Comparison of the inhibitory mechanisms of diethyl citrate, sodium citrate, and phosphonoformic acid on calcification induced by high inorganic phosphate contents in mouse aortic smooth muscle cells

Abstract This study aimed to investigate the differences and inhibitory effects of diethyl citrate (Et2Cit), sodium citrate (Na3Cit), and phosphonoformic acid (PFA) on calcification induced by high inorganic phosphate (Pi) contents in mouse aortic smooth muscle cells (MOVAS) and to develop drugs that can induce anticoagulation and inhibit vascular calcification (VC). Alive and fixed MOVAS were assessed for 14 days in the presence of high Pi with increasing Et2Cit, Na3Cit, and PFA concentrations. Calcification on MOVAS was measured through Alizarin red staining and the deposited calcium amount; apoptosis was detected by annexin V staining; and cell transdifferentiation was examined by measuring smooth muscle lineage gene (α-SMA) expression and alkaline phosphatase activity. Coincubation of MOVAS with Et2Cit, Na3Cit, and PFA significantly decreased Pi-induced VC in live MOVAS, and the apoptotic rate was reduced by low inhibitor concentrations. The 3 inhibitors could prevent the alkaline phosphatase activity induced by high Pi contents and increased the expression of α-smooth muscle actin genes. Thus, the transdifferentiation of MOVAS into osteoblast-like cells was blocked. Their inhibitory effects exhibited concentration dependence. The inhibitory effect of each inhibitor at the same concentration showed the following trend: PFA > Na3Cit > Et2Cit. Et2Cit, Na3Cit, and PFA prevented the calcification of MOVAS and inhibited the osteochondrocytic conversion of vascular smooth muscle cells. Thus, Et2Cit and Na3Cit as anticoagulants may alleviate VC in clinical applications.