Masahiro Koide

Replicative senescence of vascular smooth muscle cells enhances the calcification through initiating the osteoblastic transition

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 klotho(-/-) aging mouse model demonstrated in vivo emergence of osteoblast-like 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 P(i)-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.

Paracrine Osteogenic Signals via Bone Morphogenetic Protein-2 Accelerate the Atherosclerotic Intimal Calcification In Vivo

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, &agr;SM-actin-positive cells, presumably dedifferentiated VSMCs, expressed BMP-2. We generated BMP-2-transgenic mice using &agr;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. Conclusion—Our findings collectively demonstrate an important role of dedifferentiated VSMCs in the pathophysiology of atherosclerotic calcification through activating paracrine BMP-2 osteogenic signals.

Prorenin induces ERK activation in endothelial cells to enhance neovascularization independently of the renin-angiotensin system.

Prorenin is an enzymatically inactive precursor of renin, and its biological function in endothelial cells (ECs) is unknown despite its relevance with the incidence of diabetic microvascular complications. Recently, (pro)renin receptor was identified, and the receptor-associated prorenin system has been discovered, whereas its expression as well as function in ECs remain unclear. In the present study, we found that ECs express the (pro)renin receptor, and that prorenin provoked ERK activation through (pro)renin receptor independently of the renin-angiotensin system (RAS). Prorenin stimulated the proliferation, migration and tube-formation of ECs, while it inhibited endothelial apoptosis induced by serum and growth factor depletion. MEK inhibitor abrogated these proangiogenic effects of prorenin, while AT1 receptor antagonist or angiotensin-converting enzyme inhibitor failed to block them. In vivo neovascularization in the Matrigel-plugs implanted into mouse flanks was significantly enhanced by prorenin, in which significant ERK activation was detected in ECs. Furthermore, tumor xenografts stably transfected with prorenin demonstrated the significantly accelerated growth rate concomitantly with enhanced intratumoral neovascularization. Our data demonstrated that the RAS-independent (pro)renin receptor-mediated signal transduction plays a pivotal role in the regulation of ECs function as well as in the neovascularization, and thus prorenin is potentially involved in the pathophysiology of diabetic microvascular complications as well as cancers.

Identification of ARIA regulating endothelial apoptosis and angiogenesis by modulating proteasomal degradation of cIAP-1 and cIAP-2

Endothelial apoptosis is a pivotal process for angiogenesis during embryogenesis as well as postnatal life. By using a retrovirus-mediated 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 α-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.

Apoptosis regulator through modulating IAP expression (ARIA) controls the PI3K/Akt pathway in endothelial and endothelial progenitor cells

Endothelial and endothelial progenitor cells (ECs and EPCs) play a fundamental role in angiogenesis that is essential for numerous physiological and pathological processes. The phosphatase and tensin homolog (PTEN)/ phosphoinositide 3-kinase (PI3K) pathway has been implicated in angiogenesis, but the mechanism in the regulation of this pathway in ECs and EPCs is poorly understood. Here we show that ARIA (apoptosis regulator through modulating IAP expression), a transmembrane protein that we recently identified, regulates the PTEN/PI3K pathway in ECs and EPCs and controls developmental and postnatal angiogenesis in vivo. We found that ARIA is abundantly expressed in EPCs and regulates their angiogenic functions by modulating PI3K/Akt/endothelial nitric oxide synthase (eNOS) signaling. Genetic deletion of ARIA caused nonfatal bleeding during embryogenesis, in association with increased small vessel density and altered expression of various vascular growth factors including angiopoietins and VEGF receptors. Postnatal neovascularization induced by critical limb ischemia was substantially enhanced in ARIA-null mice, in conjunction with more bone marrow (BM)-derived ECs detected in ischemic muscles. Administration of PI3K or NO synthase inhibitor completely abolished the enhanced neovascularization in ARIA−/− mice. Mechanistically, we identified that ARIA interacts with PTEN at the intracellular domain independently of the PTEN phosphorylation in its C-terminal tail. Overexpressed ARIA increased PTEN in the membrane fraction, whereas ARIA-silencing reduced the membrane-associated PTEN, resulting in modified PI3K/Akt signaling. Taken together, our findings establish a previously undescribed mode of regulation of the PTEN/PI3K/Akt pathway by ARIA, and reveal a unique mechanism in the control of angiogenesis. These functions of ARIA might offer a unique therapeutic potential.

Loss of Bcl-2 During the Senescence Exacerbates the Impaired Angiogenic Functions in Endothelial Cells by Deteriorating the Mitochondrial Redox State

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.

Manipulation of Cardiac Phosphatidylinositol 3-Kinase (PI3K)/Akt Signaling by Apoptosis Regulator through Modulating IAP Expression (ARIA) Regulates Cardiomyocyte Death during Doxorubicin-induced Cardiomyopathy

Background: The PI3K/Akt signaling regulates many aspects of cardiomyocyte homeostasis. Results: ARIA regulates cardiac PI3K/Akt signaling and modifies cardiomyocyte death and stress-induced cardiac dysfunction. Conclusion: ARIA is a novel factor involved in the regulation of cardiac PI3K/Akt signals. Significance: ARIA-mediated manipulation of cardiac PI3K/Akt signaling is an intriguing therapeutic target to treat cardiac dysfunction. PI3K/Akt signaling plays an important role in the regulation of cardiomyocyte death machinery, which can cause stress-induced cardiac dysfunction. Here, we report that apoptosis regulator through modulating IAP expression (ARIA), a recently identified transmembrane protein, regulates the cardiac PI3K/Akt signaling and thus modifies the progression of doxorubicin (DOX)-induced cardiomyopathy. ARIA is highly expressed in the mouse heart relative to other tissues, and it is also expressed in isolated rat cardiomyocytes. The stable expression of ARIA in H9c2 cardiac muscle cells increased the levels of membrane-associated PTEN and subsequently reduced the PI3K/Akt signaling and the downstream phosphorylation of Bad, a proapoptotic BH3-only protein. When challenged with DOX, ARIA-expressing H9c2 cells exhibited enhanced apoptosis, which was reversed by the siRNA-mediated silencing of Bad. ARIA-deficient mice exhibited normal heart morphology and function. However, DOX-induced cardiac dysfunction was significantly ameliorated in conjunction with reduced cardiomyocyte death and cardiac fibrosis in ARIA-deficient mice. Phosphorylation of Akt and Bad was substantially enhanced in the heart of ARIA-deficient mice even after treatment with DOX. Moreover, repressing the PI3K by cardiomyocyte-specific expression of dominant-negative PI3K (p110α) abolished the cardioprotective effects of ARIA deletion. Notably, targeted activation of ARIA in cardiomyocytes but not in endothelial cells reduced the cardiac PI3K/Akt signaling and exacerbated the DOX-induced cardiac dysfunction. These studies, therefore, revealed a previously undescribed mode of manipulating cardiac PI3K/Akt signaling by ARIA, thus identifying ARIA as an attractive new target for the prevention of stress-induced myocardial dysfunction.

Ecscr regulates insulin sensitivity and predisposition to obesity by modulating endothelial cell functions

Insulin resistance is closely associated with obesity and is one of the earliest symptoms of type-2 diabetes. Endothelial cells are involved in the pathogenesis of insulin resistance through their role in insulin delivery and adipose tissue angiogenesis. Here we show that Ecscr (endothelial cell surface expressed chemotaxis and apoptosis regulator; also known as ARIA), the transmembrane protein that regulates endothelial cell signalling, is highly expressed in white and brown adipose tissues, and regulates energy metabolism and glucose homeostasis by modulating endothelial cell functions. Ecscr-deficient mice fed a normal chow show improved glucose tolerance and enhanced insulin sensitivity. We demonstrate that Ecscr deletion enhances the insulin-mediated Akt/endothelial nitric oxide synthase activation in endothelial cells, which increases insulin delivery into the skeletal muscle. Ecscr deletion also protects mice on a high-fat diet from obesity and obesity-related metabolic disorders by enhancing adipose tissue angiogenesis. Conversely, targeted activation of Ecscr in endothelial cells impairs glucose tolerance and predisposes mice to diet-induced obesity. Our results suggest that the inactivation of Ecscr enhances insulin sensitivity and may represent a new therapeutic strategy for treating metabolic syndrome.

Cardio-Cerebrovascular Protective Effects of Valsartan in High-Risk Hypertensive Patients With Coronary Artery Disease (from the Kyoto Heart Study)

The objective was to examine whether previous coronary artery disease (CAD) influences the add-on effects of the angiotensin II receptor blocker (ARB) valsartan on cardio-cerebrovascular morbidity and mortality in high-risk hypertensive patients who participated in the Kyoto Heart Study. The primary end point was the same as in the main study: a composite of new-onset and/or worsening of cardiovascular and cerebrovascular events. Median follow-up was 3.27 years. According to the presence of previous CAD at baseline, the study population was divided into 2 groups (with CAD, n = 707; without CAD, n = 2,324) in which primary end-point events occurred more frequently in patients with CAD than in patients without CAD (15.1% vs 5.6%, hazard ratio [HR] 2.68, 95% confidence interval [CI] 2.11 to 3.42). Add-on valsartan significantly decreased the occurrence of the primary end-point events in patients with CAD (11.3% vs 19.0%, HR 0.59, 95% CI 0.41 to 0.85) and without CAD (3.7% vs 7.6%, HR 0.49, 95% CI 0.34 to 0.70) compared to non-ARB treatment. In the presence of previous CAD, patients with valsartan add-on treatment had a significantly lower prevalence of angina pectoris and stroke than those with non-ARB treatment, whereas the valsartan add-on effects on angina and stroke were not significant in the absence of CAD. Changes in blood pressure during the follow-up period did not differ significantly between study subgroups. In conclusion, in the presence or absence of previous CAD, valsartan add-on treatment prevented more cardio-cerebrovascular events than conventional non-ARB treatment in high-risk hypertensive patients. In addition, valsartan add-on treatment conferred not only an antianginal effect but also stroke prevention exclusively in hypertensive patients with CAD compared to those without CAD.

WITHDRAWN: Cardio‐cerebrovascular protective effects of valsartan in high-risk hypertensive patients with overweight/obesity: A post-hoc analysis of the KYOTO HEART Study

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