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Vascular calcification is a major cause of mortality in patients with diabetes, chronic kidney disease and atherosclerosis. It results from the osteogenic differentiation of vascular smooth muscle cells (VSMCs), which is driven by several factors including BMPs. The activity of many of these factors is regulated by binding to glycosaminoglycans (GAGs). This study tests the hypothesis that GAGs [specifically heparan sulphate (HS)] regulate vascular calcification.VSMCs were cultured in the presence of β-glycerophosphate (3 mM) to induce mineralisation. Calcification was confirmed by staining with alizarin red; osteogenic differentiation was confirmed by the up-regulation of Runx2 and Msx2, and down-regulation of αSMA mRNA. Cells cultured in the absence of β-glycerophosphate were used as controls. As VSMCs undergo osteogenic differentiation and mineralisation, the mRNA expression levels of the HS proteoglycan syndecan 4 (5 ± 0.8-fold increase; P < 0.001) and specific HS biosynthetic enzymes [NDST1, 2.4 ± 0.2-fold increase; NDST2, 1.6 ± 0.2-fold increase (both P < 0.001)] were up-regulated compared to controls. To correlate the glycomic transcription profile of mineralising VSMCs with the GAGs synthesised by these cells, 3H-GAGs were isolated from confluent and mineralising VSMCs and characterised using specific scission agents. The ratio of chondroitin sulphate to dermatan sulphate is decreased in mineralising VSMCs, and the disaccharide composition of HS chains is subtly altered. To determine the role of HS, and syndecan 4 specifically, in VSMC mineralisation, siRNA was used. Knock-down of HS synthesis increased VSMC mineralisation compared to control siRNA-treated cells (2-fold increase; P < 0.001). Syndecan 4 siRNA also increased VSMC mineralisation compared to controls (2 ± 0.5-fold increase; P < 0.05).These studies demonstrate for the first time that HS, and syndecan 4 specifically, play a role in regulating the deposition of a mineralised matrix by VSMCs. Studies are underway to determine the effects of over-expressing syndecan 4 in VSMC mineralisation and to identify the signalling pathways regulated by syndecan 4 during this process.