Extracellular regulation of VEGF: Isoforms, proteolysis, and vascular patterning

Abstract

The regulation of vascular endothelial growth factor A (VEGF) is critical to neovascularization in numerous tissues under physiological and pathological conditions. VEGF has multiple isoforms, created by alternative splicing or proteolytic cleavage, and characterized by different receptor-binding and matrix-binding properties. These isoforms are known to give rise to a spectrum of angiogenesis patterns marked by differences in branching, which has functional implications for tissues. In this review, we detail the extensive extracellular regulation of VEGF and the ability of VEGF to dictate the vascular phenotype. We explore the role of VEGF-releasing proteases and soluble carrier molecules on VEGF activity. While proteases such as MMP9 can ‘release’ matrix-bound VEGF and promote angiogenesis, for example as a key step in carcinogenesis, proteases can also suppress VEGF's angiogenic effects. We explore what dictates pro- or anti-angiogenic behavior. We also seek to understand the phenomenon of VEGF gradient formation. Strong VEGF gradients are thought to be due to decreased rates of diffusion from reversible matrix binding, however theoretical studies show that this scenario cannot give rise to lasting VEGF gradients in vivo. We propose that gradients are formed through degradation of sequestered VEGF. Finally, we review how different aspects of the VEGF signal, such as its concentration, gradient, matrix-binding, and NRP1-binding can differentially affect angiogenesis. We explore how this allows VEGF to regulate the formation of vascular networks across a spectrum of high to low branching densities, and from normal to pathological angiogenesis. A better understanding of the control of angiogenesis is necessary to improve upon limitations of current angiogenic therapies.

Introduction

VEGF-A is a key member of the VEGF family of cytokines, along with VEGF-B, -C, -D, and PlGF [1], [2]. VEGF-A mediates angiogenesis, the expansion of an existing vascular bed by sprouting of new blood vessels [3]. Angiogenesis typically occurs as a response to a stimulus such as tissue hypoxia, and results in improved perfusion and increased oxygen delivery. Other stimuli can induce angiogenesis, including shear stress [4] and genetic transformation in tumor cells [3]. Angiogenesis is important for organ development [5] as well as for physiological processes including wound closure and exercise training [6], [7]. It is upregulated but disorganized in pathological processes such as diabetic retinopathy and solid organ tumorigenesis [8], [9], [10], where vasculature is needed to supply the tumor's rapid consumption of glucose and oxygen beyond the limits of diffusion.

The vegfa gene is translated into a number of splice isoforms, the most notable in humans being VEGF₁₂₁, VEGF₁₆₅, and VEGF₁₈₉ (Fig. 1). These isoforms have differences in biochemical properties such as their affinities for VEGF receptors and heparan sulfate proteoglycans (HSPGs), resulting in strikingly different effects on vessel growth. A major focus of the current review is the extracellular regulation of VEGF (Sections 3 Local availability and activity of VEGF: ECM, proteases and inhibitors, 4 ). In normal healthy situations, VEGF isoforms are differentially sequestered by heparan sulfate proteoglycans (HSPGs) in the ECM (Section 3.1) and are subject to various VEGF inhibitors (Section 3.2), e.g. sVEGFR1, a secreted isoform of the membrane VEGF receptor VEGFR1 [11]; these inhibitors are involved in establishing vascular quiescence [12]. During inflammation and tumorigenesis, sequestered VEGF can be released by proteases, such as the zinc-dependent matrix metalloproteinases (MMPs). Extracellular proteases can act on VEGF in several ways (Section 3.3) including cleavage of the ECM, cleavage of VEGF generating new isoforms such as VEGF₁₁₄, and also cleavage of the soluble inhibitors of VEGF. These can lead to different biological outcomes. Proteases such as MMP9 are typically thought to release VEGF and induce angiogenesis, but in other situations may reduce angiogenesis activity, e.g. by cleavage of VEGF [13]. We will explore what dictates whether proteolytic release of VEGF is pro- or anti-angiogenic, and the roles of specific proteases.

The spatial distribution of VEGF is a key regulator of angiogenesis and is itself regulated by both matrix binding and proteolytic release (Section 4). For example, VEGF isoforms that bind strongly to the ECM, such as VEGF₁₆₅ and VEGF₁₈₉, have a steep gradient [14], [15] and tight pericellular sequestration [15], [16], [17], [18]. Gradient formation has been commonly thought to be due to a restriction of the rate of diffusion by ECM binding (Section 4.2). However, using computational modeling, we have shown that HSPG binding alone cannot explain most aspects of VEGF gradients [19]. This and other differences between experimental and theoretical results require us to revisit the underlying mechanics of VEGF transport in vivo (Sections 4.3 Combined sequestration and degradation can explain VEGF gradient formation, 4.4 Proteolytic release of VEGF increases VEGF spatial range by reducing degradation). Recent advances have indicated that soluble VEGF inhibitors also play an important role in VEGF patterning [20], [21], [22].

Different tissues express different ratios of the VEGF isoforms (Fig. 2) and this may serve to produce vascular networks that match the specific needs of each tissue [23]. Mice expressing only VEGF₁₂₀ instead of the full range of VEGF isoforms have significant defects in cardiac and pulmonary development due to defective angiogenesis [24], [25]. On the other hand, tumor growth appears to be most rapid in tumors that express VEGF₁₆₄ [16], [26]. We review how VEGF, via its spatial distribution and receptor signaling, regulates angiogenesis. Heparin-binding VEGF isoforms produce a branching network with narrow vessels, while VEGF₁₂₀ (the murine equivalent of VEGF₁₂₁) results in poorly branching, tortuous, leaky vessels [14], [15], [27], [28] (Section 5.2). We explore the specific mechanisms by which VEGF isoforms can cause these different vascularization states (Section 5.3). VEGF is a mediator of sprouting angiogenesis, but in some situations high levels of VEGF can result in a highly proliferative, dysregulated state and lack of sprouting [14], [29]. We explore how these pathological angiogenesis states can arise (Section 5.4).

This review aims to provide a comprehensive overview of the biochemistry, physical transport, and biology of the splice and proteolytic isoforms of VEGF. We highlight several uncertainties in our understanding of VEGF, which may be avenues for future research.