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 ...

These data indicate that medial calcification in MS lesions is an active process potentially orchestrated by phenotypically modified VSMCs.

Objectives: To determine the mechanisms that promote mineralization of VSMC-MVs in response to calcium stress. Methods and Results: Transmission electron microscopy showed that both nonmineralized and mineralizedMVs were abundantly deposited in the extracellular matrix at sites of calcification. Using cultured human VSMCs, we showed that MV mineralization is calcium dependent and can be inhibited by BAPTA-AM. MVs released by VSMCs in response to extracellular calcium lacked the key mineralization inhibitor matrix Gla protein and showed enhanced matrix metalloproteinase-2 activity. Proteomics revealed that VSMC-MVs share similarities with chondrocyte-derived MVs, including enrichment of the calcium-binding proteins annexins (Anx) A2, A5, and A6. Biotin cross-linking and flow cytometry demonstrated that in response to calcium, AnxA6 shuttled to the plasma membrane and was selectively enriched in MVs. AnxA6 was also abundant at sites of vascular calcification in vivo, and small interfering RNA depletion of AnxA6 reduced VSMC mineralization. Flow cytometry showed that in addition to AnxA6, calcium induced phosphatidylserine exposure on the MV surface, thus providing hydroxyapatite nucleation sites. Conclusions:In contrast to the coordinated signaling response observed in chondrocyte MVs, mineralization of VSMC-MVs is a pathological response to disturbed intracellular calcium homeostasis that leads to inhibitor depletion and the formation of AnxA6/phosphatidylserine nucleation complexes. (Circ Res. 2011;109:e1-e12.) Key Words: matrix vesicles Ⅲ annexin Ⅲ calcification Ⅲ vascular smooth muscle cells Ⅲ calcium Ⅲ proteomics V ascular calcification is the deposition of apatite mineral in the medial or intimal layers of the vessel wall and is a clinically significant pathology in atherosclerosis, diabetes, chronic kidney disease, and aging. Once established, vascular calcification is progressive, particularly in association with raised levels of extracellular mineral ions such as calcium and phosphate. 1 Recent nuclear magnetic resonance studies have shown that the structural organization of the molecular components of vascular mineralizations are identical to those in bone. 2,3 This implies mechanistic similarities during the earliest phases of initiation of mineral nucleation in both tissues.During developmental osteogenesis/chondrogenesis, specialized membrane-bound bodies called matrix vesicles (MVs), which originate from the plasma membrane of chondrocytes and osteoblasts, serve as nucleation sites for hydroxyapatite. 4 In cartilage, MV production occurs throughout the growth plate, but MVs are "mineralization competent" only in the hypertrophic zone. 4 This transition is induced by an intracellular calcium signal that initiates changes in gene transcription and the subsequent release of MVs that are able to nucleate mineral to form hydroxyapatite nanocrystals. 5 Mineralization-competent MVs are enriched with the calcium-binding annexins (Anx) A2, A5, and A6 and surface Original received December 8, 2010; revisi...

Abstract-The mechanisms involved in the initiation of vascular calcification are not known, but matrix vesicles, the nucleation sites for calcium crystal formation in bone, are likely candidates, because similar structures have been found in calcified arteries. The regulation of matrix vesicle production is poorly understood but is thought to be associated with apoptotic cell death. In the present study, we investigated the role of apoptosis in vascular calcification. We report that apoptosis occurs in a human vascular calcification model in which postconfluent vascular smooth muscle cell (VSMC) cultures form nodules spontaneously and calcify after Ϸ28 days. Apoptosis occurred before the onset of calcification in VSMC nodules and was detected by several methods, including nuclear morphology, the TUNEL technique, and external display of phosphatidyl serine. Inhibition of apoptosis with the caspase inhibitor ZVAD.fmk reduced calcification in nodules by Ϸ40%, as measured by the cresolphthalein method and alizarin red staining. In addition, when apoptosis was stimulated in nodular cultures with anti-Fas IgM, there was a 10-fold increase in calcification.

Abstract-Vascular calcification is associated with an increased risk of myocardial infarction; however, the mechanisms linking these 2 processes are unknown. Studies in macrophages have suggested that calcium phosphate crystals induce the release of proinflammatory cytokines; however, no studies have been performed on the effects of calcium phosphate crystals on vascular smooth muscle cell function. In the present study, we found that calcium phosphate crystals induced cell death in human aortic vascular smooth muscle cells with their potency depending on their size and composition. Calcium phosphate crystals of approximately 1 m or less in diameter caused rapid rises in intracellular calcium concentration, an effect that was inhibited by the lysosomal proton pump inhibitor, bafilomycin A1. Bafilomycin A1 also blocked vascular smooth muscle cell death suggesting that crystal dissolution in lysosomes leads to an increase in intracellular calcium levels and subsequent cell death. These studies give novel insights into the bioactivity of calcified deposits and suggest that small calcium phosphate crystals could destabilize atherosclerotic plaques by initiating inflammation and by causing vascular smooth muscle cell death. (Circ Res. 2008;103:e28-e34.)

Abstract-The cellular and molecular events leading to calcification in atherosclerotic lesions are unknown. We and others have shown that bone-associated proteins, particularly matrix Gla protein (MGP) and osteopontin (OP), can be detected in atherosclerotic lesions, thus suggesting an active calcification process. In the present study, we aimed to determine whether human vascular smooth muscle cells (VSMCs) could calcify in vitro and to determine whether MGP and OP have a role in vascular calcification. We established that human aortic VSMCs and placental microvascular pericytes spontaneously form nodules in cell culture and induce calcification, as detected by von Kossa's method, Alizarin red S staining, and electron microscopy. The cells in calcifying nodules differed from those in monolayer cultures by expressing higher levels of the SMC markers ␣-SM actin, SM22␣, and calponin. In addition, Northern blot analysis revealed that in human VSMCs, calcification was associated with increased levels of MGP mRNA.

Summary. Background: Matrix Gla protein (MGP) is a small vitamin K-dependent protein containing five c-carboxyglutamic acid (Gla) residues that are believed to be important in binding Ca 2+, calcium crystals and bone morphogenetic protein. In addition, MGP contains phosphorylated serine residues that may further regulate its activity. In vivo, MGP has been shown to be a potent inhibitor of vascular calcification; however, the precise molecular mechanism underlying the function of MGP is not yet fully understood. Methods and results: We investigated the effects of MGP in human vascular smooth muscle cell (VSMC) monolayers that undergo calcification after exposure to an increase in Ca 2+ concentration. Increased calcium salt deposition was found in cells treated with the vitamin K antagonist warfarin as compared to controls, whereas cells treated with vitamin K 1 showed decreased calcification as compared to controls. With conformationspecific antibodies, it was confirmed that warfarin treatment of VSMCs resulted in uncarboxylated (Gla-deficient) MGP. To specifically test the effects of MGP on VSMC calcification, we used full-length synthetic MGP and MGP-derived peptides representing various domains in MGP. Full length MGP, the ccarboxylated motif (Gla) (amino acids 35-54) and the phosphorylated serine motif (amino acids 3-15) inhibited calcification. Furthermore, we showed that the peptides were not taken up by VSMCs but bound to the cell surface and to vesicle-like structures. Conclusions: These data demonstrate that both cglutamyl carboxylation and serine phosphorylation of MGP contribute to its function as a calcification inhibitor and that MGP may inhibit calcification via binding to VSMC-derived vesicles.

Abstract. Iyemere VP, Proudfoot D, Weissberg PL, Shanahan CM. (University of Cambridge, UK). Vascular smooth muscle cell phenotypic plasticity and the regulation of vascular calcification (Review).