The angiogenic factor secretoneurin induces coronary angiogenesis in a model of myocardial infarction by stimulation of vascular endothelial growth factor signaling in endothelial cells.
ABSTRACT: BACKGROUND:Secretoneurin is a neuropeptide located in nerve fibers along blood vessels, is upregulated by hypoxia, and induces angiogenesis. We tested the hypothesis that secretoneurin gene therapy exerts beneficial effects in a rat model of myocardial infarction and evaluated the mechanism of action on coronary endothelial cells. METHODS AND RESULTS:In vivo secretoneurin improved left ventricular function, inhibited remodeling, and reduced scar formation. In the infarct border zone, secretoneurin induced coronary angiogenesis, as shown by increased density of capillaries and arteries. In vitro secretoneurin induced capillary tubes, stimulated proliferation, inhibited apoptosis, and activated Akt and extracellular signal-regulated kinase in coronary endothelial cells. Effects were abrogated by a vascular endothelial growth factor (VEGF) antibody, and secretoneurin stimulated VEGF receptors in these cells. Secretoneurin furthermore increased binding of VEGF to endothelial cells, and binding was blocked by heparinase, indicating that secretoneurin stimulates binding of VEGF to heparan sulfate proteoglycan binding sites. Additionally, secretoneurin increased binding of VEGF to its coreceptor neuropilin-1. In endothelial cells, secretoneurin also stimulated fibroblast growth factor receptor-3 and insulin-like growth factor-1 receptor, and in coronary vascular smooth muscle cells, we observed stimulation of VEGF receptor-1 and fibroblast growth factor receptor-3. Exposure of cardiac myocytes to hypoxia and ischemic heart after myocardial infarction revealed increased secretoneurin messenger RNA and protein. CONCLUSIONS:Our data show that secretoneurin acts as an endogenous stimulator of VEGF signaling in coronary endothelial cells by enhancing binding of VEGF to low-affinity binding sites and neuropilin-1 and stimulates further growth factor receptors like fibroblast growth factor receptor-3. Our in vivo findings indicate that secretoneurin may be a promising therapeutic tool in ischemic heart disease.
Project description:Alternative splicing leads to the secretion of multiple forms of vascular endothelial growth factor-A (VEGF-A) that differ in their activity profiles with respect to neovascularization. FSAP (factor VII activating protease) is the zymogen form of a plasma protease that is activated (FSAPa) upon tissue injury via the release of histones. The purpose of the study was to determine if FSAPa regulates VEGF-A activity in vitro and in vivo. FSAP bound to VEGF165, but not VEGF121, and VEGF165 was cleaved in its neuropilin/proteoglycan binding domain. VEGF165 cleavage did not alter its binding to VEGF receptors but diminished its binding to neuropilin. The stimulatory effects of VEGF165 on endothelial cell proliferation, migration, and signal transduction were not altered by FSAP. Similarly, proliferation of VEGF receptor-expressing BAF3 cells, in response to VEGF165, was not modulated by FSAP. In the mouse matrigel model of angiogenesis, FSAP decreased the ability of VEGF165, basic fibroblast growth factor (bFGF), and their combination, to induce neovascularization. Lack of endogenous FSAP in mice did not influence neovascularization. Thus, FSAP inhibited VEGF165-mediated angiogenesis in the matrigel model in vivo, where VEGF's interaction with the matrix and its diffusion are important.
Project description: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.
Project description:Neuropilin (NRP) 1, previously identified as a neuronal receptor that mediates repulsive growth cone guidance, has been shown recently to function also in endothelial cells as an isoform-specific receptor for vascular endothelial growth factor (VEGF)(165) and as a coreceptor in vitro of VEGF receptor 2. However, its potential role in pathologic angiogenesis remains unknown. In the present study, we first show that VEGF selectively up-regulates NRP1 but not NRP2 via the VEGF receptor 2-dependent pathway. By NRP1 binding analysis, we showed that its induction by VEGF accompanies functional receptor expression. Endothelial proliferation stimulated by VEGF(165) was inhibited significantly by antibody perturbation of NRP1. In a murine model of VEGF-dependent angioproliferative retinopathy, intense NRP1 mRNA expression was observed in the newly formed vessels. Furthermore, selective NRP1 inhibition in this model suppressed neovascular formation substantially. These results suggest that VEGF cannot only activate endothelial cells directly but also can contribute to robust angiogenesis in vivo by a mechanism that involves up-regulation of its cognate receptor expression.
Project description:Neuropilin-2 is a transmembrane receptor involved in lymphangiogenesis and neuronal development. In adults, neuropilin-2 and its homologous protein neuropilin-1 have been implicated in cancers and infection. Molecular determinants of the ligand selectivity of neuropilins are poorly understood. We have identified and structurally characterized a zinc ion binding site on human neuropilin-2. The neuropilin-2-specific zinc ion binding site is located near the interface between domains b1 and b2 in the ectopic region of the protein, remote from the neuropilin binding site for its physiological ligand, i.e. vascular endothelial growth factor. We also present an X-ray crystal structure of the neuropilin-2 b1 domain in a complex with the C-terminal sub-domain of VEGF-A. Zn(2+) binding to neuropilin-2 destabilizes the protein structure but this effect was counteracted by heparin, suggesting that modifications by glycans and zinc in the extracellular matrix may affect functional neuropilin-2 ligand binding and signalling activity.
Project description:Using human MSCs (mesenchymal stem cells) lacking VEGF (vascular endothelial growth factor) receptors, we show that the pro-angiogenic receptor neuropilin-1 associates with phosphorylated PDGFRs [PDGF (platelet-derived growth factor) receptors], thereby regulating cell signalling, migration, proliferation and network assembly. Neuropilin-1 co-immunoprecipitated and co-localized with phosphorylated PDGFRs in the presence of growth factors. Neuropilin-1 knockdown blocked PDGF-AA-induced PDGFRalpha phosphorylation and migration, reduced PDGF-BB-induced PDGFRbeta activation and migration, blocked VEGF-A activation of both PDGFRs, and attenuated proliferation. Neuropilin-1 prominently co-localized with both PDGFRs within MSC networks assembled in Matrigel and in the chorioallantoic membrane vasculature microenvironment, and its knockdown grossly disrupted network assembly and decreased PDGFR signalling. Thus neuropilin-1 regulates MSCs by forming ligand-specific receptor complexes that direct PDGFR signalling, especially the PDGFRalpha homodimer. This receptor cross-talk may control the mobilization of MSCs in neovascularization and tissue remodelling.
Project description:Vascular endothelial growth factor (VEGF) is critical for physiological and pathological angiogenesis. Within the tumor microenvironment, VEGF functions as an endothelial cell survival factor, permeability factor, mitogen, and chemotactic agent. The majority of these functions are mediated by VEGF-induced activation of VEGF receptor 2 (VEGFR2), a high affinity receptor tyrosine kinase expressed by endothelial cells and other cell types in the tumor microenvironment. VEGF can also ligate other cell surface receptors including VEGFR1 and neuropilin-1 and -2. However, the importance of VEGF-induced activation of these receptors in tumorigenesis is still unclear. We report the development and characterization of r84, a fully human monoclonal antibody that binds human and mouse VEGF and selectively blocks VEGF from interacting with VEGFR2 but does not interfere with VEGF:VEGFR1 interaction. Selective blockade of VEGF binding to VEGFR2 by r84 is shown through ELISA, receptor binding assays, receptor activation assays, and cell-based functional assays. Furthermore, we show that r84 has potent anti-tumor activity and does not alter tissue histology or blood and urine chemistry after chronic high dose therapy in mice. In addition, chronic r84 therapy does not induce elevated blood pressure levels in some models. The ability of r84 to specifically block VEGF:VEGFR2 binding provides a valuable tool for the characterization of VEGF receptor pathway activation during tumor progression and highlights the utility and safety of selective blockade of VEGF-induced VEGFR2 signaling in tumors.
Project description:Neuropilin 1 (NRP1) is a receptor for class 3 semaphorins and vascular endothelial growth factor (VEGF) A and is essential for cardiovascular development. Biochemical evidence supports a model for NRP1 function in which VEGF binding induces complex formation between NRP1 and VEGFR2 to enhance endothelial VEGF signalling. However, the relevance of VEGF binding to NRP1 for angiogenesis in vivo has not yet been examined. We therefore generated knock-in mice expressing Nrp1 with a mutation of tyrosine (Y) 297 in the VEGF binding pocket of the NRP1 b1 domain, as this residue was previously shown to be important for high affinity VEGF binding and NRP1-VEGFR2 complex formation. Unexpectedly, this targeting strategy also severely reduced NRP1 expression and therefore generated a NRP1 hypomorph. Despite the loss of VEGF binding and attenuated NRP1 expression, homozygous Nrp1(Y297A/Y297A) mice were born at normal Mendelian ratios, arguing against NRP1 functioning exclusively as a VEGF164 receptor in embryonic angiogenesis. By overcoming the mid-gestation lethality of full Nrp1-null mice, homozygous Nrp1(Y297A/Y297A) mice revealed essential roles for NRP1 in postnatal angiogenesis and arteriogenesis in the heart and retina, pathological neovascularisation of the retina and angiogenesis-dependent tumour growth.
Project description:Neuropilin is an essential cell surface receptor that functions in both semaphorin-dependent axon guidance and vascular endothelial growth factor (VEGF)-dependent angiogenesis. The interplay between these two seemingly distinct pathways is a source of considerable interest. Indeed, several semaphorin family members have been shown to have potent anti-angiogenic activity in vivo. However, reports about whether semaphorin and VEGF competitively bind to neuropilin conflict. Previous work has demonstrated that all known ligands and inhibitors of neuropilin interact with the b1 domain of neuropilin via a C-terminal arginine. No semaphorin family member possesses a C-terminal arginine, leading to uncertainty with regard to the physical mechanism of interaction between the C-terminal domain of semaphorin and the b1 domain of neuropilin. Semaphorin 3F (Sema3F) possesses an RXRR furin recognition site in its C-terminus, and we demonstrate that it is proteolytically processed. This processing is found to be essential for the interaction of the C-terminus of Sema3F with the b1 domain of neuropilin. We further demonstrate that furin activation of the C-terminus of Sema3F produces a species that potently inhibits the binding of VEGF to neuropilin. These studies provide a mechanistic basis for understanding the anti-angiogenic activity of semaphorin as well as the physical interaction and competition between neuropilin ligands.
Project description:Neuropilin-1 is a type 1 membrane protein with three distinct functions. First, it can mediate cell adhesion via a heterophilic molecular interaction. Second, in neuronal cells, neuropilin-1 binds the class 3 semaphorins, which are neuronal chemorepellents, and plays a role in the directional guidance of axons. Neuropilin-1 is expected to form complexes with the plexinA subfamily members and mediate the semaphorin-elicited inhibitory signals into neurons. Third, in endothelial cells, neuropilin-1 binds a potent endothelial cell mitogen, vascular endothelial growth factor (VEGF)(165), and regulates vessel formation. Though the binding sites in neuropilin-1 for the class 3 semaphorins and VEGF(165) have been analyzed, the sites involved in cell adhesion activity of the molecule have not been identified. In this study, we produced a variety of mutant neuropilin-1s and tested their cell adhesion activity. We showed that the b1 and b2 domains within the extracellular segment of neuropilin-1 were required for the cell adhesion activity, and peptides with an 18-amino acid stretch in the b1 and b2 domains were sufficient to induce the cell adhesion activity. In addition, we demonstrated that the cell adhesion ligands for neuropilin-1 were proteins and distributed in embryonic mesenchymal cells but distinct from the class 3 semaphorins, VEGF, or plexins.
Project description:Matrix-binding isoforms and non-matrix-binding isoforms of vascular endothelial growth factor (VEGF) are both capable of stimulating vascular remodeling, but the resulting blood vessel networks are structurally and functionally different. Here, we develop and validate a computational model of the binding of soluble and immobilized ligands to VEGF receptor 2 (VEGFR2), the endosomal trafficking of VEGFR2, and site-specific VEGFR2 tyrosine phosphorylation to study differences in induced signaling between these VEGF isoforms. In capturing essential features of VEGFR2 signaling and trafficking, our model suggests that VEGFR2 trafficking parameters are largely consistent across multiple endothelial cell lines. Simulations demonstrate distinct localization of VEGFR2 phosphorylated on Y1175 and Y1214. This is the first model to clearly show that differences in site-specific VEGFR2 activation when stimulated with immobilized VEGF compared to soluble VEGF can be accounted for by altered trafficking of VEGFR2 without an intrinsic difference in receptor activation. The model predicts that Neuropilin-1 can induce differences in the surface-to-internal distribution of VEGFR2. Simulations also show that ligated VEGFR2 and phosphorylated VEGFR2 levels diverge over time following stimulation. Using this model, we identify multiple key levers that alter how VEGF binding to VEGFR2 results in different coordinated patterns of multiple downstream signaling pathways. Specifically, simulations predict that VEGF immobilization, interactions with Neuropilin-1, perturbations of VEGFR2 trafficking, and changes in expression or activity of phosphatases acting on VEGFR2 all affect the magnitude, duration, and relative strength of VEGFR2 phosphorylation on tyrosines 1175 and 1214, and they do so predictably within our single consistent model framework.