Hydroxyapatite nanoparticles promote mitochondrial-based pyroptosis via activating calcium homeostasis and redox imbalance in vascular smooth muscle cells

Hydroxyapatite nanoparticles (HAP) have been widely used in various fields because of their natural biological origin and functional properties. The emerging evidence on their toxicities has attracted research interest. HAP-induced vascular smooth muscle cell (VSMC) damage is a key step in vascular calcification (VC), particularly in patients with chronic kidney disease. However, the injury effects and mechanism of action of HAP on VSMCs have not been extensively investigated. This study comprehensively characterized commercially available HAP and investigated its adverse biological effects in cultured A7R5 cells.In vitroexperiments revealed that internalized HAP was localized in lysosomes, followed by the release of Ca2+owing to the low pH microenvironment. Upon Ca2+homeostasis, Ca2+enters the mitochondria, leading to the simultaneous generation of reactive oxygen species (ROS). ROS subsequently attack mitochondrial transmembrane potentials, promote mitochondrial ROS production, and oxidize mitochondrial DNA (Ox-mtDNA). Mitochondrial permeability-transition pores open, followed by the release of more Ox-mtDNA from the mitochondria into the cytosol due to the redox imbalance. This activates NLRP3/caspase-1/gasdermin D-dependent pyroptosis and finally excretes inflammatory factors to induce VC; an antioxidant could rescue this process. It has been suggested that HAP could induce an imbalance in intracellular Ca2+homeostasis in A7R5 cells, followed by the promotion of mitochondrial dysfunction and cell pyroptosis, finally enhancing VC. To detect thein vivotoxicity of HAP, mice were treated with Cy7-labelled HAP NPs for 24 h.In vivoresults also demonstrated that HAP accumulated in the kidneys, accompined with increased Ca concentration, upregulated oxidative stress-related factor and kidney damage. Overall, our research elucidates the mechanism of calcium homeostasis and redox imbalance, providing insights into the prevention of HAP-induced cell death.