Vascular calcification is the formation of bone and cartilage within blood vessels. It involves the osteogenic differentiation of vascular smooth muscle cells (VSMCs), VSMC apoptosis, calcifying matrix vesicle release and extracellular matrix mineralisation. This disease process has the potential to be regulated by proteoglycans (PGs), which comprise a protein core and glycosaminoglycan (GAG) chains. Syndecan 4 is a transmembrane PG, which has been previously implicated in the regulation of growth factor signalling and apoptosis, both of which play an important role in vascular calcification. This study tests the hypothesis that GAGs, and syndecan 4 specifically, regulate vascular calcification.
VSMCs were cultured with 3 mM β-glycerophosphate (β-GP) to induce mineralisation. Controls were cultured without β-GP. Osteogenic differentiation was confirmed by the up-regulation of Runx2, Msx2 and BMP-2, and down-regulation of αSMA and SM22α mRNA. During VSMC osteogenic differentiation and mineralisation, the mRNA levels of specific enzymes involved in the biosynthesis of heparan sulphate (HS) (including NDST1, P < 0.001) and chondroitin/dermatan sulphate (CS/DS) (including C4ST1 and DSEL, both P < 0.001) were up-regulated in β-GP-treated VSMCs 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 DS to CS, and CS/DS 4-O-sulphation was increased in mineralising VSMCs. VSMC mineralisation was also associated with shortening of HS chains (˜50% decrease).
Syndecan 4 displays HS and/or CS/DS chains. The mRNA expression levels of syndecan 4 were significantly increased (˜5-fold; P < 0.001) in β-GP-treated VSMCs at the late stages of mineralisation. Knocking-down syndecan 4 expression using two different siRNAs significantly increased VSMC mineralisation in the presence of β-GP (˜2-fold; P < 0.01). Syndecan 4 regulates FGF-2 signalling, and interestingly, FGF-2 mRNA was also increased (˜19-fold; P < 0.001) in β-GP-treated VSMCs at the late stages of mineralisation. In response to exogenous FGF-2 (50 ng/ml), Akt activation was reduced in syndecan 4 siRNA VSMCs when compared to controls. FGF-2 inhibits VSMC mineralisation, and this ability was partially lost in the absence of syndecan 4.
Together, these studies demonstrate that VSMC mineralisation is associated with changes in CS/DS chain composition. We also demonstrate for the first time that syndecan 4 plays a role in VSMC mineralisation. Studies are now underway to characterise syndecan 4 expression in human calcified aortic sections.
- Vascular calcification
- Syndecan 4
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