Vascular calcification is a major cause of morbidity and mortality; particularly in patients with end-stage kidney disease in whom elevated phosphate levels promote mineralisation. We previously identified that Gas6/Axl signalling inhibits vascular smooth muscle cell (VSMC) mineralisation. To understand the mechanism by which this inhibition occurs, we have now investigated its effect on key events during calcification, namely VSMC osteogenic differentiation and apoptosis. In short-term assays addition of increasing concentrations of phosphate (0.9 €“ 3.4 mM) results in increased VSMC death. Treatment with Gas6 (400 ng/ml) reduces VSMC death and the amount of active caspase-3, demonstrating that Gas6 prevents phosphate-induced VSMC apoptosis. VSMC mineralisation was induced by adding 5 mM β-glycerophosphate to the culture medium. To determine the temporal pattern of expression of osteogenic markers during mineralisation, RNA was isolated every 2–3 days and qPCR was performed. These studies demonstrated that osteogenic transcription factors (Msx2, Twist and Runx2) are transiently upregulated during the early stages of calcium deposition by VSMC. To investigate the effect of Gas6/Axl signalling on VSMC osteogenic differentiation, Axl was overexpressed in VSMC using lentiviruses and Gas6 (100 ng/ml) was added. This resulted in a reduction in osteogenic marker expression and levels of active caspase-3, increased α smooth muscle actin expression and reduced calcium deposition. This study demonstrates that Axl/Gas6 signalling suppresses both VSMC osteogenic differentiation and apoptosis during phosphate-induced mineralisation and further highlights the pathway as a potential therapeutic target for the treatment of vascular calcification.
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