EXPRESSION OF SPARC DURING DEVELOPMENT OF THE CHICKEN CHORIOALLANTOICMEMBRANE - EVIDENCE FOR REGULATED PROTEOLYSIS IN-VIVO

SPARC is a secreted glycoprotein that has been shown to disrupt focal adhesions and to regulate the proliferation of endothelial cells in vi tro. Moreover, peptides resulting from the proteolysis of SPARC exhibi t angiogenic activity. Here we describe the temporal synthesis, turnov er, and angiogenic potential of SPARC in the chicken chorioallantoic m embrane. Confocal immunofluorescence microscopy revealed specific expr ession of SPARC protein in endothelial cells, and significantly higher levels of SPARC were observed in smaller newly formed blood vessels i n comparison to larger, developmentally older vessels. SPARC mRNA was detected at the earliest stages of chorioallantoic membrane morphogene sis and reached maximal levels at day 13 of embryonic development. Int erestingly, steady-state levels of SPARC mRNA did not correlate direct ly with protein accumulation; moreover, the protein appeared to underg o limited degradation during days 10-15. Incubation of [I-125]-SPARC w ith chorioallantoic membranes of different developmental ages confirme d that extracellular proteolysis occurred during days 9-15, but not at later stages (e.g., days 17-21). Comparison of peptides produced by i ncubation with chorioallantoic membranes with those generated by plasm in showed an identical pattern of proteolysis. Plasmin activity was pr esent throughout development, and in situ zymography identified sites of plasminogen activator activity that corresponded to areas exhibitin g high levels of SPARC expression. Synthetic peptides from a plasmin-s ensitive region of SPARC, between amino acids 113-130, stimulated angi ogenesis in the chorioallantoic membrane in a dose-dependent manner; i n contrast, intact SPARC was inactive in similar assays. We have shown that SPARC is expressed in endothelial cells of newly formed blood ve ssels in a manner that is both temporally and spatially restricted. Be tween days 9 and 15 of chorioallantoic membrane development, the prote in undergoes proteolytic cleavage that is mediated, in part, by plasmi n. SPARC peptides released specifically by plasmin induce angiogenesis in vivo. We therefore propose that SPARC acts as an intrinsic regulat or of angiogenesis in vivo.