Vascular calcification is an established pathological process that contributes to several forms of cardiovascular morbidity—notably, atherosclerosis. The molecular mechanisms involved continue to be explored. Hepatocyte growth factor (HGF) signalling, via its receptor c-MET, has been identified in association with atherosclerotic plaque development. We have demonstrated that overexpression of HGF in human smooth muscle cells (hSMC) accelerates their mineralisation. Reports demonstrating upregulation of the notch ligand δ, and the presence of a feedback loop linked to the c-MET pathway, raise the possibility that the effects of HGF on mineralisation may be mediated via notch signalling. We aim to test the hypothesis that notch signalling is involved in Ad-HGF-induced in vitro mineralisation of hSMCs. We demonstrate accelerated mineralisation in response to adenoviral-mediated HGF overexpression, confirmed by alizarin red staining, calcium incorporation and increased alkaline phosphatase activity. In addition, we show upregulation and phosphorylation of c-MET and reduction of the mineralisation inhibitor osteopontin. We identify upregulation of the notch-3 intracellular domain via western blot analysis and, using immunocytochemistry, show an altered distribution of notch-3 in Ad-HGF-infected cells. Finally, we show (i) an attenuation of mineralisation in hSMCs following overexpression of NK4, the c-MET antagonist and (ii) that treatment of hSMCs with DAPT, the notch inhibitor, also decreased the rate of mineralisation compared with Ad-HGF infected cells and controls. These findings suggest a link with the notch pathway as a possible downstream effector of HGF and further elucidate a novel mechanism underpinning vascular calcification.
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