P710Differential regulation of vascular calcification by neutral and acid sphingomyelinases

Vascular calcification is the formation of mineralised tissue within the walls of arteries and is associated with increased cardiovascular 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 sphingolipid, ceramide, is required for normal bone formation and development. The levels of ceramide are controlled by secretory and lysosomal acid sphingomyelinases (aSMase) and the plasma membrane-located neutral sphingomyelinase (nSMase). Our aim was to investigate whether ceramide and sphingomyelinases are involved in VSMC matrix mineralisation. Bovine aortic VSMCs were cultured ± elevated phosphate to induce osteogenic differentiation and matrix mineralisation. Osteogenic differentiation was confirmed by downregulation of VSMC markers (αSMA, SM22α) and upregulation of osteogenic markers (Runx2, Msx2). Matrix mineralisation was detected using alizarin red and quantified by dye elution. mRNA expression of sphingomyelinases were quantified by Q-PCR and activity changes were determined using a fluorescent in vitro assay. Mass spectroscopy was used to investigate changes in ceramide species. nSMase activity was inhibited by GW4869 (1-10 μM) and lysosomal aSMase activity was inhibited by desipramine (1-10 μM). Q-PCR demonstrated that at the onset of matrix mineralisation and osteogenic differentiation the mRNA expression of nSMase 1 and 3 increased two-fold (P<0.05); no changes in aSMase expression were detected. The activity of both nSMase and lysosmal aSMase significantly increased (P<0.05) at the onset of matrix mineralisation. Mass spectroscopy revealed that the levels of C14, C18 and C20 ceramide species increased during matrix mineralisation. Inhibition of nSMase activity with GW4869 significantly increased matrix mineralisation in a dose-dependent manner (1 μM P<0.05; 10 μM P<0.01). Conversely, the inhibition of lysosomal aSMase with desipramine completely prevented matrix mineralisation (P<0.01). These data demonstrate for the first time that sphingomyelinases modulate VSMC matrix mineralisation. The pharmacological inhibition of sphingomyelinases has shown that nSMase is an inhibitor of VSMC matrix mineralisation, whereas lysosmal aSMase is a promoter. Collectively, these findings suggest that the sphingomyelinase-regulated production of ceramide in different sub-cellular locations may differentially regulate vascular calcification.