VEGF recruits lactosylceramide to induce endothelial cell adhesion molecule expression and angiogenesis in vitro and in vivo

Angiogenesis is largely driven by vascular endothelial growth factor (VEGF). However, the role of lipid second messengers such as lactosylceramide (LacCer) and LacCer synthase in angiogenesis is not well understood. We have determined the distribution of various LacCer synthase mRNA transcripts using sequential analysis of gene expression (SAGE). Endothelial cells from colon cancer tissues had a 4.5-fold increase in a LacCer synthase transcript (beta 1,4GalT-V) as compared to normal colon tissue endothelial cells. Consequently, our focus turned to understanding the role of this enzyme in regulating VEGF-induced angiogenesis in vitro and in vivo. Herein, we show that in human endothelial cells, VEGF-induced angiogenesis is mitigated by dimethylsphingosine and suramin; inhibitors of sphingosine kinase 1(SphK-1) and sphingosine1-phosphate receptor 1(S1P (1)), respectively, and this were bypassed by LacCer but not by S1P. VEGF and basic fibroblast growth factor-induced angiogenesis was mitigated by PDMP; an inhibitor of glucosylceramide synthase and LacCer synthase in human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells (HAEC). Likewise, GalT-V gene ablation using corresponding siRNA also mitigated VEGF-induced angiogenesis. In Matrigel plug angiogenesis assay in nude mice, angiogenesis was markedly inhibited by D-PDMP with concordantly diminished LacCer synthase activity. Mechanistic studies revealed that the use of LY294002, a PI3 kinase inhibitor, mitigated VEGF-induced expression of platelet-endothelial cell adhesion molecule (PECAM-1/CD31); the trans-endothelial migration of a monocyte cell line (U-937) and angiogenesis in HAEC cells. Since this enzyme is a target for VEGF action and LacCer serves as a lipid second messenger in inducing angiogenesis in vitro and in vivo, novel therapeutic approaches may be developed using our findings to mitigate colon cancer.