Bo-Kyung Son

Statins Protect Human Aortic Smooth Muscle Cells From Inorganic Phosphate-Induced Calcification by Restoring Gas6-Axl Survival Pathway

Vascular calcification is clinically important in the development of cardiovascular disease. It is reported that hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (statins) inhibited vascular calcification in several clinical trials. However, the mechanism is poorly understood. Recently, it has been suggested that apoptosis is one of the important processes regulating vascular smooth muscle cell (VSMC) calcification. In this study, we investigated the effect of statins on VSMC calcification by testing their effect on apoptosis, focusing in particular on regulation of the survival pathway mediated by growth arrest-specific gene 6 (Gas6), a member of the vitamin K–dependent protein family, and its receptor, Axl. In human aortic smooth muscle cells (HASMC), statins significantly inhibited inorganic phosphate (Pi)-induced calcification in a concentration-dependent manner (reduced by 49% at 0.1 &mgr;mol/L atorvastatin). The inhibitory effect of statins was mediated by preventing apoptosis, which was increased by Pi in a concentration-dependent manner, and not by inhibiting sodium-dependent phosphate cotransporter (NPC) activity, another mechanism regulating HASMC calcification. Furthermore, the antiapoptotic effect of statins was dependent on restoration of Gas6, whose expression was downregulated by Pi. Restoration of Gas6 mRNA by statins was mediated by mRNA stabilization, and not by an increase in transcriptional activity. Suppression of Gas6 using small interfering RNA and the Axl-extracellular domain abolished the preventive effect of statins on Pi-induced apoptosis and calcification. These data demonstrate that statins protected HASMC from Pi-induced calcification by inhibiting apoptosis via restoration of the Gas6-Axl pathway.

Androgen Receptor-Dependent Activation of Endothelial Nitric Oxide Synthase in Vascular Endothelial Cells: Role of Phosphatidylinositol 3-Kinase/Akt Pathway

The mechanisms of testosterone-induced vasodilatation are not fully understood. This study investigated the effect of testosterone on nitric oxide (NO) synthesis and its molecular mechanism using human aortic endothelial cells (HAEC). Testosterone at physiological concentrations (1-100 nm) induced a rapid (15-30 min) increase in NO production, which was associated with phosphorylation and activation of endothelial NO synthase (eNOS). Then, the involvement of the androgen receptor (AR), which is abundantly expressed in HAEC, was examined. The effect of testosterone on eNOS activation and NO production were abolished by pretreatment with an AR antagonist nilutamide and by transfection with AR small interference RNA. In contrast, testosterone-induced eNOS phosphorylation was unchanged by pretreatment with an aromatase inhibitor or by transfection with ERalpha small interference RNA. 5alpha-Dihydrotestosterone, a nonaromatizable androgen, also stimulated eNOS phosphorylation. Next, the signaling cascade that leads to eNOS phosphorylation was explored. Testosterone stimulated rapid phosphorylation of Akt in a time- and dose-dependent manner, with maximal response at 15-60 min. The rapid phosphorylation of eNOS or NO production induced by testosterone was inhibited by Akt inhibitor SH-5 or by phosphatidylinositol (PI) 3-kinase inhibitor wortmannin. Co-immunoprecipitation assays revealed a testosterone-dependent interaction between AR and the p85alpha subunit of PI3-kinase. In conclusion, testosterone rapidly induces NO production via AR-dependent activation of eNOS in HAEC. Activation of PI3-kinase/Akt signaling and the direct interaction of AR with p85alpha are involved, at least in part, in eNOS phosphorylation.

Sirtuin 1 Retards Hyperphosphatemia-Induced Calcification of Vascular Smooth Muscle Cells

Objective— Arterial calcification is associated with cardiovascular disease as a complication of advanced atherosclerosis. Aged vascular cells manifest some morphological features of a senescent phenotype. Recent studies have demonstrated that mammalian sirtuin 1 (SIRT1), a histone deacetylase, is an exciting target for cardiovascular disease management. Here, we investigated the role of SIRT1 in a calcification model of vascular smooth muscle cells (SMCs). Methods and Results— In adenine-induced renal failure rats with hyperphosphatemia, massive calcification was induced in the aortic media. Senescence-associated &bgr;-galactosidase (SA&bgr;-gal) activity, a marker of cellular senescence, in medial SMCs was significantly increased, and its induction was positively associated with the degree of calcification. In cultured SMCs, inorganic phosphate (Pi) stimulation dose-dependently increased SA&bgr;-gal-positive cells, and Pi-induced senescence was associated with downregulation of SIRT1 expression, leading to p21 activation. The activation via SIRT1 downregulation was blunted by inhibition of Pi cotransporter. Activation of SIRT1 by resveratrol significantly reduced the senescence-associated calcification. Conversely, SIRT1 knockdown by small interfering RNA accelerated the Pi-induced SMC senescence and subsequent calcification. In addition, SIRT1 knockdown induced phenotypic change from a differentiated state to osteoblast-like cells. The senescence-related SMC calcification was completely prevented by p21 knockdown. In addition to Pi-induced premature senescence, SMCs with replicative senescence were also more sensitive to Pi-induced calcification compared with young SMCs, and this finding was attributable to augmented p21 expression. Conclusion— SIRT1 plays an essential role in preventing hyperphosphatemia-induced arterial calcification via inhibition of osteoblastic transdifferentiation. In addition, Pi-induced SMC calcification may be associated with both premature and replicative cellular senescence.

Aortic arch calcification detectable on chest X-ray is a strong independent predictor of cardiovascular events beyond traditional risk factors

OBJECTIVES Arterial calcification makes the management of hemodynamics more difficult. Some reports have previously shown that simple assessment of aortic calcification using plain radiography is associated with cardiovascular (CV) events; however, these studies simply assessed whether aortic calcification was present or absent only, without considering its extent. Here, we evaluated validity of grading aortic arch calcification (AAC) to predict new CV events. METHODS AND RESULTS We retrospectively reviewed chest X-rays in 239 asymptomatic out-patients who underwent measurement of endothelial function at the 1994-2000 without past history of CV events. The extent of AAC was divided into four grades (0-3). Among these subjects, the follow-up of CV events in 209 patients was completed. At baseline, AAC grade was positively related to age, pulse pressure, diabetes and renal dysfunction. Impairment of endothelial function, as determined by flow-mediated dilation (FMD), was also correlated to increasing AAC grade. Fifty-seven CV events in total occurred during a mean follow-up period of 69+/-45 months. With multivariate adjustment, Kaplan-Meier analysis showed that the incidence was significantly higher in patients with higher AAC grade (grades 2 and 3) than in those with grade 0 or 1 (p<0.01, log-rank test). Two kinds of multivariate Cox-proportional hazards analyses showed the predictive values of AAC grade were significant (hazard ratio, 2.49; p=0.01, 2.56; p<0.01, respectively), and the predictive power was superior to that of renal dysfunction or FMD. In addition, the prediction was valuable even in patients without CKD. CONCLUSIONS AAC detectable on chest X-ray is a strong independent predictor of CV events beyond traditional risk factors including endothelial dysfunction. Risk stratification by assessment of AAC may provide important information for management of atherosclerotic disease.

Adiponectin Antagonizes Stimulatory Effect of Tumor Necrosis Factor-α on Vascular Smooth Muscle Cell Calcification: Regulation of Growth Arrest-Specific Gene 6-Mediated Survival Pathway by Adenosine 5′-Monophosphate-Activated Protein Kinase

Adiponectin exhibits diverse protective effects against atherogenesis and antagonizes many effects of TNFalpha. Here, we investigated the effect of adiponectin and TNFalpha on vascular calcification, a critical event in the development and progression of vascular disease. In human aortic smooth muscle cells (HASMC), TNFalpha augmented inorganic phosphate (Pi)-induced calcification, whereas adiponectin significantly suppressed it and abolished the stimulatory effect of TNFalpha in a concentration-dependent manner. Similarly, adiponectin ameliorated the accelerating effect of TNFalpha on Pi-induced apoptosis, the essential process of HASMC calcification. Furthermore, these effects of TNFalpha and adiponectin were associated with AMP-activated protein kinase (AMPK)-dependent growth arrest-specific gene 6 (Gas6) expression and Akt signaling. The AMPK activator, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), induced phosphorylation of AMPK and significantly inhibited Pi-induced calcification in HASMC. Conversely, pharmacological inhibition of AMPK by compound C blocked both AMPK activation and the inhibitory effect of adiponectin on calcification, providing evidence that AMPK plays a regulatory role in vascular calcification. Reporter assay revealed that adiponectin restored Gas6 promoter activity decreased by TNFalpha, and the effect of adiponectin was abrogated by compound C. These results demonstrate that adiponectin antagonizes the stimulatory effect of TNFalpha on vascular calcification by restoration of the AMPK-dependent Gas6-mediated survival pathway.

Androgen receptor-dependent transactivation of growth arrest-specific gene 6 mediates inhibitory effects of testosterone on vascular calcification

Recent epidemiological studies have found that androgen deficiency is associated with a higher incidence of cardiovascular disease in men. However, little is known about the mechanism underlying the cardioprotective effects of androgens. Here we show the inhibitory effects of testosterone on vascular calcification and a critical role of androgen receptor (AR)-dependent transactivation of growth arrest-specific gene 6 (Gas6), a key regulator of inorganic phosphate (Pi)-induced calcification of vascular smooth muscle cells (VSMC). Testosterone and nonaromatizable androgen dihydrotestosterone inhibited Pi-induced calcification of human aortic VSMC in a concentration-dependent manner. Androgen inhibited Pi-induced VSMC apoptosis, an essential process for VSMC calcification. The effects on VSMC calcification were mediated by restoration of Pi-induced down-regulation of Gas6 expression and a subsequent reduction of Akt phosphorylation. These effects of androgen were blocked by an AR antagonist, flutamide, but not by an estrogen receptor antagonist, ICI 182,780. We then explored the mechanistic role of the AR in Gas6 expression and found an abundant expression of AR predominantly in the nucleus of VSMC and two consensus ARE sequences in the Gas6 promoter region. Dihydrotestosterone stimulated Gas6 promoter activity, and this effect was abrogated by flutamide and by AR siRNA. Site-specific mutation revealed that the proximal ARE was essential for androgen-dependent transactivation of Gas6. Furthermore, chromatin immunoprecipitation assays demonstrated ligand-dependent binding of the AR to the proximal ARE of Gas6. These results indicate that AR signaling directly regulates Gas6 transcription, which leads to inhibition of vascular calcification, and provides a mechanistic insight into the cardioprotective action of androgens.

A novel organ culture model of aorta for vascular calcification.

Vascular calcification is a characteristic feature of aging, atherosclerosis, diabetes mellitus, and end-stage renal disease. The use of organ culture provides complementary information that may bridge the gap between traditional cell culture and animal models, and establishes easily controlled experimental conditions. Therefore, we investigated whether organ culture of the aorta could be used as a model of vascular calcification, applying it to animal models of other conditions. Thoracic aortas were dissected from C57BL/6 mice and cultured. To induce vascular calcification, stimulation with inorganic phosphate (Pi) was performed. Morphometric assessment of medial calcium deposition was quantitatively performed, and the amount of dissolved calcium was measured. Pi-stimulation induced calcium deposition in medial layers in a time- and dose-dependent manner. To investigate the phenotypic change of vascular smooth muscle cells (VSMC), the expression of Runx2, osterix, osteocalcin, and ALP activity were determined. Finally, to investigate the influence of Pi-stimulation on the cultured aorta in other models, aortas from streptozotocin (STZ)-induced diabetic mice, aged mice, and Sirt1 knockout (+/-) mice were dissected. These cultures showed a greater tendency for aortic calcification by Pi-stimulation than did control cultures. These results indicate that organ culture of the aorta from mice reflects the state of calcification and suggests that this model will be useful to explore the molecular mechanisms of vascular calcification and the pathology of senescence.

Thrombomodulin, a novel molecule regulating inorganic phosphate-induced vascular smooth muscle cell calcification

Hyperphosphatemia has emerged as a cardiovascular risk factor that stimulates calcification in vessels. We explored molecules that were induced by inorganic phosphate (Pi) at an early stage in vascular smooth muscle cells (VSMC). In the present study, we examined the role of thrombomodulin (TM) in Pi-induced VSMC calcification based on the results of DNA microarray analysis. Both mRNA and protein expression of TM were markedly augmented in Pi-induced calcification. Conversely, knockdown of TM by siRNA significantly inhibited calcification, in addition to Pi-induced apoptosis which plays critical roles in VSMC calcification. We further found that TM suppressed both of mRNA and protein expression of growth arrest-specific gene 6 (Gas6), a key molecule regulating apoptosis. Recombinant extracellular epidermal growth factor (EGF)-repeat domain of TM exaggerated calcification and this effect was abrogated by a neutralizing antibody for EGF receptor, suggesting that the cleaved and secreted form of TM may activate EGF receptor. We also found that downregulation of Gas6 by TM/EGF receptor axis was mediated by ERK in VSMC calcification. In the aorta of adenine-fed rat, a typical medial calcification model with hyperphosphatemia, we found that TM expression was increased. Furthermore, in human calcified aorta, increased TM expression was also observed. These results indicate that TM is a novel molecule that promotes apoptosis and vascular calcification by regulation of Gas6, presumably via EGF receptor/ERK axis.

Protective effects of estrogen against vascular calcification via estrogen receptor α-dependent growth arrest-specific gene 6 transactivation

Vascular calcification is one of the major complications of cardiovascular disease and is an independent risk factor for myocardial infarction and cardiac death. Postmenopausal women have a higher prevalence of vascular calcification compared with premenopausal women, suggesting protective effects of estrogen (E2). However, the underlying mechanisms of its beneficial effects remain unclear. In the present study, we examined the inhibitory effects of E2 on vascular smooth muscle cell (VSMC) calcification, and found that growth arrest-specific gene 6 (Gas6), a crucial molecule in vascular calcification, is transactivated by estrogen receptor α (ERα) in response to E2. In human aortic smooth muscle cells, physiological levels of E2 inhibited inorganic phosphate (Pi)-induced calcification in a concentration-dependent manner. This inhibitory effect was significantly abolished by MPP, an ERα-selective antagonist, and ERα siRNA, but not by PHTPP, an ERβ-selective antagonist, and ERβ siRNA, implicating an ERα-dependent action. Apoptosis, an essential process for Pi-induced VSMC calcification, was inhibited by E2 in a concentration-dependent manner and further, MPP abolished this inhibition. Mechanistically, E2 restored the inhibited expression of Gas6 and phospho-Akt in Pi-induced apoptosis through ERα. Furthermore, E2 significantly activated Gas6 transcription, and MPP abrogated this E2-dependent Gas6 transactivation. E2-BSA failed to activate Gas6 transcription and to inhibit Ca deposition in VSMC, suggesting beneficial actions of genomic signaling by E2/nuclear ERα. Taken together, these results indicate that E2 exerts inhibitory effects on VSMC apoptosis and calcification through ERα-mediated Gas6 transactivation. These findings indicate a potential therapeutic strategy for the prevention of vascular calcification, especially in postmenopausal women.

Suppressive Role of PPARγ-Regulated Endothelial Nitric Oxide Synthase in Adipocyte Lipolysis

Introduction Metabolic syndrome causes insulin resistance and is associated with risk factor clustering, thereby increasing the risk of atherosclerosis. Recently, endothelial nitric oxide synthase deficient (eNOS-/-) mice have been reported to show metabolic disorders. Interestingly, eNOS has also been reported to be expressed in non-endothelial cells including adipocytes, but the functions of eNOS in adipocytes remain unclear. Methods and Results The eNOS expression was induced with adipocyte differentiation and inhibition of eNOS/NO enhanced lipolysis in vitro and in vivo. Furthermore, the administration of a high fat diet (HFD) was able to induce non-alcoholic steatohepatitis (NASH) in eNOS-/- mice but not in wild type mice. A PPARγ antagonist increased eNOS expression in adipocytes and suppressed HFD-induced fatty liver changes. Conclusions eNOS-/- mice induce NASH development, and these findings provide new insights into the therapeutic approach for fatty liver disease and related disorders.