Human Vascular Smooth Muscle Cells Undergo Vesicle-Mediated Calcification in Response to Changes in Extracellular Calcium and Phosphate Concentrations

Abstract

Abstract. Patients with ESRD have a high circulating calcium (Ca) ϫ phosphate (P) product and develop extensive vascular calcification that may contribute to their high cardiovascular morbidity. However, the cellular mechanisms underlying vascular calcification in this context are poorly understood. In an in vitro model, elevated Ca or P induced human vascular smooth muscle cell (VSMC) calcification independently and synergistically, a process that was potently inhibited by serum. Calcification was initiated by release from living VSMC of membrane-bound matrix vesicles (MV) and also by apoptotic bodies from dying cells. Vesicles released by VSMC after prolonged exposure to Ca and P contained preformed basic calcium phosphate and calcified extensively. However, vesicles released in the presence of serum did not contain basic calcium phosphate, co-purified with the mineralization inhibitor fetuin-A and calcified minimally. Importantly, MV released under normal physiologic conditions did not calcify, and VSMC were also able to inhibit the spontaneous precipitation of Ca and P in solution. The potent mineralization inhibitor matrix Gla protein was found to be present in MV, and pretreatment of VSMC with warfarin markedly enhanced vesicle calcification. These data suggest that in the context of raised Ca and P, vascular calcification is a modifiable, cell-mediated process regulated by vesicle release. These vesicles contain mineralization inhibitors derived from VSMC and serum, and perturbation of the production or function of these inhibitors would lead to accelerated vascular calcification.Patients with ESRD develop extensive medial calcification, or Monckeberg's sclerosis, that causes increased arterial stiffness and contributes to the high cardiovascular mortality (1,2). Calciphylaxis is an increasingly common and life-threatening form of calcification characterized by destructive calcification in the media of subcutaneous arterioles, leading to occlusion and subsequent widespread tissue necrosis (2,3). The precise pathophysiology of vascular calcification in ESRD is unknown, but risk factors include age, hypertension, time on dialysis, and, most significant, abnormalities in calcium (Ca) and phosphate (P) metabolism (4,5). Normal serum concentrations of Ca and inorganic P ions are metastable with respect to basic calcium phosphate (BCP; a mixture of octacalcium phosphate, dicalcium phosphate dihydrate, and apatite) precipitation but can support growth of nascent crystals. In ESRD, systemic Ca and inorganic P concentrations typically exceed 2.4 and 2.0 mM, respectively (4). Consequently, calcification in ESRD has traditionally been ascribed to supersaturation and subsequent precipitation of mineral ions. This has led to therapeutic measures to reduce the Ca/P product aimed mostly at reduction of P.Recent studies have shown that vascular calcification is a regulated process similar to bone formation (6,7). VSMC in the normal artery wall constitutively express potent inhibitors of calcification, such as matrix Gla ...

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