Diethyl citrate and sodium citrate reduce the cytotoxic effects of nanosized hydroxyapatite crystals on mouse vascular smooth muscle cells

OBJECTIVE: This study aimed to investigate the damage mechanism of nanosized hydroxyapatite (nano-HAp) on mouse aortic smooth muscle cells (MOVASs) and the injury-inhibiting effects of diethyl citrate (Et(2)Cit) and sodium citrate (Na(3)Cit) to develop new drugs that can simultaneously induce anticoagulation and inhibit vascular calcification. METHODS: The change in cell viability was evaluated using a cell proliferation assay kit, and the amount of lactate dehydrogenase (LDH) released was measured using an LDH kit. Intracellular reactive oxygen species (ROS) and mitochondrial damage were detected by DCFH-DA staining and JC-1 staining. Cell apoptosis and necrosis were detected by Annexin V staining. Intracellular calcium concentration and lysosomal integrity were measured using Fluo-4/AM and acridine orange, respectively. RESULTS: Nano-HAp decreased cell viability and damaged the cell membrane, resulting in the release of a large amount of LDH. Nano-HAp entered the cells and damaged the mitochondria, and then induced cell apoptosis by producing a large amount of ROS. In addition, nano-HAp increased the intracellular Ca(2+) concentration, leading to lysosomal rupture and cell necrosis. On addition of the anticoagulant Et(2)Cit or Na(3)Cit, cell viability and mitochondrial membrane potential increased, whereas the amount of LDH released, ROS, and apoptosis rate decreased. Et(2) Cit and Na(3)Cit could also chelate with Ca(+) to inhibit the intracellular Ca(2+) elevations induced by nano-HAp, prevent lysosomal rupture, and reduce cell necrosis. High concentrations of Et(2)Cit and Na(3)Cit exhibited strong inhibitory effects. The inhibitory capacity of Na(3)Cit was stronger than that of Et(2)Cit at similar concentrations. CONCLUSION: Both Et(2)Cit and Na(3)Cit significantly reduced the cytotoxicity of nano-HAp on MOVASs and inhibited the apoptosis and necrosis induced by nano-HAp crystals. The chelating function of citrate resulted in both anticoagulation and binding to HAp. Et(2)Cit and Na(3)Cit may play a role as anticoagulants in reducing injury to the vascular wall caused by nano-HAp.