No abstract

Vascular endothelial growth factor A (VEGF) drives endothelial cell maintenance and angiogenesis. Endothelial cell behavior is altered by the stiffness of the substrate the cells are attached to suggesting that VEGF activity might be influenced by the mechanical cellular environment. We hypothesized that extracellular matrix (ECM) stiffness modifies VEGF-cell-matrix tethering leading to altered VEGF processing and signaling. We analyzed VEGF binding, internalization, and signaling as a function of substrate stiffness in endothelial cells cultured on fibronectin (Fn) linked polyacrylamide gels. Cell produced extracellular matrices on the softest substrates were least capable of binding VEGF, but the cells exhibited enhanced VEGF internalization and signaling compared to cells on all other substrates. Inhibiting VEGF-matrix binding with sucrose octasulfate decreased cell-internalization of VEGF and, inversely, heparin pre-treatment to enhance Fn-matrix binding of VEGF increased cell-internalization of VEGF regardless of matrix stiffness. β1 integrins, which connect cells to Fn, modulated VEGF uptake in a stiffness dependent fashion. Cells on hard surfaces showed decreased levels of activated β1 and inhibition of β1 integrin resulted in a greater proportional decrease in VEGF internalization than in cells on softer matrices. Extracellular matrix binding is necessary for VEGF internalization. Stiffness modifies the coordinated actions of VEGF-matrix binding and β1 integrin binding/activation, which together are critical for VEGF internalization. This study provides insight into how the microenvironment may influence tissue regeneration and response to injury and disease. J. Cell. Physiol. 231: 2026-2039, 2016. © 2016 Wiley Periodicals, Inc.

Angiogenesis is a highly regulated process orchestrated, in large part, by the vascular endothelial growth factor-A (VEGF-A) system of ligands and receptors. Considerable effort has been invested in finding optimal ways to modulate VEGF-A activity to treat disease, however, the mechanisms by which the various components interact remain poorly understood. This is in part because of the difficulty of analyzing the various interactions in an intercomparable manner. In the present study, we established conditions to allow for the detailed characterization of the molecular interactions between VEGF and its receptors and the co-receptor NRP-1 using surface plasmon resonance (SPR). We found that VEGF dissociated 25-times faster from its major signaling receptor, VEGF receptor-2 (VEGFR-2) than from its “decoy” receptor, VEGF receptor-1 (VEGFR-1). Using a systematic approach, we obtained kinetic parameters for each individual interaction under a consistent set of experimental conditions allowing for comparison between various receptors. The set of quantitative kinetic parameters and experimental conditions reported herein will provide valuable tools for developing comprehensive models of the VEGF system.

Angiogenesis is a highly regulated process orchestrated by the complex vascular endothelial growth factor (VEGF) system, which is comprised of multiple isoforms and cell surface receptors. In addition, heparin/heparan sulfate (HS) proteoglycans and neuropilins (NRP) have been described as co‐receptors. In the present study we have characterized molecular interactions between the two most commonly expressed VEGF isoforms and their receptors and co‐receptors, using surface plasmon resonance (SPR). Our data suggest that heparin/HS plays different roles in the regulation of VEGF‐VEGFR complex formation depending on the receptor type. VEGFR2 is able to interact with VEGF165 bound by heparin, but interacts very weakly with heparin. VEGFR1 and NRP1 on the contrary, bind heparin independently of VEGF165. It appears heparin/HS play selective roles within each complex and could be key players in determining VEGF cellular response. NRP1 on the other hand, can interact directly with VEGFR1 and 2, alone or as a complex with VEGF165. Our results are also correlated to MAPK pathway activation in response to VEGF in endothelial cells. We propose that HS/heparin and NRP1 dictate the specific receptor type activated by VEGF and ultimately determine the biological response of the VEGF system. Supported by HL088572 and AHAF M2012014.