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 ...

Objective-Mineralization-regulating proteins are found deposited at sites of vascular calcification. However, the relationship between the onset of calcification in vivo and the expression of genes encoding mineralization-regulating proteins is unknown. This study aimed to determine the temporal and spatial pattern of expression of key bone and cartilage proteins as atherosclerotic calcification progresses. Methods and Results-Using reverse transcription-polymerase chain reaction on a panel of noncalcified and calcified human arterial samples, two classes of proteins could be identified: (1) Matrix Gla protein, osteonectin, osteoprotegerin, and aggrecan were constitutively expressed by vascular smooth muscle cells (VSMCs) in the normal vessel media but downregulated in calcified arteries whereas (2) alkaline phosphatase, bone sialoprotein, osteocalcin, and collagen II were expressed predominantly in the calcified vessel together with Cbfa1, Msx2, and Sox9, transcription factors that regulate expression of these genes. In the calcified plaque in situ hybridization identified subsets of VSMCs expressing osteoblast and chondrocyte-like gene expression profiles whereas osteoclast-like macrophages were present around sites of calcification. Conclusions-These observations suggest a sequence of molecular events in vascular calcification beginning with the loss of expression by VSMCs, of constitutive inhibitory proteins, and ending with expression by VSMCs and macrophages of chondrocytic, osteoblastic, and osteoclastic-associated proteins that orchestrate the calcification process. Key Words: calcification Ⅲ atherosclerosis Ⅲ osteoblast Ⅲ cartilage Ⅲ vascular smooth muscle cell Ⅲ macrophage V ascular calcification occurs as a complication of atherosclerosis and involves the nucleation of hydroxyapatite (HA) on membrane-bound vesicles and the local expression/ deposition of bone-associated, mineralization-regulating proteins. [1][2][3] Thus, it shares fundamental similarities with developmental osteogenesis and a feature of many end-stage calcified lesions is the presence of bone trabeculae and/or cartilage-like cells in the vessel wall. 4,5 Until recently little was known of the function of bone-associated proteins in the vasculature, but gene knockout (KO) and in vitro studies have demonstrated that many of them regulate vascular smooth muscle cell (VSMC) phenotype and/or inhibit HA crystal growth. 6 -8 Moreover, the vascular phenotypes of the matrix Gla protein (MGP) and osteoprotegerin (OPG) KOs suggest that, in the normal vascular media, calcification is actively inhibited. 8,9 Studies of human medial calcification (Monckeberg's sclerosis) in diabetes and aging have suggested that the VSMCs that predominate in these lesions lose expression of calcification inhibitors, such as MGP, and begin to express "late" differentiation markers of both osteoblasts (bone sialoprotein; BSP) and osteocalcin (bone Gla protein; BGP) and chondrocytes (collagen II; COLII). 4 This implies that vascular calcification may be caused by ph...

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...

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