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 (Et₂Cit) and sodium citrate (Na₃Cit) 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 Ca²⁺ concentration, leading to lysosomal rupture and cell necrosis. On addition of the anticoagulant Et₂Cit or Na₃Cit, cell viability and mitochondrial membrane potential increased, whereas the amount of LDH released, ROS, and apoptosis rate decreased. Et₂Cit and Na₃Cit could also chelate with Ca²⁺ to inhibit the intracellular Ca²⁺ elevations induced by nano-HAp, prevent lysosomal rupture, and reduce cell necrosis. High concentrations of Et₂Cit and Na₃Cit exhibited strong inhibitory effects. The inhibitory capacity of Na₃Cit was stronger than that of Et₂Cit at similar concentrations.
Conclusion: Both Et₂Cit and Na₃Cit 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. Et₂Cit and Na₃Cit may play a role as anticoagulants in reducing injury to the vascular wall caused by nano-HAp.
Keywords: vascular calcification, hydroxyapatite, diethyl citrate, sodium citrate, cardiovascular disease