Vascular calcification is the formation of bone-like structures in the vessel wall. The pathology is characterised by osteogenic differentiation of vascular smooth muscle cells (VSMCs) and mineralisation of the extracellular matrix. Tumour necrosis factor-stimulated gene-6 (TSG-6) is an inflammation-associated protein that has been implicated in the inhibition of bone formation and thus, we hypothesised, may also influence vascular calcification.
Confluent VSMCs were cultured with 3 mM beta-glycerophosphate (BGP) to induce mineralisation. Calcification was confirmed by staining with alizarin red. Osteogenic differentiation was demonstrated by the up-regulation of osteogenic markers (e.g. Runx2 and Msx2) and down-regulation of smooth muscle cell markers (e.g. aSMA) by qPCR. TSG-6 mRNA expression is significantly up-regulated (6-fold, p < 0.0005) during the early stages of BGP-induced VSMC mineralisation, in comparison to control. TSG-6 was knocked-down using siRNA and reduced expression (>86%) confirmed using qPCR and western blotting. Loss of TSG-6 expression significantly increased mineralisation (1.6-fold, p < 0.001) and accelerated osteogenic differentiation. Knock-down of basal TSG-6 expression in VSMCs promoted a mature smooth muscle phenotype, as shown by up-regulation of contractile markers (smooth muscle myosin heavy chain, aSMA, SM22a and calponin) using qPCR and western blotting. On-going studies aim to determine the mechanism by which these effects occur.
In conclusion, TSG-6 is a novel regulator of vascular calcification, which can modulate both mineralisation and osteogenic differentiation of VSMCs. Our data also suggest that basal TSG-6 expression is crucial in maintaining the VSMC synthetic state, as loss of basal TSG-6 promotes a mature, contractile VSMC phenotype.