Calcium phosphate crystals observed during atherosclerosis have a cytotoxic effect that results in loss of calcium homeostasis

Smooth muscle cells (SMCs) promote the stability of atherosclerotic plaques. SMCs synthesize collagen that surrounds, and protects, the fibrous plaque cap. SMC death occurs within atherosclerotic plaques, contributing to plaque instability. Previous observations (1) suggest that calcium phosphate CaP crystals formed within a plaque are cytotoxic to SMCs and cause rapid necrosis that coincides with a loss of cellular calcium homeostasis. We examined changes in intracellular calcium homeostasis and cell viability in Fura-2-loaded A7r5. We observed four distinct types of response following addition of CaP crystals: 1) non-responding cells, 2) cells with transient calcium oscillations that survived 3) cells with calcium oscillations that proceeded an irreversible calcium rise and cell death and 4) an irreversible, calcium rise without any preceding oscillations. Transfection of A7r5 cells with annexins prevented the cytotoxic effect of the CaP crystals. In addition, treaments that reduced either endoplasmic reticulum calcium sequestration, or lysosomal uptake, or mitochondrial calcium uptake, reduced the cytotoxic effect of the CaP crystals. Our data supports a hypothesis where endocytosis of CaP crystals, or CaP crystal-induced cell membrane rupture, leads to a rapid loss of calcium homeostasis and cell death. Annexin-mediated membrane repair may help SMCs avoid CaP-induced cell death.