-1. These results show that the adherens junction is an important regulatory component of TGF-beta1-VEGF interaction in endothelial cells.     

VEGF receptor signaling in vertebrate development

The secreted glycoprotein vascular endothelial growth factor A (VEGF or VEGFA) affects many different cell types and modifies a wide spectrum of cellular behaviors in tissue culture models, including proliferation, migration, differentiation and survival. The versatility of VEGF signaling is reflected in the complex composition of its cell surface receptors and their ability to activate a variety of different downstream signaling molecules. A major challenge for VEGF research is to determine which of the specific signaling pathways identified in vitro control development and homeostasis of tissues containing VEGF-responsive cell types in vivo.     

Phosphorylation of Dab2 is involved in inhibited VEGF‐VEGFR‐2 signaling induced by downregulation of syndecan‐1 in glomerular endothelial cell

Disabled‐2 (Dab2) and PAR‐3 (partitioning defective 3) are reported to play critical roles in maintaining retinal microvascular endothelial cells biology by regulating VEGF‐VEGFR‐2 signaling. The role of Dab2 and PAR‐3 in glomerular endothelial cell (GEnC) is unclear. In this study, we found that, no matter whether with vascular endothelial growth factor (VEGF) treatment or not, decreased expression of Dab2 could lead to cell apoptosis by preventing activation of VEGF‐VEGFR‐2 signaling in GEnC, accompanied by reduced membrane VEGFR‐2 expression. And silencing of PAR‐3 gene expression caused increased apoptosis of GEnC by inhibiting activation of VEGF‐VEGFR‐2 signaling and membrane VEGFR‐2 expression. In our previous research, we found that the silencing of syndecan‐1 gene expression inhibited VEGF‐VEGFR‐2 signaling by modulating internalization of VEGFR‐2. And our further research demonstrated that downregulation of syndecan‐1 lead to no significant change in the expression of Dab2 and PAR‐3 both at messenger RNA and protein levels in GEnC, while phosphorylation of Dab2 was significantly increased in GEnC transfected with Dab2 small interfering RNA (siRNA) compared with control siRNA. Atypical protein kinase C (aPKC) could induce phosphorylation of Dab2, thus negatively regulating VEGF‐VEGFR‐2 signaling. And we found that decreased expression of syndecan‐1 lead to activation of aPKC, and aPKC inhibitor treatment could block phosphorylation of Dab2 in GEnC. Besides, aPKC inhibitor treatment could activate VEGF‐VGEFR‐2 signaling in GEnC transfected with syndecan‐1 siRNA in a dose‐dependent manner. In conclusion, we speculated that phosphorylation of Dab2 is involved in preventing activation of VEGF‐VEGFR‐2 signaling in GEnC transfected with syndecan‐1 siRNA. This provides a new target for the therapy of GEnC injury and kidney disease.     

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.     

EphrinB2/EphB4 signaling regulates non‐sprouting angiogenesis by VEGF

Vascular endothelial growth factor (VEGF) is the master regulator of angiogenesis, whose best‐understood mechanism is sprouting. However, therapeutic VEGF delivery to ischemic muscle induces angiogenesis by the alternative process of intussusception, or vascular splitting, whose molecular regulation is essentially unknown. Here, we identify ephrinB2/EphB4 signaling as a key regulator of intussusceptive angiogenesis and its outcome under therapeutically relevant conditions. EphB4 signaling fine‐tunes the degree of endothelial proliferation induced by specific VEGF doses during the initial stage of circumferential enlargement of vessels, thereby limiting their size and subsequently enabling successful splitting into normal capillary networks. Mechanistically, EphB4 neither inhibits VEGF‐R2 activation by VEGF nor its internalization, but it modulates VEGF‐R2 downstream signaling through phospho‐ERK1/2. In vivo inhibitor experiments show that ERK1/2 activity is required for EphB4 regulation of VEGF‐induced intussusceptive angiogenesis. Lastly, after clinically relevant VEGF gene delivery with adenoviral vectors, pharmacological stimulation of EphB4 normalizes dysfunctional vascular growth in both normoxic and ischemic muscle. These results identify EphB4 as a druggable target to modulate the outcome of VEGF gene delivery and support further investigation of its therapeutic potential.     

VEGF signaling has distinct spatiotemporal roles during heart valve development

Heart valve malformations are one of the most common types of birth defects, illustrating the complex nature of valve development. Vascular endothelial growth factor (VEGF) signaling is one pathway implicated in valve formation, however its specific spatial and temporal roles remain poorly defined. To decipher these contributions, we use two inducible dominant negative approaches in mice to disrupt VEGF signaling at different stages of embryogenesis. At an early step in valve development, VEGF signals are required for the full transformation of endocardial cells to mesenchymal cells (EMT) at the outflow tract (OFT) but not atrioventricular canal (AVC) endocardial cushions. This role likely involves signaling mediated by VEGF receptor 1 (VEGFR1), which is highly expressed in early cushion endocardium before becoming downregulated after EMT. In contrast, VEGFR2 does not exhibit robust cushion endocardium expression until after EMT is complete. At this point, VEGF signaling acts through VEGFR2 to direct the morphogenesis of the AVC cushions into mature, elongated valve leaflets. This latter role of VEGF requires the VEGF-modulating microRNA, miR-126. Thus, VEGF roles in the developing valves are dynamic, transitioning from a differentiation role directed by VEGFR1 in the OFT to a morphogenetic role through VEGFR2 primarily in the AVC-derived valves.     

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.     

VEGF receptor signaling in vertebrate development

The secreted glycoprotein vascular endothelial growth factor A (VEGF or VEGFA) affects many different cell types and modifies a wide spectrum of cellular behaviors in tissue culture models, including proliferation, migration, differentiation and survival. The versatility of VEGF signaling is reflected in the complex composition of its cell surface receptors and their ability to activate a variety of different downstream signaling molecules. A major challenge for VEGF research is to determine which of the specific signaling pathways identified in vitro control development and homeostasis of tissues containing VEGF-responsive cell types in vivo.

Note: Previously published in VEGF in Development, edited by Christiana Ruhrberg. Landes Bioscience and Springer Science+Business Media 2008; pp. 14-29.     

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.