Heme oxygenase is a rate-limiting enzyme in heme degradation, leading to the generation of free iron, biliverdin, and carbon monoxide. Induction of heme oxygenase-1 is implicated in the antioxidant defense mechanism and can modulate vascular function. To test the association of microsatellite polymorphism in the promoter region of human HO-1 gene with the risk of coronary artery disease (CAD) in type 2 diabetic patients, we examined the allele frequencies of (GT) (n) repeats in HO-1 gene in 474 patients with CAD and in 322 controls. A transient-transfection assay with HO-1 promoter/luciferase fusion constructs carrying various lengths of (GT) (n) repeats was performed to explore the regulatory effect of (GT) (n) repeats on HO-1 gene expression in cultured rat aortic smooth muscle cells. Serum thiobarbituric acid-reactive substances (TBARs), a measure of lipid peroxidation, was significantly higher in subjects carrying the L/L genotype (> or =32 repeats). Among type 2 diabetic subjects, the frequencies of the L alleles and proportion of genotypes with L alleles were significantly higher in those with CAD than in those without CAD. The adjusted odds ratio for CAD in type 2 diabetic patients with L alleles was 4.7 (95% confidence interval, 1.9-12.0, P=0.001). Transfection experiments in aortic smooth muscle cells revealed that HO-1 promoter/luciferase fusion constructs containing longer (GT) (n) repeats exhibited lower transcriptional activity. These results imply that the length polymorphism in the HO-1 gene promoter modulate the transcription of the gene in vascular cells. Type 2 diabetics carrying longer (GT) (n) repeats might have higher oxidative stress and increased susceptibility to the development of CAD.

OBJECTIVE-Endothelial progenitor cells (EPCs) are impaired in diabetes. This study aimed to investigate the direct effects of high glucose on EPCs.RESEARCH DESIGN AND METHODS-Mononuclear cells isolated from healthy subjects were incubated with glucose/ mannitol or drugs for EPC study. After 4 days of culture, attached early EPCs appeared. The monolayer late EPCs with cobblestone shape appeared at 2-4 weeks. Various immunofluroscence stainings were used to characterize the early and late EPCs. Senescence assay and the activity of endothelial nitric oxide synthase (eNOS) were determined. Migration and tube formation assay were done to evaluate the capacity for vasculogenesis in late EPCs.RESULTS-Chronic incubation with high glucose but not mannitol (osmotic control) dose-dependently reduced the number and proliferation of early and late EPCs, respectively. High glucose enhanced EPC senescence and impaired the migration and tube formation of late EPCs. High glucose also decreased eNOS, FoxO1, and Akt phosphorylation and bioavailable nitric oxide (NO) in both EPCs. The effects of high glucose could be ameliorated by coincubation with NO donor sodium nitroprusside or p38 mitogen-activated protein kinase inhibitor and deteriorated by eNOS inhibitor or PI3K (phosphatidylinositol 3Ј-kinase) inhibitor. Antioxidants including vitamin C, N-acetylcysteine-and polyethylene glycol (PEG)-conjugated superoxide dismutase, and PEG-catalase had no effects, whereas pyrrolidine dithiocarbamate, diphenyleneiodonium, apocynin, and rotenone even deteriorated the downregulation of both EPCs. CONCLUSIONS-High

Background: This study examines the predictive value of a novel systemic immuneinflammation index (SII, platelet × neutrophil/lymphocyte ratio) in coronary artery disease (CAD) patients. Methods: A total of 5602 CAD patients who had undergone a percutaneous coronary intervention (PCI) were enrolled. They were divided into two groups by baseline SII score (high SII vs low SII) to analyse the relationship between SII groups and the long-term outcome. The primary outcomes were major cardiovascular events (MACE) which includes nonfatal myocardial infarction (MI), nonfatal stroke and cardiac death. Secondary outcomes included a composite of MACE and hospitalization for congestive heart failure. Results: An optimal SII cut-off point of 694.3 × 10 9 was identified for MACE in the CAD training cohort (n = 373) and then verified in the second larger CAD cohort (n = 5602). Univariate and multivariate analyses showed that a higher SII score (≥694.3) was independently associated with increased risk of developing cardiac death (HR: 2.02; 95% CI: 1.43-2.86), nonfatal MI (HR: 1.42; 95% CI: 1.09-1.85), nonfatal stroke (HR: 1.96; 95% CI: 1.28-2.99), MACE (HR: 1.65; 95% CI: 1.36-2.01) and total major events (HR: 1.53; 95% CI: 1.32-1.77). In addition, the SII significantly improved risk stratification of MI, cardiac death, heart failure, MACE and total major events than conventional risk factors in CAD patients by the significant increase in the C-index (P < .001) and reclassification risk categories by significant NRI (P < .05) and IDI (P < .05). Conclusions: SII had a better prediction of major cardiovascular events than traditional risk factors in CAD patients after coronary intervention. K E Y W O R D Scoronary artery disease, inflammation, percutaneous coronary intervention 2 of 11 | YANG et Al.

Apoptosis is regulated by specific intracellular signaling pathways. The development of cardiomyopathy involves the apoptosis of cardiomyocytes; however, the details of their apoptotic signaling are not yet known. Insulin-like growth factor I (IGF I) is an important survival growth factor for myocardium and other tissues, but the effects of IGF I on apoptotic signaling remain largely unknown. To study apoptotic signaling pathways in cardiomyocytes and to understand IGF I actions on the apoptotic signaling of cardiac muscle cells, we have defined the effects of IGF I on Bcl-2, Bax, caspase 3, DNA fragmentation, and cell survival in primary cardiomyocytes. Compared with Bax levels, the levels of Bcl-2 were found to be quite low in these cells. Serum withdrawal and doxorubicin reduced cell viability, increased fragmentation of DNA, increased cellular contents of Bax, and activated caspase 3. IGF I enhanced cell viability, suppressed DNA fragmentation, attenuated Bax induction, and suppressed caspase 3 activation. The levels of Bcl-2-associated Bax were increased after serum withdrawal and incubation with doxorubicin and were reduced by IGF I. Thus, cardiomyocyte apoptosis induced by serum withdrawal and doxorubicin likely results, in part, from the induction of Bax and activation of caspase 3, but IGF I may inhibit cardiomyocyte apoptosis by attenuating Bax induction and caspase 3 activation. These findings provide new insight into the mechanisms of cardiomyocytes apoptosis and may help elucidate how IGF I modulates apoptotic signaling in cardiac muscle.

ObjectivesPossible association between diabetes mellitus (DM) and Alzheimer’s disease (AD) has been controversial. This study used a nationwide population-based dataset to investigate the relationship between DM and subsequent AD incidence.MethodsData were collected from Taiwan’s National Health Insurance Research Database, which released a cohort dataset of 1,000,000 randomly sampled people and confirmed it to be representative of the Taiwanese population. We identified 71,433 patients newly diagnosed with diabetes (age 58.74±14.02 years) since January 1997. Using propensity score, we matched them with 71,311 non-diabetic subjects by time of enrollment, age, gender, hypertension, hyperlipidemia, and previous stroke history. All the patients were followed up to December 31, 2007. The endpoint of the study was occurrence of AD.ResultsOver a maximum 11 years of follow-up, diabetic patients experienced a higher incidence of AD than non-diabetic subjects (0.48% vs. 0.37%, p<0.001). After Cox proportional hazard regression model analysis, DM (hazard ratio [HR], 1.76; 95% confidence interval [CI], 1.50–2.07, p<0.001), age (HR, 1.11; 95% CI, 1.10–1.12, p<0.001), female gender (HR, 1.24; 95% CI, 1.06–1.46, p = 0.008), hypertension (HR, 1.30; 95% CI, 1.07–1.59, p = 0.01), previous stroke history (HR, 1.79; 95% CI, 1.28–2.50, p<0.001), and urbanization status (metropolis, HR, 1.32; 95% CI, 1.07–1.63, p = 0.009) were independently associated with the increased risk of AD. Neither monotherapy nor combination therapy with oral antidiabetic medications were associated with the risk of AD after adjusting for underlying risk factors and the duration of DM since diagnosis. However, combination therapy with insulin was found to be associated with greater risk of AD (HR, 2.17; 95% CI, 1.04–4.52, p = 0.039).ConclusionNewly diagnosed DM was associated with increased risk of AD. Use of hypoglycemic agents did not ameliorate the risk.

Background Oxidative stress activates endothelial innate immunity and disrupts endothelial functions, including eNOS-derived NO bioavailability. Here, we postulated that oxidative stress induces sterol regulatory element binding protein 2 (SREBP2) and microRNA-92a (miR-92a), which in turn activate endothelial innate immune response, leading to dysfunctional endothelium. Methods and Results Using cultured endothelial cells (ECs) challenged by diverse oxidative stresses, hypercholesterolemic zebrafish, and Ang II-infused or aged mice, we demonstrated that SREBP2 transactivation of microRNA-92a (miR-92a) is oxidative stress-inducible. The SREBP2-induced miR-92a targets key molecules in endothelial homeostasis, including Sirtuin 1, Krüppel-like factor 2 (KLF2), and KLF4, leading to NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome activation and eNOS inhibition. In EC-specific SREBP2 transgenic mice, locked nucleic acid (LNA)-modified antisense miR-92a (LNA-92a) attenuates inflammasome, improves vasodilation, and ameliorates Ang II-induced and aging-related atherogenesis. In patients with coronary artery disease, the level of circulating miR-92a is inversely correlated with EC-dependent, flow-mediated vasodilation and is positively correlated with serum level of IL-1β. Conclusions Our findings suggest that SREBP2-miR-92a-inflammasome exacerbates endothelial dysfunction during oxidative stress. Identification of this mechanism may help in diagnosis and/or treatment of disorders associated with oxidative stress, innate immune activation, and endothelial dysfunction.

Attachment to, and migration of leukocytes into the vessel wall is an early event in atherogenesis. Expression of cell adhesion molecules by the arterial endothelium may play a major role in atherosclerosis. It has been suggested that antioxidants inhibit the expression of adhesion molecules and may thus attenuate the processes leading to atherosclerosis. In the present study, the effects of a potent water-soluble antioxidant, salvianolic acid B (Sal B), and an aqueous ethanolic extract (SME), both derived from a Chinese herb, Salvia miltiorrhiza, on the expression of endothelial-leukocyte adhesion molecules by tumor necrosis factor-alpha (TNF-alpha)-treated human aortic endothelial cells (HAECs) were investigated. When pretreated with SME (50 and 100 microg/ml), the TNF-alpha-induced expression of vascular adhesion molecule-1 (VCAM-1) was notably attenuated (77.2 +/- 3.2% and 80.0 +/- 2.2%, respectively); and with Sal B (1, 2.5, 5, 10, and 20 microg/ml), 84.5 +/- 1.9%, 78.8 +/- 1.2%, 58.9 +/- 0.4%, 58.7 +/- 0.9%, and 57.4 +/- 0.3%, respectively. Dose-dependent lowering of expression of intercellular cell adhesion molecule-1 (ICAM-1) was also seen with SME or Sal B. In contrast, the expression of endothelial cell selectin (E-selectin) was not affected. SME (50 microg/ml) or Sal B (5 microg/ml) significantly reduced the binding of the human monocytic cell line, U937, to TNF-alpha-stimulated HAECs (45.7 +/- 2.5% and 55.8 +/- 1.2%, respectively). SME or Sal B significantly inhibited TNF-alpha-induced activation of nuclear factor kappa B (NF-kappaB) in HAECs (0.36- and 0.48-fold, respectively). These results demonstrate that SME and Sal B have anti-inflammatory properties and may explain their anti-atherosclerotic properties. This new mechanism of action of Sal B and SME, in addition to their previously reported inhibition of LDL, may help explain their efficacy in the treatment of atherosclerosis.

Objective-Both matrix metalloproteinases (MMPs) and endothelial progenitor cells (EPCs) have been implicated in the process of neovascularization. Here we show that the impaired neovascularization in mice lacking MMP-9 is related to a defect in EPC functions in vasculogenesis. Methods and Results-Hindlimb ischemia surgery was conducted in MMP-9 Ϫ/Ϫ mice and wild-type (MMP-9 ϩ/ϩ ) mice. Blood flow recovery was markedly impaired in MMP-9 Ϫ/Ϫ mice when compared with that in wild-type mice as determined by laser Doppler imaging. Flow cytometry demonstrated that the number of EPC-like cells (Sca-1 ϩ /Flk-1 ϩ ) in peripheral blood increased in wild-type mice after hindlimb ischemia surgery and exogenous vascular endothelial growth factor stimulation, but not in MMP-9 Ϫ/Ϫ mice. Plasma levels and bone marrow concentrations of soluble Kit-ligand (sKitL) were significantly elevated in wild-type mice in response to tissue ischemia, but not in MMP-9 Ϫ/Ϫ mice. C-kit positive bone marrow cells of MMP-9 Ϫ/Ϫ mice have attenuated adhesion and migration than those isolated from wild-type mice. In in vitro studies, incubation with selective MMP-9 inhibitor suppressed the colony formation, migration, and tube formation capacities of EPC. Transplantation of bone marrow cells from wild-type mice restored collateral flow formation in MMP-9 Ϫ/Ϫ mice. Conclusions-These findings suggest that MMP-9 deficiency impairs ischemia-induced neovascularization, and these effects may occur through a reduction in releasing the stem cell-active cytokine, and EPC mobilization, migration, and vasculogenesis functions.