Abstract
The splice isoforms of vascular endothelial growth A (VEGF) each have different affinities for the extracellular matrix (ECM) and the coreceptor NRP1, which leads to distinct vascular phenotypes in model systems expressing only a single VEGF isoform. ECM-immobilized VEGF can bind to and activate VEGF receptor 2 (VEGFR2) directly, with a different pattern of site-specific phosphorylation than diffusible VEGF. To date, the way in which ECM binding alters the distribution of isoforms of VEGF and of the related placental growth factor (PlGF) in the body and resulting angiogenic signaling is not well-understood. Here, we extend our previous validated cell-level computational model of VEGFR2 ligation, intracellular trafficking, and site-specific phosphorylation, which captured differences in signaling by soluble and immobilized VEGF, to a multi-scale whole-body framework. This computational systems pharmacology model captures the ability of the ECM to regulate isoform-specific growth factor distribution distinctly for VEGF and PlGF, and to buffer free VEGF and PlGF levels in tissue. We show that binding of immobilized growth factor to VEGF receptors, both on endothelial cells and soluble VEGFR1, is likely important to signaling in vivo. Additionally, our model predicts that VEGF isoform-specific properties lead to distinct profiles of VEGFR1 and VEGFR2 binding and VEGFR2 site-specific phosphorylation in vivo, mediated by Neuropilin-1. These predicted signaling changes mirror those observed in murine systems expressing single VEGF isoforms. Simulations predict that, contrary to the ‘ligand-shifting hypothesis,’ VEGF and PlGF do not compete for receptor binding at physiological concentrations, though PlGF is predicted to slightly increase VEGFR2 phosphorylation when over-expressed by 10-fold. These results are critical to design of appropriate therapeutic strategies to control VEGF availability and signaling in regenerative medicine applications.
Highlights
Angiogenesis, the growth of new capillaries from the existing vasculature, is critical for maintenance of health and response to injury, as well as being a component of many diseases
While phosphorylation of tyrosine Y1175, which leads to ERK1/2 activation and cell proliferation, is similar whether vascular endothelial growth factor (VEGF) is immobilized or presented in solution, phosphorylation of Y1214, which leads to phosphorylation of p38 and cell migration, increases when VEGF is immobilized [18, 19]. This shift in signaling, which parallels the phenotypes seen with single VEGF isoform expression, can be explained by reduced internalization of VEGF-receptor 2 (VEGFR2) bound to immobilized VEGF, altering the exposure of VEGFR2 to specific phosphatases, as we recently demonstrated via a computational model of VEGFR2 signaling in vitro [20]
As VEGF-receptor 1 (VEGFR1) can bind to NRP1 without ligand, and the NRP1- and heparin-binding domains of VEGFR1 overlap, we examine the impact of allowing matrix-bound soluble VEGFR1 (sR1) to bind VEGF121 and PlGF1 in the interstitial space, allowing these non-matrix-binding ligands to be sequestered
Summary
Angiogenesis, the growth of new capillaries from the existing vasculature, is critical for maintenance of health and response to injury, as well as being a component of many diseases. Regulation of angiogenesis is highly complex [1], and not fully understood. This complexity is a key reason for the lack of approved, effective therapies to promote angiogenesis for tissue engineering applications [2,3,4], for wound healing [5], or for ischemic diseases such as peripheral artery disease (PAD) [6], despite much research and multiple clinical trials [7]. VEGF-A (hereafter referred to as VEGF), considered the primary pro-angiogenesis VEGF ligand, has multiple splice isoforms, the most prevalent in humans being VEGF121, VEGF165, and VEGF189. Constitutive dimers of these splice isoforms bind to VEGF-receptor 1 (VEGFR1) and VEGF-receptor 2 (VEGFR2). VEGF receptors dimerize, transphosphorylate, and initiate downstream signaling [8,9,10]
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