Angiogenesis, the growth of new blood vessels from extant capillaries, is a continuous process throughout most of the embryonic and mature lifespan of vertebrates. It is also necessary for remodeling and tumor growth. The angiogenic process is thought to proceed in discrete stages that include migration, proliferation, and remodeling of the extracellular matrix (ECM) by endothelial cells, although the regulation of these events is poorly understood. We have recently defined a group of secreted macromolecules that modulate interactions between cells and ECM. Several of these components are major products of endothelial cells undergoing angiogenesis in vitro and in vivo. In this proposal we focus on SPARC (secreted protein, acidic and rich in cysteine), a Cu+2 and Ca+2-binding protein which modulates the shape, adhesion, cell-cycle progression, ECM production, and migration of cultured endothelial cells. The expression of SPARC in tissues exhibiting morphogenesis and remodeling, as well as the interaction of SPARC with angiogenesis factors such as platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF), indicate potential functions for SPARC as an endothelial morphogen. We have advanced the hypothesis that SPARC alters the relationship among endothelial cells, mitogens, and ECM and thereby predisposes endothelium toward an activated state of remodeling and neovascularization. In 7 Aims we address the function of SPARC at specific stages of angiogenesis in vivo and in vitro. Sequences of SPARC protein with growth-modulatory activity will be mutated, and recombinant proteins will be used to identify signaling pathways in endothelial cells that are responsive to native SPARC. The ability of SPARC to modulate endothelial cell proliferation and migration, through high-affinity binding to PDGF or indirectly through a serum factor that alters responses of cells to bFGF, will be characterized by identifying a) a binding site(s) on SPARC for PDGF, and b) the factor in serum or plasma responsible for the subversion of bFGF activity. A Cu+2-binding sequence in SPARC will be tested for angiogenic activity in vivo and in vitro, and its proteolytic release from SPARC will be characterized; mutations in this region will target critical residues. A potential role for SPARC in the modulation of matrix integrity will be examined during vasculogenesis and angiogenesis in embryonic mice that lack type I collagen, a protein expressed at high levels during capillary formation. Related experiments will test the effect of SPARC on the contraction of collagen gels by angiogenic endothelial cells that form cords and tubes in vitro. Finally, we will attempt to ascertain how the SPARC gene is regulated in angiogenic cells by performing transient transfections with regions of the SPARC promoter and first intron. Since 2 genes related to SPARC have been isolated from embryonic brain, we will attempt to clone similar genes from tissues rich in capillaries with probes corresponding to bioactive regions of SPARC. Through these aims we hope to elucidate how blood vessels grow int he context of signals mediated by SPARC, a dynamic participant in the extracellular regulation of cell behavior.
Showing the most recent 10 out of 127 publications
