Abstract-Thrombospondin-1 (TSP-1) is a matricellular protein that is expressed in negligible amounts in normal blood vessels but is markedly upregulated in vascular injury. Although TSP-1 can act as a pleiotropic regulator for human vascular smooth muscle cells (HVSMCs), the intracellular signaling pathways stimulated by this protein remain obscure. In cultured HVSMCs derived from saphenous vein, TSP-1 induces tyrosine phosphorylation of a number of cellular proteins, with a complex temporal pattern of activation. Immunoprecipitation techniques have identified the early tyrosine-phosphorylated signals as being the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-K) and focal adhesion kinase (FAK). Tyrosine phosphorylation of the p85 subunit of PI 3-K showed a biphasic response to TSP-1 stimulation, which corresponded to a biphasic activation of the lipid kinase. Treatment with both wortmannin and LY294002 inhibited PI 3-K activity of HVSMCs but did not affect tyrosine phosphorylation of the p85 regulatory subunit. TSP-1-stimulated FAK phosphorylation, however, was substantially reduced by these inhibitors, as was the TSP-1-induced chemotaxis of these cells. These results suggest that activation of PI 3-K is an early signal induced by TSP-1 and is critical for chemotaxis. Activation of this kinase precedes and may occur upstream from FAK phosphorylation, although the nature of the interaction between these 2 enzymes remains obscure. Key Words: thrombospondin-1 Ⅲ focal adhesion kinase Ⅲ phosphatidylinositol 3-kinase Ⅲ human vascular smooth muscle T hrombospondin-1 (TSP-1), a large, homotrimeric glycoprotein, is a member of the TSP family of matricellular proteins. This family currently consists of 5 isoforms: TSP-1 through TSP-4 and TSP-5/cartilage oligomeric matrix protein, 1 which, although implicated in a wide variety of biological processes, 2 show differential tissue expression both temporally and spatially. 3,4 TSP-1 was originally described as a product of platelet ␣-granules 5 but has since been shown to be synthesized by a number of different cell types, including vascular endothelial and smooth muscle cells. 6,7 TSP-1 is present in negligible amounts in normal human blood vessels, but the expression of this protein is markedly upregulated in injured and diseased vasculature, 8 -10 suggesting a possible role for TSP-1 in the abnormal cellular response to vascular injury.Literature evidence would suggest that TSP-1 levels are regulated by growth factors. Both platelet-derived growth factor (PDGF) and transforming growth factor-␤ (TGF-␤) have been shown to induce TSP-1 secretion from vascular smooth muscle cells (VSMCs). 11,12 TSP-1 mRNA levels have been shown to be similarly regulated. Basic fibroblast growth factor upregulates TSP-1 mRNA in 3T3 cells, 13 and PDGF induces upregulation in a manner similar to that of c-myc and c-fos, with TSP-1 being classified as an immediate early response gene. 14 These data, in conjunction with the role of TSP-1 in potentiating the chemotactic response of VS...

Thrombospondin-1 is a large matricellular protein that acts as a pleiotropic growth factor for human vascular smooth muscle cells, and may play a role in the progression of vascular disease. Although we have previously demonstrated the dependence of both thrombospondin-1-stimulated cell chemotaxis and proliferation on tyrosine kinases, the receptor mechanisms involved remain obscure. This investigation aims to determine the nature of the receptor(s) involved in the cellular responses to thrombospondin-1. Cellular signals were identified by western blotting following cell stimulation, while cellular responses were assessed by measuring DNA synthesis and chemotaxis. These data demonstrate that thrombospondin-1-induced cell chemotaxis can be inhibited by a peptide containing the Arg-Gly-Asp motif, a function-blocking αvβ3 antibody, a function-blocking integrin-associated protein (IAP) antibody and pertussis toxin, while thrombospondin-1-stimulated DNA synthesis is inhibited by a function-blocking α3β1 antibody. Similarly the Arg-Gly-Asp-containing peptide inhibits tyrosine phosphorylation of focal adhesion kinase and the p85 regulatory subunit of phosphatidylinositol 3-kinase, but does not significantly affect tyrosine phosphorylation, or activation, of extracellular-regulated kinase. These data suggest that soluble thrombospondin-1 interacts with human vascular smooth muscle cells via two independent and separable receptor-binding sites, to differentially stimulate cell chemotaxis and DNA synthesis.