Recent studies have identified factors responsible for angiogenesis within developing tumors, but mediators of vessel formation at sites of trauma, injury, and wound healing are not clearly established. Here we show that sphingosine 1-phosphate (S1P) released by platelets during blood clotting is a potent, specific, and selective endothelial cell chemoattractant that accounts for most of the strong endothelial cell chemotactic activity of blood serum, an activity that is markedly diminished in plasma. Preincubation of endothelial cells with pertussis toxin inhibited this effect of S1P, demonstrating the involvement of a Galphai-coupled receptor. After S1P-induced migration, endothelial cells proliferated avidly and differentiated forming multicellular structures suggestive of early blood vessel formation. S1P was strikingly effective in enhancing the ability of fibroblast growth factor to induce angiogenesis in the avascular mouse cornea. Our results show that blood coagulation initiates endothelial cell angiogenic responses through the release of S1P, a potent endothelial cell chemoattractant that exerts its effects by activating a receptor-dependent process.

Angiogenesis, the formation of new blood vessels, is an important component of restoration of hematopoiesis after BMT, but the mediators involved in hematopoietic angiogenesis have not been identified. We examined the influence of the lipid growth factors, phosphatidic acid (PA), lysophosphatidic acid (LPA), and sphingosine 1-phosphate (S1P), on several angiogenic properties of endothelial cells, including migration and stabilization of vascular barrier integrity. In a previous study, PA was found to disrupt the permeability of established endothelial monolayers, an early event in the angiogenic response that liberates cells for subsequent mobilization. In the present study, both PA and LPA weakly induced the chemotactic migration of endothelial cells from an established monolayer. The chemotactic response induced by PA and LPA was similar in intensity to that observed with optimal levels of the known protein endothelial cell chemoattractants, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). A markedly greater chemotactic response was effected by nanomolar concentrations of S1P, indicating that this platelet-derived factor plays an important role in a key aspect of angiogenesis, chemotactic migration of endothelial cells. The chemotactic response to S1P was completely inhibited by preincubation of endothelial cells with antisense oligonucleotides to the high-affinity S1P receptor, Edg-1. In addition, chemotaxis of endothelial cells to S1P was inhibited by preincubation of cells with specific inhibitors of tyrosine kinases, but inhibitors of phosphatidylinositol 3' kinase had little effect. Finally, LPA effectively stabilized endothelial monolayer barrier function, a late event in angiogenesis. Thus, the phospholipid growth factors, PA, S1P, and LPA, display divergent and potent effects on angiogenic properties of endothelial cells and angiogenic differentiation of endothelial cells potentially act in tandem to effectively induce neovascularization. These mediators may thus exert important roles in restoration of hematopoiesis, as they facilitate blood vessel formation at sites of transplanted stem cells, allowing the progeny of engrafted progenitors to move from marrow sinusoids to the peripheral vasculature.

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