Extracellular Matrix Stiffness Controls VEGF Signaling and Processing in Endothelial Cells

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.

Matrix stiffness enhances VEGFR-2 internalization, signaling, and proliferation in endothelial cells

“…However, others have observed decreased VEGFR-2 internalization in bovine aortic endothelial cells cultured on compliant substrates [37]. Strikingly, this study observed reduced β1 integrin activation with increasing matrix stiffness, whereas many others report heightened integrin activation and signaling as a function of ECM stiffness [10,14,42,64], which could contribute to the differences in results.…”

“…While some have begun to delineate the pathways of these chemical and mechanical cues and identify crosstalk mechanisms [30–37], most are not yet fully understood. Here, the combined effects of matrix rigidity and VEGF stimulation on endothelial cell behavior were investigated.…”

Cell autonomous ANTXR1-mediated regulation of extracellular matrix components in primary fibroblasts

“…Therefore, elevated Hif1-α levels may contribute to the increased VEGF expression, resulting in up-regulation of ECM components in Antxr1 deficient fibroblasts. Such regulatory effects of VEGF on collagen production are consistent with previous reports demonstrating a striking inductive effect of VEGF on collagen expression in cultured fibroblasts and in skin fibrosis [19–22]. It was also shown that VEGF induces fibronectin secretion in human airway smooth muscle cells in an ERK1/2 dependent manner [32].…”

“…High levels of VEGF were shown to aggravate fibrosis in experimental animal models and stimulate collagen production by cultured fibroblasts isolated from the skin in patients with systemic sclerosis [19–22]. To ascertain potential signaling pathways involved in cell autonomous mechanisms of Antrx1 -dependent regulation of ECM we asked whether levels of VEGF in fibroblasts are affected by loss of ANTXR1.…”

“…ERK1/2 activity, a major downstream target of VEGF-dependent signaling is activated in skin of Antrx1 knockout mice [5]. Both CTGF and VEGF are involved in regulation of ECM production and a shift in the balance between CTGF and VEGF is associated with a switch from angiogenesis to fibrosis [19–22, 33]. It has also been reported that overexpression of CTGF has an inhibitory effect on VEGF levels resulting in severe fibrotic scaring and reduced VEGF-dependent angiogenesis [34].…”