Analysis of a broad range of MMP mutants shows that embryonic vascular development proceeds normally, yet defects are observed in both normal and pathological postnatal vascular remodelling and angiogenesis (). These data imply that MMPs might have a specific role in postnatal refinement, remodelling and neoangiogenesis, but not in the original construction of the embryonic vascular networks.
-mutant mice show defects in angiogenesis at the growth plate of long bones36
. Similarly, Mmp14
-mutant mice lack appropriate vascular invasion at secondary ossification centres, despite normal levels of VEGF and its receptor VEGFR2 (also known as FLK1) (REF. 43
). This lack of vascular growth is recapitulated in an experimentally induced corneal angiogenesis assay43
, which induces new blood-vessel growth in control mice. Blood-vessel growth is not observed in Mmp14
, whereas it is reduced but not eliminated in Mmp2
. In a laser-induced injury model of retinal degeneration, neovascularization is reduced in single Mmp2
mutants and is strongly reduced in the Mmp2 Mmp9
double mutant, indicating that these MMPs function redundantly56
. Mmp2 Mmp14
double mutants also have developmental vascular defects, including capillaries with extremely small lumens. These defective blood vessels are sufficient to support embryonic growth and development, but not postnatal survival.
How do MMPs contribute to vascular remodelling? Likely mechanisms include proteolysis of type I collagen, modification of platelet-derived growth factor (PDGF) signalling, regulation of perivascular cells and processing of VEGF. Invading blood vessels in postnatal tissues encounter ECM that is rich in type I collagen, a protein that is not highly expressed in embryos. When control aortic explants are embedded in three-dimensional collagen matrices57
, they sprout neovessels and the endothelial cells invade the collagen matrix in a growth-factor-dependent manner. Strikingly, Mmp14
-null explants do not sprout neovessels or invade the collagen matrix, whereas explants from Mmp2
- and Mmp9
-null mice are indistinguishable from control explants. However, when Mmp14
-null explants are embedded in a fibrin matrix (similar to the provisional ECM at a wound site) they form capillaries, demonstrating matrix specificity. So, MMP14 contributes to postnatal vascular development by cleaving type I collagen; the relative absence of type I collagen in the embryo might partially explain the lack of embryonic vascular defects.
MMP9 and MMP14 have another effect on the vasculature: the perivascular (or smooth muscle) cells that ensheath the endothelial cells of the blood vessels are missing or their density is significantly decreased in normal vessels and during tumour neoangiogenesis57–59
. This defect is especially evident in the smaller arterioles of the brain, and many of the remaining perivascular cells have irregular morphology. This phenotype is similar to that of mice that carry weak PDGF-B alleles. Signalling that occurs downstream of PDGF is attenuated in Mmp14
-null mice, and PDGF receptor-β (PDGFRβ) co-immunoprecipitates with MMP14, demonstrating that they form a physical complex60
. These data indicate a novel and direct function for MMP14 in PDGF signalling.
MMPs and VEGF signaling
MMP processing of VEGF might have an important role in physiological and tumour angiogenesis. VEGF is stored extracellularly: after secretion, VEGF binds to the ECM, from where it must be released to initiate angiogenesis61
. Small insulinomas form constitutively in the RIP1-Tag (rat insulin promoter 1–T-antigen) mouse pancreatic islet cancer model, but only 1–2% of these develop into angiogenic adenomas and carcinomas62
. Despite continuous expression of VEGF and its receptor, VEGFR2, VEGF availability is limited and cannot bind to its receptor in pre-angiogenic tumours. MMP9 mobilizes VEGF and initiates angiogenesis. Importantly, when the Mmp9
mutation is crossed into the RIP1-Tag background, fewer tumours become angiogenic, supporting the role of MMP9 in mobilizing VEGF63
MMPs can also cleave VEGF, separating the matrix-binding domain from the receptor-binding domain. Uncleaved VEGF is enriched in at least one MMP9-mutant postnatal tissue, the hypertrophic chondrocyte zone38
, which indicates that VEGF is an important downstream effector, and possibly a substrate, of MMP9. Truncated VEGF has different effects on tumour blood vessels than does uncleavable VEGF; truncated VEGF increases vessel diameter, whereas uncleavable VEGF increases vessel sprouting64
-null mice display defective post-embryonic neovasculature, suggesting that in wild-type mice, postnatal blood vessels respond differently to cleaved and uncleaved forms of VEGF. Perhaps the sprouting-initiation function of uncleaved VEGF is the only VEGF function required in embryos; however, this function of VEGF is unaffected in Mmp9