Abstract P277: Ceramide Modulates GPCR And TK Receptor Signaling To Impact Endothelial Functions And Blood Pressure

Abstract

Ceramides are sphingolipids that modulate a variety of cellular processes, via two major modes of actions: by functioning as second messengers and by regulating the formation of lipid raft, important signaling platform. Receptor-mediated signaling orchestrates multiple pathophysiological processes, hence more than 50% of drugs clinically prescribed target mainly GPCRs. Alterations of ceramide levels are implicated in endothelial dysfunction, a key event in many cardiovascular diseases, including hypertension and atherosclerosis. However, specific molecular mechanisms of how ceramides modulate the structure and the function of GPCRs and TK receptors to impact endothelial functions remains unknown and unexplored. Thus, we generated mice lacking the endothelial sphingolipid de novo biosynthesis, by deleting the serine palmitoyltransferase long subunit 2, of the first enzyme of the pathway, named ECKO-Sptlc2. Systolic blood pressure was markedly increased in ECKO-Sptlc2 vs. control mice (122.1± 1.9 vs. 103.6±0.7 mmHg, n=9). Vasodilation of mesenteric arteries to acetylcholine and histamine (Gq-coupled receptors) and the NO-signaling downstream was preserved suggesting that altered membrane sphingolipid levels did not affect Gq-coupled receptor activation. On the contrary, vasodilation induced by the activation of tyrosine kinase receptors, i.e. VEGFR2 (Emax 32.8±3.5 vs. 55.7±3.6 % vasodilation, n=5), Gi-coupled GPCRs, i.e. S1PR1 (Emax 9.0±11.0 vs. 58.7 ±3.5 % vasodilation, n=5) and flow were markedly reduced. C16-, C24- and C24:1-ceramide are the most abundant ceramides in the endothelium and were markedly reduced by the loss of Sptlc2. Interestingly, the treatment of the mice and endothelial cells in culture with C16-, C24- and C24:1-ceramides, showed that C16-ceramide play a specific role in VEGFR2, IR, S1PR1 and flow mediated vasodilation. Mechanistically, C16-ceramide modulates VEGFR2 phosphorylation and downstream signaling in a concentration-dependent manner. The finding that C16-ceramide affects specific the function of Gi-coupled, TK but not Gq-coupled receptor is of great significance considering that alterations in C16-ceramide strongly correlate with CV conditions, such as CAD and heart failure.