Katsuya Iijima

Sirt1 inhibitor, Sirtinol, induces senescence-like growth arrest with attenuated Ras–MAPK signaling in human cancer cells

The induction of senescence-like growth arrest has emerged as a putative contributor to the anticancer effects of chemotherapeutic agents. Clinical trials are underway to evaluate the efficacy of inhibitors for class I and II histone deacetylases to treat malignancies. However, a potential antiproliferative effect of inhibitor for Sirt1, which is an NAD+-dependent deacetylase and belongs to class III histone deacetylases, has not yet been explored. Here, we show that Sirt1 inhibitor, Sirtinol, induced senescence-like growth arrest characterized by induction of senescence-associated β-galactosidase activity and increased expression of plasminogen activator inhibitor 1 in human breast cancer MCF-7 cells and lung cancer H1299 cells. Sirtinol-induced senescence-like growth arrest was accompanied by impaired activation of mitogen-activated protein kinase (MAPK) pathways, namely, extracellular-regulated protein kinase, c-jun N-terminal kinase and p38 MAPK, in response to epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I). Active Ras was reduced in Sirtinol-treated senescent cells compared with untreated cells. However, tyrosine phosphorylation of the receptors for EGF and IGF-I and Akt/PKB activation were unaltered by Sirtinol treatment. These results suggest that inhibitors for Sirt1 may have anticancer potential, and that impaired activation of Ras–MAPK pathway might take part in a senescence-like growth arrest program induced by Sirtinol.

Cilostazol Inhibits Oxidative Stress–Induced Premature Senescence Via Upregulation of Sirt1 in Human Endothelial Cells

Objective—Cilostazol, a selective inhibitor of PDE3, has a protective effect on endothelium after ischemic vascular damage, through production of nitric oxide (NO). The purpose of the present study was to clarify the molecular mechanisms underlying the preventive effect of treatment with cilostazol on oxidative stress–induced premature senescence in human endothelial cells. Methods and Results—Prematurely senescent human umbilical vein endothelial cells (HUVECs) were induced by treatment with hydrogen peroxide (H2O2) as judged by senescence-associated &bgr;-galactosidase assay (SA-&bgr;gal), cell morphological appearance, and plasminogen activator inhibitor-1 (PAI-1) expression. Treatment with H2O2 caused 93% of the cells to be SA-&bgr;gal positive, whereas 46% of cilostazol (100 &mgr;mol/L)-treated cells were positive. HUVECs treated with other cAMP-elevating agents and DETA-NO showed a reduction of SA-&bgr;gal–positive cells as well. Cilostazol increased phosphorylation of Akt at Ser473 and of endothelial nitric oxide synthase (eNOS) at Ser1177, with a dose-dependent increase in Sirt1 expression. Moreover, the effect of cilostazol on premature senescence was abrogated through inhibition of Sirt1. Conclusions—Our results indicated that cilostazol exerted protective effects against endothelial senescence and dysfunction, and enhancement of NO production is a key mediator in upregulation of Sirt1.

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.

Low Testosterone Level Is an Independent Determinant of Endothelial Dysfunction in Men

We investigated whether a low plasma testosterone level is related to endothelial dysfunction in men with coronary risk factors. One hundred and eighty-seven consecutive male outpatients (mean age±SD: 47±15 years) who underwent measurement of flow-mediated vasodilation (FMD) of the brachial artery using ultrasonography were enrolled. The relationship between plasma hormones and FMD was analyzed. Total and free testosterone and dehydroepiandrosterone-sulfate (DHEA-S) were significantly correlated with %FMD (r=0.261, 0.354 and 0.295, respectively; p<0.001), while estradiol and cortisol were not. %FMD in the highest quartile of free testosterone was 1.7-fold higher than that in the lowest quartile. Multiple regression analysis revealed that total and free testosterone were related to %FMD independent of age, body mass index, hypertension, hyperlipidemia, diabetes mellitus and smoking (β=0.198 and 0.247, respectively; p<0.01), and were independent of age, body mass index, systolic blood pressure, total cholesterol, high-density lipoprotein cholesterol, fasting plasma glucose, smoking and nitroglycerin-induced dilation (β=0.196 and 0.227, respectively; p<0.01). DHEA-S was not significantly related to %FMD in multivariate analysis. In conclusion, a low plasma testosterone level was associated with endothelial dysfunction in men independent of other risk factors, suggesting a protective effect of endogenous testosterone on the endothelium.

Amelioration of Vascular Endothelial Dysfunction in Obstructive Sleep Apnea Syndrome by Nasal Continuous Positive Airway Pressure

BACKGROUND Asymmetric NG,NG-dimethylarginine (ADMA) is an endogenous inhibitor of endothelial nitric oxide (NO) synthase and its plasma concentration is elevated in patients with cardiovascular risk factors, including hyperlipidemia, hypertension, diabetes, and hyperhomocysteinemia. Obstructive sleep apnea syndrome (OSAS) has been attracting attention as a risk factor for cardiovascular disorders because it often accompanies hypertension, obesity, glucose impairment, and dyslipidemia, all of which are factors in metabolic syndrome and risk factors for cardiovascular disease. METHODS AND RESULTS In the present study, flow-mediated vasodilatation (FMD) of the brachial artery and plasma concentrations of ADMA were measured before and after nasal continuous positive airway pressure (nCPAP) therapy, which abrogates apnea, in 10 male patients aged 36-69 years old, who were given a diagnosis of OSAS by polysomnography. The percent FMD (%FMD) improved significantly from 3.3+/-0.3% to 5.8+/-0.4% (p<0.01) and 6.6+/-0.3% (p<0.01), before, 1 week, and 4 weeks after nCPAP, respectively. At the same time, the plasma NOx concentrations, metabolites of NO, tended to increase, but the plasma ADMA concentration decreased inversely to %FMD and NOx. A negative correlation between %FMD and plasma ADMA concentration, and a positive correlation between %FMD and plasma NOx concentrations were observed. CONCLUSION Nasal CPAP improves endothelial function, in part by the decreasing ADMA concentration, thereby potentiating NO production.

Induction of Endothelial Nitric Oxide Synthase, SIRT1, and Catalase by Statins Inhibits Endothelial Senescence Through the Akt Pathway

Objective—Statins (3-hydroxy-3-methylglutaryl–coenzyme A reductase inhibitors) have pleiotropic vascular protective effects besides cholesterol lowering. Recently, experimental and clinical studies have indicated that senescence of endothelial cells is involved in endothelial dysfunction and atherogenesis. Therefore, the present study was performed to determine whether statins would reduce endothelial senescence and to clarify the molecular mechanisms underlying the antisenescent property of statins. Methods and Results—Senescent human umbilical vein endothelial cells were induced by hydrogen peroxide (H2O2), as judged by senescence-associated &bgr;-galactosidase assay and cell morphological appearance. Atorvastatin, pravastatin, and pitavastatin inhibited the oxidative stress induced-endothelial senescence. These statins phosphorylated Akt at Ser473 and subsequently led to increased expression of endothelial nitric oxide synthase (eNOS), SIRT1, and catalase. Treatment with LY294002 or Akt short interfering RNA decreased the eNOS activation, SIRT1 expression, and antisenescent property of atorvastatin. Moreover, in streptozotocin-diabetic mice, administration of pitavastatin increased eNOS, SIRT1, and catalase expression and decreased endothelial senescence, but levels remained unaltered in Sirt1 knockout mice. Conclusion—Our results indicate that treatment with statins inhibits endothelial senescence and that enhancement of SIRT1 plays a critical role in prevention of endothelial senescence through the Akt pathway, a direct target of statins.

Low testosterone level as a predictor of cardiovascular events in Japanese men with coronary risk factors

OBJECTIVE Recent epidemiological studies have found that testosterone deficiency is associated with higher mortality largely due to cardiovascular (CV) disease in community-dwelling older men. We investigated whether a low plasma testosterone level could predict cardiovascular events in middle-aged Japanese men with coronary risk factors. METHODS One hundred and seventy-one male outpatients (30-69 years old, mean+/-SD=48+/-13 years) who had any coronary risk factor (hypertension, diabetes, dyslipidemia, smoking, and obesity) without a previous history of CV disease were followed up. At baseline, the subjects underwent examination of coronary risk factors, measurement of flow-mediated dilation (FMD) of the brachial artery as an indicator of vascular endothelial function and assays of plasma total testosterone, dehydroepiandrosterone-sulfate (DHEA-S), estradiol and cortisol. RESULTS During the mean follow-up period of 77 months, a total of 20 CV events occurred. Kaplan-Meier survival analysis by tertile of plasma hormone levels revealed that the subjects with the lowest testosterone tertile were more likely to develop CV events than those with the highest tertile (P<0.01 by log-rank test). Cox proportional hazards models showed that the subjects with the lowest tertile of plasma testosterone (<14.2 nmol/L) had an approximately 4-fold higher CV event risk compared to those with the higher testosterone tertiles after adjustment for coronary risk factors including medication and FMD (unadjusted hazard ratio, 3.61; 95% CI, 1.47-8.86: multivariate-adjusted hazard ratio, 4.61; 95% CI, 1.02-21.04). Multivariate analysis did not show any significant association of DHEA-S, estradiol or cortisol with CV events. CONCLUSIONS A low plasma testosterone level is associated with CV events in middle-aged Japanese men, independent of coronary risk factors and endothelial function. This is the first report to show the relationship between endogenous testosterone and CV events in Asian population.

Impact of Blood Pressure Variability on Cardiovascular Events in Elderly Patients with Hypertension

Blood pressure variability is one of the characteristic features of hypertension in the elderly. However, its clinical significance remains to be determined. We therefore examined the impact of blood pressure variability on the development of cardiovascular events in elderly hypertensive patients. A total of 106 consecutive hypertensive patients aged more than 60 years old (mean age, 73.9±8.1 years old; male, 54%), all of whom underwent 24-h ambulatory blood pressure monitoring, were followed up (median, 34 months; range, 3−60 months). During the follow-up period, 39 cardiovascular events were observed, including 14 cases of cerebral infarction and 7 cases of acute myocardial infarction. The coefficient of variation (CV) of 24-h systolic blood pressure (SBP) values was used as an index of blood pressure variability. The patients showed a mean CV value of 10.6%, and were divided into two groups according to this mean value as a cut-off point: a high CV group (n=46) and a low CV group (n=60). Although baseline clinical characteristics were similar in the two groups, Kaplan-Meier plots for event-free survival revealed that the rate of cardiovascular events was significantly higher in high CV group than in low CV group (p<0.05). Coxs proportional hazards analysis showed that increased blood pressure variability (a high CV value of 24-h SBP) was an independent predictive variable for cardiovascular events. The CV value of daytime SBP and the SD value of both 24-h SBP and daytime SBP also had positive correlations with the onset of cardiovascular events. These results suggest that increased blood pressure variability may be an independent risk factor for cardiovascular events in elderly hypertensive patients.

Polypharmacy as a risk for fall occurrence in geriatric outpatients

Objective:  To investigate the predictors of falls, such as comorbidity and medication, in geriatric outpatients in a longitudinal observational study.

Development of a simple screening test for sarcopenia in older adults

To develop a simple screening test to identify older adults at high risk for sarcopenia.