Cellular Responses to Calcified Particles, their Pathological Consequences, and Investigations into How They Can Be Avoided

Sharma, Kumar (2021). Cellular Responses to Calcified Particles, their Pathological Consequences, and Investigations into How They Can Be Avoided. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.00099481

Abstract

Smooth muscle cells are critical in maintaining the stability of atherosclerotic plaques. Necrosis of smooth muscle cells was found to be increased in advanced atherosclerotic lesions concomitant with precipitation of calcium-phosphate particles (Bennett, Sinha et al. 2016). Previous studies have shown that these calcium-phosphate particles interact with smooth muscle cell and are processed intracellularly through acidic vesicles or other mechanisms (Ewence, Bootman et al. 2008). Whilst the calcium-phosphate particles are believed to be deleterious for plaque stability, the reasons are not fully clear.

This study examined the cytotoxic effect of calcium-phosphate particles on smooth muscle cells. A consistent observation was that the calcium-phosphate particles triggered rapid smooth muscle cell death by causing an irreversible increase in cytosolic calcium and membrane rupture. A key aim of this study was to find ways in which the pathological calcium signals and loss of cell integrity caused by calcium-phosphate particles could be reduced. Consequently, pharmacological modulators of cellular calcium signalling and membrane repair proteins were used to test the hypothesis that smooth muscle cells could be protected from the deleterious actions of calcium-phosphate particles.

Detailed characterisation of cellular calcium responses indicated that calcium-phosphate particles trigger four distinct responses in cells, 1) cells that did not respond to CaP during a 45-minute incubation; 2) cells that responded to CaP, but survived a 45-minute incubation; 3) cells that died during a 45-minute incubation with CaP incubation and displayed Ca²⁺ oscillation(s) before the point of death; 4) cells that died during a 45-minute incubation CaP incubation, but did not display any Ca²⁺ oscillation. The cytotoxic effect of calcium-phosphate particles in smooth muscle cells was reduced by a number of treatments, including bafilomycin, increased inositol 1, 4, 5-trisphosphate hydrolysis, and over-expression of annexins. However, inhibiting the sarco-endoplasmic reticulum pump, store-operated calcium channels, or disrupting the mitochondrial membrane potential did not alter the cytotoxic effect of calcium-phosphate particles. These data indicate that either reducing cytotoxic calcium signalling or increasing the membrane repair capacity of smooth muscle cells may plausibly enable smooth muscle cells within atherosclerotic plaques to withstand the accumulation of calcium-phosphate precipitations and thereby promote plaque stability.

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