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 ...

Objective-Vascular calcification is an important risk factor for cardiovascular diseases. Here, we investigated a role of dedifferentiated vascular smooth muscle cells (VSMCs) in the atherosclerotic intimal calcification. Methods and Results-We prepared human cultured VSMCs in either redifferentiatiated or dedifferentiated state and analyzed the gene expressions of bone-calcification regulatory factors. Expression of bone morphogenetic protein-2 (BMP-2), a potent initiator for osteoblast differentiation, was significantly enhanced in dedifferentiated VSMCs. Furthermore, endogenous BMP-2 antagonists, such as noggin, chordin, and matrix gamma-carboxyglutamic acid protein, were all downregulated in the dedifferentiated VSMCs. Conditioned medium from dedifferentiated VSMCs, but not from redifferentiated VSMCs, stimulated the osteoblastic differentiation of the mesenchymal progenitor C2C12 cells, which was abolished by BMP-2 knockdown. In atherosclerotic intima from apolipoprotein (apo)E-deficient mice, ␣SM-actin-positive cells, presumably dedifferentiated VSMCs, expressed BMP-2. We generated BMP-2-transgenic mice using ␣SM-actin promoter and crossed them with apoE-deficient mice (BMP-2-transgenic/apoE-knockout).Significantly accelerated atherosclerotic intimal calcification was detected in BMP-2-transgenic/apoE-knockout mice, although serum lipid concentration and atherosclerotic plaque size were not different from those in apoE-knockout mice. Enhanced calcification appeared to be associated with the frequent emergence of osteoblast-like cells in atherosclerotic intima in BMP-2-transgenic/apoE-knockout mice. V ascular calcification has been an important risk factor for cardiovascular diseases as well as all-causal mortality. 1,2 There are several types of vascular calcification, such as calcification in intima associated with atherosclerosis and calcification in tunica media (medial calcification), which is often observed in elderly people and patients with diabetes mellitus and/or chronic kidney disease. 3 Recently, we have reported an important role of senescent vascular smooth muscle cells (VSMCs) in the formation of medial calcification associated with aging. 4 However, the molecular mechanism(s) governing atherosclerotic intimal calcification remains to be elucidated. Atherosclerotic calcification of coronary artery is a significant risk for the unsuccessful coronary intervention and balloon-induced coronary artery dissection. 5 Calcification score of coronary arteries assessed by electron beam computer tomography has been reported to correlate well with the incidence of cardiovascular diseases. 6 Furthermore, calcification was found to be a reliable marker of plaque instability, defined as plaques that have undergone rupture using autopsy specimens. 7 However, there is a controversy around this with arguments describing calcification as a marker of plaque stability as well. Negative correlation between extensive calcification and plaque instability was shown, and the pattern of calcification rather than the vo...

Endothelial apoptosis is a pivotal process for angiogenesis during embryogenesis as well as postnatal life. By using a retrovirusmediated signal sequence trap method, we identified a previously undescribed gene, termed ARIA (apoptosis regulator through modulating IAP expression), which regulates endothelial apoptosis and angiogenesis. ARIA was expressed in blood vessels during mouse embryogenesis, as well as in endothelial cells both in vitro and in vivo. ARIA is a unique protein with no homology to previously reported conserved domain structures. Knockdown of ARIA in HUVECs by using small interfering RNA significantly reduced endothelial apoptosis without affecting either cell migration or proliferation. ARIA knockdown significantly increased inhibitor of apoptosis (cIAP)-1 and cIAP-2 protein expression, although their mRNA expression was not changed. Simultaneous knockdown of cIAP-1 and cIAP-2 abolished the antiapoptotic effect of ARIA knockdown. Using yeast 2-hybrid screening, we identified the interaction of ARIA with 20S proteasome subunit ␣-7. Thereafter, we found that cIAP-1 and cIAP-2 were degraded by proteasomes in endothelial cells under normal condition. Overexpression of ARIA significantly reduced cIAP-1 expression, and this reduction was abolished by proteasomal inhibition in BAECs. Also, knockdown of ARIA demonstrated an effect similar to proteasomal inhibition with respect to not only expression but also subcellular localization of cIAP-1 and cIAP-2. In vivo angiogenesis studied by Matrigel-plug assay, mouse ischemic retinopathy model, and tumor xenograft model was significantly enhanced by ARIA knockdown. Together, our data indicate that ARIA is a unique factor regulating endothelial apoptosis, as well as angiogenesis, presumably through modulating proteasomal degradation of cIAP-1 and cIAP-2 in endothelial cells.

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Abstract-Ageing is an important risk factor for ischemic cardiovascular diseases, although its underlying molecular mechanisms remain to be elucidated. Here, we report a crucial role of Bcl-2 in the impaired angiogenic functions in senescent endothelial cells (ECs) by modulating the mitochondrial redox state. Cellular senescence impaired angiogenic functions in ECs without attenuating the mitogen-activated protein kinase or Akt signaling, and vascular endothelial growth factor receptor 2 or Tie-2 expressions. We identified that Bcl-2 expression was markedly reduced in 3 independent models for senescent ECs, and pharmacological inhibition, as well as small interfering RNA-mediated gene silencing of Bcl-2, significantly impaired the angiogenic functions in young ECs. Bcl-2 has an antioxidative role by locating the glutathione at mitochondria, and we found that mitochondrial oxidative stress was significantly augmented in senescent ECs, in association with reduced mitochondria-associated glutathione. Transfection of Bcl-2 in senescent ECs significantly reduced the mitochondrial oxidative stress, restored the mitochondrial membrane potential, and improved the angiogenic capacity. Furthermore, gene transfer of Bcl-2 using adenovirus significantly improved the in vivo angiogenesis in the Matrigel plugs implanted into aged mice, whereas the Bcl-2 inhibitor reduced the angiogenesis in the Matrigel plugs implanted into young mice. Together, Bcl-2 plays a crucial role in the regulation of the mitochondrial redox state in ECs, and, thus, loss of Bcl-2 during the senescence exacerbates the impaired angiogenesis by augmenting the mitochondrial oxidative stress.