Serum factors involved in human microvascular endothelial cell morphogenesis

Our previous studies have demonstrated that lipid and protein angiogenic factors operate in tandem to induce optimal angiogenic responses in vivo. This study was undertaken to clarify the nature of the substances in human serum that are responsible for its remarkable ability to promote capillary morphogenesis in vitro. The ability of dilute (2%) human serum to promote the morphogenic differentiation of human dermal microvascular endothelial cells on Matrigel supports was depleted by more than 50% by treatment of the serum with activated charcoal, a procedure that effectively removes biologically active lipid growth factors. The remainder of the activity within serum was lost on heating to 60°C for 60 minutes, indicating the involvement of a protein in the response. The ability of charcoal-treated serum to promote capillary morphogenesis was completely restored by the addition of sphingosine 1-phosphate (SPP, 500 nmol/L), but other lipids thought to be released into serum during clotting were ineffective. In addition, basic fibroblast growth factor (bFGF) effectively restored the ability of heat-treated serum to promote endothelial cell morphogenesis, but other protein growth factors, including vascular endothelial growth factor and platelet-derived growth factor, were ineffective. Together, SPP and bFGF were as effective as whole serum in promoting capillary morphogenesis. Responses to purified SPP were entirely sensitive to the effects of preexposure of the cells to pertussis toxin, whereas responses to bFGF were entirely pertussis toxin-resistant. Consistent with our hypothesis that two distinct factors in serum play a role in promoting capillary morphogenesis, responses induced by serum were inhibited approximately 50% by preexposure of endothelial cells to pertussis toxin. We conclude that platelet-released SPP acts in conjunction with circulating bFGF to promote capillary formation by microvascular endothelial cells. Lipid and protein growth factors apparently exert complementary roles in the angiogenic response, as demonstrated by their ability to promote chemotaxis, angiogenic differentiation, and angiogenesis in vivo.