172 The Role of Bioactive Sphingolipids in Vascular Calcification

Vascular calcification is the formation of mineralised tissue within the walls of arteries and is associated with increased cardiovascular morbidity and mortality. The pathology involves the osteogenic differentiation of vascular smooth muscle cells (VSMCs) and matrix mineralisation. The development of vascular calcification has many similarities to bone formation; although the exact mechanisms are not fully understood. Recent studies have demonstrated that the bioactive sphingolipids, ceramide and sphingosine-1-phosphate (S1P), are regulators of bone formation. The levels of ceramide are controlled by neutral sphingomyelinases (nSMase) and the levels of S1P by sphingosine kinases (SK). Our aim was to investigate whether sphingolipid signalling is involved in VSMC matrix mineralisation. Bovine aortic VSMCs were cultured in the presence of beta-glycerophosphate (BGP) to induce osteogenic differentiation and matrix mineralisation; control VSMCs were cultured minus BGP. Osteogenic differentiation was confirmed by down-regulation of VSMC markers (alpha-SMA, SM22-alpha) and up-regulation of osteogenic markers (Runx2, Msx2). Matrix mineralisation was detected using alizarin red and quantified by dye elution. mRNA expression of nSMase-1and3 and SK-1and2 isoforms was determined by Q-PCR and nSMase activity was measured using a fluorescent in vitro assay. Ceramide species and S1P levels were measured by mass spectroscopy. nSMase activity was inhibited by GW4869 (1–10 µM). Total SK activity was inhibited by Ski-II (1 µM), SK1 specific activity by SK1-I (1 µM) and SK2 specific activity by ABC294640 (5 µM). At the onset of matrix mineralisation, nSMase1, nSMase3 and SK2 mRNA expression increased significantly by 50% (P These data demonstrate for the first time that bioactive sphingolipids modulate VSMC matrix mineralisation. The pharmacological inhibition of nSMase and SK2 activity imply that bioactive sphingolipids are inhibitors of matrix mineralisation. The results of this study may identify novel therapeutic targets for the treatment of vascular calcification.