Inhibition of osteo/chondrogenic transformation of vascular smooth muscle cells by MgCl2 via calcium-sensing receptor

Objectives: The progression of vascular calcification, an active process promoted by osteo/chondrogenic transformation of vascular smooth muscle cells (VSMCs) is attenuated by activation of the calcium-sensing receptor (CASR). Recent in-vitro studies revealed that vascular calcification could be blunted by Mg2+, but the underlying mechanisms remained elusive. The present study explored whether the effects of MgCl2 on vascular calcification involve the CASR. Methods: Experiments were performed in primary human aortic smooth muscle cells (HAoSMCs) and in the mouse vascular calcification model of vitamin D3 overload. Results: Phosphate-induced calcium deposition and mRNA expression of the osteogenic markers msh homeobox 2 (MSX2), CBFA1 (core-binding factor α 1), and ALPL (tissue-nonspecific alkaline phosphatase) in HAoSMCs were blunted by additional treatment with MgCl2. MgCl2 upregulated CASR mRNA expression in HAoSMCs in a dose-dependent manner. Furthermore, the inhibitory effects of MgCl2 on phosphate-induced calcium deposition and osteogenic markers mRNA expression were mimicked by the CASR agonist GdCl3 and reversed by additional treatment with the CASR antagonist NPS-2143 or by silencing of the CASR gene in HAoSMCs. MgCl2 also blunted the osteogenic transformation of VSMCs induced by hydroxyapatite particles. High-dosed cholecalciferol treatment induced vascular calcification and upregulated aortic osteogenic markers Msx2, Cbfa1 and Alpl and collagen type I (Col1a1), collagen type III (Col3a1) and fibronectin (Fbn) mRNA expression in mice, effects reduced by additional treatment with MgCl2. These effects were paralleled by increased aortic Casr mRNA expression in cholecalciferol-treated mice, which was further augmented by MgCl2. Conclusion: The protective effects of MgCl2 on osteo/chondrogenic transformation of VSMCs and vascular calcification involve regulation of CASR and CASR-dependent signaling.