Medial artery calcification, which does not accompany lipid or cholesterol deposit, preferentially occurs in elderly population, but its underlying mechanisms remain unclear. In the present study, we investigated the potential role of senescent vascular smooth muscle cells (VSMCs) in the formation of senescence-associated medial calcification. Replicative senescence was induced by the extended passages (until passages 11-13) in human primary VSMCs, and cells in early passage (passage 6) were used as control young cells. VSMC calcification was markedly enhanced in the senescent cells compared with that in the control young cells. We identified that genes highly expressed in osteoblasts, such as alkaline phosphatase (ALP) and type I collagen, were significantly upregulated in the senescent VSMCs, suggesting their osteoblastic transition during the senescence. Knockdown of either ALP or type I collagen significantly reduced the calcification in the senescent VSMCs. Of note, runt-related transcription factor-2 (RUNX-2), a core transcriptional factor that initiates the osteoblastic differentiation, was also upregulated in the senescent VSMCs. Knockdown of RUNX-2 significantly reduced the ALP expression and calcification in the senescent VSMCs, suggesting that RUNX-2 is involved in the senescence-mediated osteoblastic transition. Furthermore, immunohistochemistry of aorta from the klothoaging mouse model demonstrated in vivo emergence of osteoblastlike cells expressing RUNX-2 exclusively in the calcified media. We also found that statin and Rho-kinase inhibitor effectively reduced the VSMC calcification by inhibiting Pi-induced apoptosis and potentially enhancing matrix Gla protein expression in the senescent VSMCs. These findings strongly suggest an important role of senescent VSMCs in the pathophysiology of senescence-associated medial calcification, and the inhibition of osteoblastic transition could be a new therapeutic approach for the prevention of senescence-associated medial calcification.runt-related transcription factor-2; statin VASCULAR CALCIFICATION is widespread in patients with coronary artery disease and peripheral artery disease (21) and is closely associated with the incidence of cardiovascular events as well as all-cause mortality (3,27,34). Calcification in the tunica media is often observed in elderly people and is highly correlated with their morbidity and mortality (8).Many recent findings have suggested that vascular calcification is regulated by the machinery similar to bone formation, which is accomplished through the extracellular matrix (ECM) calcification (16,21,34). During the ECM calcification, hydroxyapatite crystals that contain calcium and inorganic phosphate precipitate within the collagen fibrils (32). Many key players in the ECM calcification, such as matrix Gla protein (MGP) and alkaline phosphatase (ALP), have been identified (22,32). Inorganic pyrophosphate, a small molecule made of two phosphate ions, and MGP prevent incorporation of mineral crystals into the collagen fibrils ...

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