Novel role of lactosylceramide in vascular endothelial growth factor-mediated angiogenesis in human endothelial cells

Vascular endothelial growth factor ( VEGF) has been implicated in angiogenesis associated with coronary heart disease, vascular complications in diabetes, inflammatory vascular diseases, and tumor metastasis. The mechanism of VEGF-driven angiogenesis involving glycosphingolipids such as lactosylceramide ( LacCer), however, is not known. To demonstrate the involvement of LacCer in VEGF-induced angiogenesis, we used small interfering RNA ( siRNA)mediated silencing of LacCer synthase expression ( GalT-V) in human umbilical vein endothelial cells. This gene silencing markedly inhibited VEGF-induced platelet endothelial cell adhesion molecule-1 ( PECAM-1) expression and angiogenesis. Second, we used D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol ( D-PDMP), an inhibitor of LacCer synthase and glucosylceramide synthase, that significantly mitigated VEGF-induced PECAM-1 expression and angiogenesis. Interestingly, these phenotypic changes were reversed by LacCer but not by structurally related compounds such as glucosylceramide, digalactosylceramide, and ceramide. In a human mesothelioma cell line ( REN) that lacks the endogenous expression of PECAM-1, VEGF/LacCer failed to stimulate PECAM-1 expression and tube formation/angiogenesis. In REN cells expressing human PECAM-1 gene/protein, however, both VEGF and LacCer-induced PECAM-1 protein expression and tube formation/angiogenesis. In fact, VEGF-induced but not LacCer-induced angiogenesis was mitigated by SU-1498, a VEGF receptor tyrosine kinase inhibitor. Also, VEGF/LacCer-induced PECAM-1 expression and angiogenesis was mitigated by protein kinase C and phospholipase A(2) inhibitors. These results indicate that LacCer generated in VEGF-treated endothelial cells may serve as an important signaling molecule for PECAM-1 expression and in angiogenesis. This finding and the reagents developed in our report may be useful as anti-angiogenic drugs for further studies in vitro and in vivo.