Rationale: Vascular calcification is a significant contributor to cardiovascular morbidity and mortality. We recently reported that cartilage oligomeric matrix protein (COMP) is pivotal for maintaining the homeostasis of vascular smooth muscle cells (VSMCs). Whether COMP affects the process of vascular calcification is unknown.Objective: We aimed to test whether COMP modulates vascular calcification. Methods and Results: VSMC calcification in vitro was induced by calcifying media containing high inorganic phosphate or calcium. In vivo medial vessel calcification was induced in rats by 5/6 nephrectomy with a high-phosphate diet or by periadventitial application of CaCl 2 to the abdominal aorta. COMP protein level was markedly reduced in both calcified VSMCs and arteries. COMP deficiency remarkably exacerbated VSMC calcification, whereas ectopic expression of COMP greatly reduced calcification. Furthermore, COMP knockdown facilitated osteogenic markers expression by VSMCs even in the absence of calcifying media. By contrast, COMP overexpression significantly inhibited high phosphate-or high calcium-induced VSMC osteochondrogenic transition. Induction of osteogenic marker expression by COMP silencing was reversed by a soluble form of bone morphogenetic protein (BMP)-2 receptor IA, which suggests a BMP-2-dependent mechanism. Our data revealed that COMP bound directly to BMP-2 through the C terminus, inhibited BMP-2 receptor binding, and blocked BMP-2 osteogenic signaling, indicating COMP inhibits osteochondrogenic transition of VSMCs at least partially through inhibiting BMP-2. V ascular calcification is a common complication of chronic kidney disease, atherosclerosis, and diabetes mellitus. 1,2 It is directly related to cardiovascular morbidity and mortality. 3,4 Previously viewed as an inevitable, passive, and degenerative process, vascular calcification is increasingly being considered a complex and regulated process, with great similarities to skeletal mineralization. 1,5,6 These findings have led to the important question of which cell types give rise to the skeletal elements of calcified arteries and what mechanisms regulate vascular calcification. To date, substantive studies have demonstrated that vascular smooth muscle cells (VSMCs) retain multipotential capability and can transform into osteo-/chondrocytic-like cells and express genes that are typically expressed by osteoblasts and chondrocytes during osteogenesis. [7][8][9][10] Several factors facilitate osteochondrogenic transition of VSMCs and vascular calcification, including high calcium-phosphate products, oxidative stress, bone morphogenetic proteins (BMPs) (BMP-2, -4, -6), and vitamin D. Alternatively, loss of inhibitors of mineralization, such as matrix Gla protein (MGP) and osteopontin, also predispose vascular calcification. 5,11,12 A finely tuned balance between inducers and inhibitors likely controls whether calcification occurs under pathological conditions. Other important mechanisms including apoptosis, mineral imbalOriginal received Oc...
Objective-Vascular calcification significantly increases cardiovascular morbidity and mortality. We recently reported that the deficiency of cartilage oligomeric matrix protein (COMP) leads to vascular mineralization. We characterized the COMP-degrading metalloproteinase, a disintegrin and metalloproteinase with thrombospondin motifs-7 (ADAMTS-7). Here, we tested whether ADAMTS-7 facilitates vascular calcification. Methods and Results-ADAMTS-7 expression was markedly upregulated in calcifying rat vascular smooth muscle cells (VSMCs) in vitro, calcified arteries of rats with chronic renal failure in vivo, and radial arteries of uraemic patients.Silencing of ADAMTS-7 markedly reduced COMP degradation and ameliorated VSMC calcification, whereas ectopic expression of ADAMTS-7 greatly enhanced COMP degradation and exacerbated mineralization. The transcriptional activity of ADAMTS-7 promoter was not altered by high phosphate. We used bioinformatics and quantitative polymerase chain reaction analysis to demonstrate that high-phosphate upregulated ADAMTS-7 mRNA and protein via miR-29a/b repression, which directly targeted the 3′ untranslated region of ADAMTS-7 in VSMCs. MicroRNA (MiR)-29a/b mimic markedly inhibited but miR-29a/b inhibitor greatly enhanced high-phosphate-induced ADAMTS-7 expression, COMP degradation, and subsequent VSMC calcification. ADAMTS-7 silencing significantly diminished miR-29a/b repressionexaggerated VSMC calcification. Conclusion-Our data reveal a novel mechanism by which ADAMTS-7 upregulation by miR-29a/b repression mediates vascular calcification, which may shed light on preventing cardiovascular morbidity and mortality.
Key Words: smooth muscle cells Ⅲ phenotype Ⅲ COMP Ⅲ integrin Ⅲ neointima U nlike cardiac or skeletal muscle cells, vascular smooth muscle cells (VSMCs) have a unique property of plasticity and can undergo reversible changes in phenotype. 1,2 Normally, they are mainly restricted to the media of adult blood vessels, express a repertoire of contractile proteins such as smooth muscle (SM) myosin heavy chain, SM ␣-actin, SM-22␣ and calponin, and have low rate of replication. However, on various environmental cues, VSMCs can undergo transition from a quiescent, contractile/differentiated phenotype to a synthetic/dedifferentiated phenotype, with a high rate of migration/proliferation and a concomitant reduction in expression of VSMC marker proteins. 1,3 Phenotypic switching of VSMCs plays an essential role in the development of cardiovascular diseases such as atherosclerosis, postangioplastic restenosis and hypertension. Studies have demonstrated the contribution to cell dedifferentiation of growth factors, mitogenic cytokines, reactive oxygen species, stretch or injury. 2,4,5 Nevertheless, how normal VSMCs maintain the differentiated state is much less understood and has been largely ignored. Understanding the mechanisms that conserve a differentiated phenotype is critical to interfere with the development of cardiovascular diseases. MethodsAnimal care and use of carotid-artery injury model in male SpragueDawley rats were in accordance with institutional guidelines. VSMCs were isolated from the thoracic aortic arteries of rats by collagenase digestion. Small interfering (si)RNA against COMP, ␣ 7 integrin, and ␣ 8 integrin were transfected in vitro by use of Oligofectamine (Invitrogen). The adenovirus for COMP was constructed and a single exposure of 5ϫ10 8 plaque forming units was luminally delivered to balloon-injured carotid segments for in vivo studies. The promoter constructs of SM ␣-actin-luc and SM22␣-luc was transfected into VSMCs and the relative promoter activities were detected by use of a luciferase assay kit. For cell adhesion assay, nontreated 48-well plates were coated with purified COMP, fibronectin, or polymerized collagen I and incubated with neutralizing anti-␣ 7 integrin monoclonal antibody or mouse normal IgG. The contractility of VSMCs and vessel tension measurement was examined as described previously.An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org. Results COMP Level Is Decreased in Balloon-Injured Rat Carotid ArteriesUsing a rat balloon-injury model, we first examined the expression of COMP in carotid arteries after injury. COMP protein level was markedly lower in injured vessels than in sham-operated vessels 4 to 14 days after injury and was paralleled by an increase of COMP degradation fragment (Figure 1). Combined with our previous observation that degradation of COMP promoted VSMC migration and neointima formation, 6 COMP may negatively regulate VSMC activation in response to injury. Association of COMP and VSMC Markers In VitroVSMC...
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