Vascular calcification is the formation of mineralised tissue, bone and/or cartilage within blood vessels. It involves the osteogenic differentiation of vascular smooth muscle cells (VSMCs), VSMC apoptosis and extracellular matrix mineralisation. This pathology has the potential to be regulated by proteoglycans (PGs), which comprise a protein core and glycosaminoglycan (GAG) chains. This study tests the hypothesis that PGs regulate vascular calcification. VSMCs were cultured with β-glycerophosphate (β-GP) to induce mineralisation. Osteogenic differentiation was confirmed by the up-regulation of Runx2 and Msx2, and down-regulation of alphaSMA and SM22alpha mRNA. During VSMC osteogenic differentiation and mineralisation, the mRNA expression levels of syndecan 1, biglycan, decorin, versican, osteoglycin and lumican mRNAs were down-regulated. In contrast, syndecan 4 mRNA expression was increased during the late stages of VSMC mineralisation. Syndecan 4 is a transmembrane PG, and knocking-down syndecan 4 expression using siRNA increased VSMC mineralisation. Previous studies have shown that syndecan 4 regulates fibroblast growth factor-2 (FGF-2) signalling, and FGF-2 mRNA and protein expression were increased during the late stages of VSMC mineralisation. Exogenous FGF-2 inhibited VSMC mineralisation, and this ability was lost in the absence of syndecan 4, suggesting that syndecan 4 is required for the inhibitory effect of FGF-2 on VSMC mineralisation. However, over-expressing syndecan 4 also increased VSMC mineralisation, suggesting that syndecan 4 levels must be finely regulated in VSMCs.Syndecan 4 displays heparan sulphate (HS) and/or chondroitin/dermatan sulphate (CS/DS) GAG chains on its extracellular domain, and these GAGs can regulate FGF-2 signalling. The mRNA levels of specific CS/DS (C4ST1, C4ST2, DS epimerase-1, -2) biosynthetic enzymes were up-regulated in mineralising VSMCs. 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 ratios of DS to CS and CS/DS 4-O-sulphation were increased in mineralising VSMCs. In contrast, there was no change in HS disaccharide composition. Knocking-down EXT1 (a crucial HS biosynthetic enzyme) expression using siRNA increased VSMC mineralisation and reduced FGF-2-induced Akt activation. The cytoplasmic domain of syndecan 4 also regulates FGF-2 signalling through its interaction with protein kinase Calpha (PKCalpha). Inhibiting PKCalpha activity with Gö6976, or knocking-down PKCalpha expression using siRNA, increased VSMC mineralisation. The HS chains displayed on syndecan 4 (and possibly other PGs) and syndecan 4's interaction with PKCalpha may therefore be required for the inhibitory effect of syndecan 4 on VSMC mineralisation.Overall these studies suggest that syndecan 4 expression is increased in mineralising VSMC to maintain FGF-2 signalling, and in turn, slow down the mineralisation process.