Cleavage of the Matricellular Protein SPARC by Matrix Metalloproteinase 3 Produces Polypeptides That Influence Angiogenesis

Abstract

SPARC, a matricellular protein that affects cellular adhesion and proliferation, is produced in remodeling tissue and in pathologies involving fibrosis and angiogenesis. In this study we have asked whether peptides generated from cleavage of SPARC in the extracellular milieu can regulate angiogenesis. Matrix metalloproteinase (MMP)-3, but not MMP-1 or 9, showed significant activity toward SPARC. Limited digestion of recombinant human (rhu)SPARC with purified catalytic domain of rhuMMP-3 produced three major fragments, which were sequenced after purification by HPLC. Three synthetic peptides (Z-1, Z-2, and Z-3) representing motifs from each fragment were tested in distinct assays of angiogenesis. Peptide Z-1 (3.9 kDa, containing a Cu 2؉ -binding sequence KHGK) exhibited a biphasic effect on [ 3 H]thymidine incorporation by cultured endothelial cells and stimulated vascular growth in the chick chorioallantoic membrane (CAM). In contrast, peptides Z-2 (6.1 kDa, containing Ca 2؉ -binding EF hand-1) and Z-3 (2.2 kDa, containing neither Cu 2؉ -binding motifs nor EF hands), inhibited cell proliferation in a concentration-dependent manner and exhibited no effects on vessel growth in the CAM. Reciprocal results were obtained in a migration assay in native collagen gels: peptide Z-1 was ineffective over a range of concentrations, whereas Z-2 or Z-3 stimulated cell migration. Therefore, proteolysis of SPARC by MMP-3 produced peptides that regulate endothelial cell proliferation and/or migration in vitro in a mutually exclusive manner. One of these peptides containing KHGK also demonstrated a concentration-dependent effect on angiogenesis.

X-ray fluorescence microscopy reveals large-scale relocalization and extracellular translocation of cellular copper during angiogenesis

“…It has been noted that simply adding exogenous copper to a cell system can initiate angiogenesis (6); translocation of intracellular copper to the extracellular space as demonstrated here may be serving the same purpose. Alternatively, peptides resulting from the proteolytic degradation of extracellular matrix proteins such as SPARC/osteonectin (33) may be activated by excreted copper and behave as proangiogenics in an autocrine manner (34).…”

Extracellular influences on tumour angiogenesis in the aged host

“…Intact SPARC inhibits the angiogenic response by impairing proper collagen alignment and blocking pro-angiogenic growth factors (Kupprion et al, 1998). At the same time it has been reported that cleaved SPARC might facilitate vessel growth by enhancing endothelial cell proliferation (Sage et al, 2003). In aged tissues, the complexities surrounding the role of SPARC in the angiogenic response are obviated by age-related deficits in the levels of growth factors and other pro-angiogenic mediators (Reed et al, 2005).…”

The Novel SPARC Family Member SMOC-2 Potentiates Angiogenic Growth Factor Activity

“…This is also suggested by the binding of VEGF by SPARC and reduced VEGF receptor activation in the presence of SPARC (11). However, it has been observed that cleavage of SPARC by matrix metalloproteinases and plasmin releases peptides that are pro-angiogenic (15,16). These peptides are found within the follistatin-like domain of SPARC (8,15).…”