220 Regulation of Vascular Calcification by Bioactive Sphingolipids

Vascular calcification is the formation of mineralised tissue within the vascular wall and is an independent risk factor for future cardiovascular events. The pathology has many similarities to embryonic bone formation and involves the osteogenic differentiation of vascular smooth muscle cells and matrix mineralisation. However, the exact mechanisms are not fully understood and no treatment is available to prevent or reverse the condition. Recent studies have demonstrated that the bioactive sphingolipids, ceramide and sphingosine-1-phosphate (S1P), can regulate embryonic bone formation. Ceramide can be generated by lysosomal acid-sphingomyelinase (L-SMase) and be converted to sphingosine by acid ceramidase (ACDase) and subsequently to S1P by sphingosine kinases (SK 1u00262). The aim of this study was to investigate whether ceramide and S1P also regulate vascular calcification. Aortic VSMCs were cultured with 3 mM beta-glycerophosphate (BGP) to induce osteogenic differntiation and mineralisation; control VSMCs were cultured in the absence of BGP. Matrix mineralisation was detected with alizarin red and quantified by dye elution. Changes in L-SMase and SK activity were determined using an in vitro assay and mRNA levels of L-SMase, SK1 and SK2 with Q-PCR. Mass spectroscopy was used to measure the levels of ceramide species, sphingosine and S1P. Inhibition of L-SMase and ACDase was achieved using desipramine (1–10 µM). In comparison to control VSMCs, when mineralisation was widespread the levels of C18 and C20-ceramide increased by 75%, sphingosine decreased by 33% and S1P increased three-fold. In addition, L-SMase activity decreased by 30% although no changes in mRNA expression were detected; SK activity increased 66%, SK2 mRNA increased but SK1 was unchanged. Desipramine dose-dependently inhibited mineralisation when compared to VSMCs cultured with BGP alone. Furthermore, desipramine increased total ceramide two-fold, decreased sphingosine 33% and prevented the BGP-induced increase of S1P. To further investigate the role of bioactive sphingolipids during mineralisation, exogenous C2-ceramide (10 µM) and S1P (10 µM) were added to cultured VSMCs. C2-ceramide decreased BGP-induced mineralisation, whereas S1P increased mineralisation. All changes were significant, n = 3 (p In conclusion, SK activity and S1P levels are increased during phosphate-induced VSMC mineralisation. Inhibition of L-SMase/ACDase leads to ceramide accumulation, prevents S1P production and attenuates mineralisation; identifying ceramide as an inhibitor and S1P as a promoter of matrix mineralisation. These novel findings demonstrate an important role for sphingolipids in VSMC mineralisation and may identify potential therapeutic targets for the treatment of vascular calcification.