5-aza-2′-Deoxycytidine, a DNA Methyltransferase Inhibitor, Facilitates the Inorganic Phosphorus-Induced Mineralization of Vascular Smooth Muscle Cells

Aim: Vascular calcification, an independent risk factor for cardiovascular disease in patients with chronic kidney disease (CKD), refers to the mineralization of vascular smooth muscle cells (VSMCs) caused by phenotypic changes toward osteoblast-like cells. DNA methylation, mediated by DNA methyltransferases (DNMTs), plays an important role in the differentiation of osteoblasts. We herein assessed the effects of a DNMT inhibitor on phenotypic changes in VSMCs and the development of vascular calcification. Methods: The effects of 5-aza-2'-deoxycytidine (5-aza-dC), a DNMT inhibitor, on human aortic smooth muscle cells (HASMCs) were evaluated. The expression and DNA methylation status of osteogenic genes were determined using RT-qPCR and bisulfite sequencing, respectively. Mineralization of HASMCs was induced by high concentrations of inorganic phosphate (Pi), as confirmed by quantitation of the calcium levels and von Kossa staining. Moreover, we examined the effects of the suppression of DNMT1 and/or alkaline phosphatase (ALP) on the mineralization of HASMCs. Results: 5-aza-dC increased the expression and activity of ALP and reduced the DNA methylation levels of the ALP promoter region in the HASMCs. In addition, both treatment with 5-aza-dC and downregulation of the DNMT1 expression promoted the Pi-induced mineralization of HASMCs. Moreover, both treatment with phosphonoformic acid (PFA), a sodium-dependent phosphate transporter inhibitor, and suppression of the ALP expression inhibited the 5-aza-dC-promoted mineralization of HASMCs. Conclusions: The present study showed that DNMT inhibitors facilitate the Pi-induced development of vascular calcification via the upregulation of the ALP expression along with a reduction in the DNA methylation level of the ALP promoter region.