Vascular calcification is highly correlated with cardiovascular disease mortality, often seen in type II diabetes and attributed to over 422 million cases worldwide. It is now widely accepted that VC is an active process with similarities to bone ossification, occurring within the smooth muscle cell (SMC) layer, involving the transition of SMCs from the cell cycle into a senescent phenotype, activation of osteogenic transcription factors and the loss of mineralisation inhibitors. Recent evidence demonstrates the beneficial role of Sirtuin 1 (SIRT1), an NAD+ -dependant deacetylase, in insulin sensitivity and glucose homeostasis, and suggests a link between smooth muscle cell senescence, hyperglycaemia and SIRT1 downregulation. The current study aims to investigate the therapeutic role of SIRT1 activation for the prevention of vascular calcification.Methods
Human coronary artery SMCs (hCASMCs) were incubated in high (25 mM) glucose to reflect a diabetic milieu and compared to low glucose (5 mM) and osteogenic media containing β-glycerophosphate and calcium chloride. SIRT1 was inhibited by Sirtinol and activated by SRT1720. Cellular calcification was confirmed via alizarin red staining, alkaline phosphatase activity, qPCR and western blot analysis. B-galactosidase staining and qPCR confirmed cellular senescence. SIRT1 levels within diabetic patients was confirmed via ELISA of diabetic serum and correlated with tissue histology.Results
hCASMCs, cultured in high glucose osteogenic conditions in the presence of SRT1720, showed a significant decrease in ALP activity (p<0.05) at day 4, which was sustained until day 7. A 3-fold reduction in alizarin red staining was observed (p<0.05), alongside a decline in the osteogenic transcription factor RUNX2 mRNA expression to a tenth of its control levels (p<0.05), and a reduction in RUNX2 protein by a half. Additionally, calcification associated senescence within hCASMCs was shown to significantly decline with the addition of SRT1720 (p<0.05) with P16 expression also shown to be decreased by tenfold (p<0.05). Conversely, SIRT1 inhibition via Sirtinol significantly increased ALP activity (p<0.05) with an increase in alizarin red staining by day 21 in both osteogenic and control treatments (p<0.05). RUNX2 activity was significantly increased at mRNA level (p<0.001) and at protein level, with downstream osteocalcin significantly increased (p<0.01). Sirtinol increased X-Gal staining by 20% (p<0.01) alongside P16 expression (p<0.05), the results of which are confirmed with corresponding SIRT1 siRNA knockdown.Conclusions
SIRT1 activation via small a molecule activator attenuates deposition of a calcified matrix in hCASMCs grown in diabetic conditions, via the downregulation of the RUNX2 transcription factor and subsequent downstream calcifying markers. These data suggest an essential role of SIRT1 in the protection against vascular calcification, which may be compromised within diabetic patients.