VEGF signaling is essential for specification, morphogenesis, differentiation and homeostasis of vessels during development and in the adult (Damert et al., 2002; Ferrara et al., 2003; Gerber et al., 1999; Gerber et al., 2002; Helmlinger et al., 2000). Furthermore, this signaling pathway is an integral component of pathological angiogenesis during tumor expansion (Inoue et al., 2002). In fact, alterations in VEGF levels or in receptor phosphorylation results in suppression of vascular expansion and concomitant reduction of tumor growth and metastasis (Ferrara et al., 2004; Kim et al., 1993).
Levels of this VEGF are under exquisite transcriptional and translational control and alterations in as much as half can have devastating effects during development. In fact, unlike most mammalian genes, inactivation of only one allele results in embryonic lethality at mid-gestation due to severe cardiovascular defects (Carmeliet et al., 1996; Ferrara et al., 1996). Interestingly, organ-specific increase of VEGF by 2-fold can also lead to lethality (Miquerol et al., 2000). Thus, both decrease and increase in VEGF levels translate in significant pathological effects to the vasculature and to the organism as a whole.
VEGF-A (also known as simply VEGF) exists as four different isoforms termed according to the number of amino acids that constitute the monomer (they are: 121, 165, 189 and 206 in humans - in mouse the forms
are identical, but lack one amino acid). These forms are generated by
alternative splicing of a single pre-mRNA and differ in their ability to bind to heparan sulfate and extracellular matrix molecules. The gene encoding VEGF-A comprises 14kb and contains eight exons. Exons 3, 4 and
5 code for the receptor binding sites, while exons 6a, 6b and 7 code for residues that bind to matrix proteins. These last three exons can be selectively spliced generating the different protein isoforms. The last exon (exon 8) is present in all isoforms. Only the VEGF121 lacks exons 6a, 6b and 7 and is the only highly soluble form. All other variants bind to extracellular matrix proteins restricting access of the growth factor to receptors on target cells. While much emphasis has been placed in understanding the transcriptional, post-transcriptional regulation and stability of VEGF transcripts, little attention has been given to its translated product once outside the cell. Not less important than intracellular control, the regulation of VEGF in the extracellular environment has been implicated in the angiogenic switch facilitating the transition from hyperplastic to malignant tumor (Bergers et al., 2000).
VEGF release from matrix stores is thought to involve matrix metalloproteinases and possibly other extracellular proteases. These proteases either cleave the extracellular matrix proteins themselves or, they directly process the growth factor intramolecularly releasing an active form (Lee et al., 2005). Our recent findings indicate that a specific cohort of MMPs can cleave VEGF severing the receptor-binding domain from the matrix-binding motif. We have identified the cleavage site and determined that the soluble form can phosphorylate VEGF receptors and induce angiogenesis in vivo. Thus, regardless of the spliced isoform, extracellular exposure to certain MMPs can alter the affinity of VEGF to matrix proteins by proteolytic processing.
Exploration of the biological significance of VEGF processing event revealed that matrix-bound and soluble VEGF have alternative outcomes that impact vascular morphogenesis. In fact, we found that soluble VEGF is less effective in inducing neovascularization resulting in the formation of large and poorly branched vessels. In contrast, MMP-resistant VEGF leads to the formation of thin and highly branched
vessels. As result, soluble VEGF does not support tumor growth as
effectively as matrix-bound VEGF. Consequently understanding the regulation of matrix-bound versus soluble VEGF holds strong significance to cancer progression.
We also found that matrix-bound VEGF is able to signal via VEGFR2 and that the cellular responses to those signaling events are quite different from those transmitted by soluble VEGF. While cleaved VEGF
(VEGF-113) induces growth of endothelial cells in sheets; matrix-tethered VEGF (VEGF- delta 108-118) induces the formation of cord-like structures. The different cellular responses to bound and soluble VEGF suggest alternative signaling events downstream VEGFR2. We have explored some of the downstream signaling molecules known to be activated by VEGFR2 and found that soluble VEGF can induce phosphorylation of p38 more effectively than bound VEGF. In contrast, Akt was more readily activated by matrix-bound VEGF. Together these findings suggest that presentation of VEGF (soluble or matrix-bound) results in differential molecular cascades with consequences to vascular morphogenesis.
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