Copper impedes calcification of human aortic vascular smooth muscle cells through inhibition of osteogenic transdifferentiation and promotion of extracellular matrix stability

Abstract

Vascular calcification (VC), a common pathological condition, is a strong predictor of cardiovascular events and associated mortality. Development and progression of VC heavily rely on vascular smooth muscle cells (VSMCs) and are closely related to oxidative stress, inflammation, and remodelling of extracellular matrix (ECM). Copper (Cu), an essential microelement, participates in these processes, however its involvement in pathophysiology of VC and VSMCs physiology remains poorly investigated. In the present study we analysed Cu impact on the calcification of human aortic primary VSMCs induced in vitro by treatment with high calcium and phosphate levels. Supplementation with physiological micromolar Cu significantly reduced the amount of calcium deposited on VSMCs as compared to moderate deficiency, Cu restriction with chelators or Cu excess. Moreover, optimal concentrations of Cu ions increased protein production by VSMCs, stimulated their metabolic activity, inhibited alkaline phosphatase activity associated with cell-conditioned medium and cellular lysates, and prevented osteogenic differentiation of VSMCs. RNA-seq results indicated that high calcium and phosphate treatments activated many pathways related to oxidative stress and inflammation in VSMCs at the initial stage of calcification. At the same time, expression of VSMC-specific markers and certain components of ECM were downregulated. Supplementation of calcifying cells with 10 M Cu prevented most of the transcriptomic alterations induced by high calcium and phosphate while chelation-mediated restriction of Cu greatly aggravated them. In summary, physiological concentration of Cu impedes in vitro calcification of VSMCs, prevents their osteogenic transition and minimises early phenotypic alterations induced by high calcium and phosphate, thereby underlining the importance of Cu homeostasis for the physiology of VSMCs, one of the cornerstones of cardiovascular health. Our data suggest that peculiarities of Cu metabolism and its status should be considered when developing preventive and therapeutic approaches for cardiovascular diseases.