Novel antiangiogenic pathway of thrombospondin-1 mediated by suppression of the cell cycle

We have recently reported that keratin 14-promoter-driven vascular endothelial growth factor (VEGF)-E(NZ-7) transgenic mice have a significant number of capillary vessels in subcutaneous tissue. However, these vessels are generated in a layer some distance from the epithelial basal cells that express VEGF-E(NZ-7), suggesting that one or more antiangiogenenic molecules may exist very near the basal cell layer. By screening keratinocyte-conditioned medium, we found that thrombospondin-1 (TSP-1) is produced from keratinocytes and suppresses human umbilical vein endothelial cells (HUVEC) growth as well as tubular formation in a HUVEC-fibroblast coculture system. Different to the known mechanism of CD36-dependent endothelial cell apoptosis, the HUVEC we used did not express CD36 at detectable levels, indicating a new mechanism for TSP-1-induced antiangiogenesis. We found that TSP-1 induces little apoptosis of endothelial cells but causes cell-cycle arrest, increasing the amounts of p21(CIP/WAF-1) and unphosphorylated retinoblastoma (Rb) in HUVEC. CD36-binding peptide in TSP-1 and CD36-neutralizing antibody did not block the TSP-1-induced cell-cycle arrest. Our results strongly suggest that TSP-1 utilizes a novel pathway for its antiangiogenic effect independent of CD36, and suppresses the cell cycle.