Tsuyoshi Tabuchi

Effect of atorvastatin on microRNA 221 / 222 expression in endothelial progenitor cells obtained from patients with coronary artery disease.

Background  Endothelial progenitor cells (EPCs) play an important role in the maintenance of vascular integrity. Lipid lowering therapy (LLT) with statins may contribute to biologically relevant activities including the proliferation of endothelial cells. The physiological role of microRNA (miR)‐221/222, a newly discovered class of small RNA, is closely linked to the proliferation of endothelial cells. We therefore investigated whether LLT with statins might affect miR‐221/222 expression in EPCs obtained from patients with coronary artery disease (CAD).

Expression of miR-146a/b is associated with the Toll-like receptor 4 signal in coronary artery disease: effect of renin–angiotensin system blockade and statins on miRNA-146a/b and Toll-like receptor 4 levels

The TLR4 (Toll-like receptor 4) signal plays an important role in immunity in CAD (coronary artery disease). miR-146a/b (where miR is microRNA) regulates the TLR4 downstream molecules IRAK1 (interleukin-1-receptor-associated kinase 1) and TRAF6 (tumour-necrosis-factor-receptor-associated factor 6). It has also been reported that statins and RAS (renin-angiotensin system) inhibition and have anti-atherosclerotic properties. In the present study, we have investigated whether miR-146a/b was expressed with the TLR4 signal in CAD patients, and whether combined treatment with a statin and RAS inhibition might affect these levels. A total of 66 patients with CAD and 33 subjects without CAD (non-CAD) were enrolled. Patients with CAD were randomized to 12 months of combined treatment with atorvastatin and telmisartan [an ARB (angiotensin II receptor blocker)] or atorvastatin and enalapril [an ACEI (angiotensin-converting enzyme inhibitor)]. PBMCs (peripheral blood mononuclear cells) were obtained from peripheral blood at baseline and after 12 months. Levels of miR-146a/b, IRAK1 mRNA, TRAF6 mRNA and TLR4 mRNA/TLR4 protein were significantly higher in the CAD group than in the non-CAD group (all P<0.01). Levels of miR-146a/b were positively correlated with IRAK1 mRNA and TRAF6 mRNA levels. After 12 months of treatment, these levels were markedly decreased in the ARB and ACEI groups, with the decrease in the ARB group being greater than that in the ACEI group (all P<0.05). In our 12-month follow-up study, high levels of miR-146a and TLR4 mRNA/TLR4 protein at baseline were independent predictors of cardiac events. The present study demonstrates that combined treatment with an ARB and a statin decreases miR-146a/b and the TLR4 signal in CAD patients, possibly contributing to the anti-atherogenic effects of ARBs and statins in this disorder.

Expression of microRNA-208 is associated with adverse clinical outcomes in human dilated cardiomyopathy.

BACKGROUND Recently, microRNA-208 (miR-208) encoded by the alpha-myosin heavy chain (MHC) gene, has been shown to be involved in pathological cardiac growth, fibrosis, and up-regulation of beta-MHC expression. A recent study has also reported 2 additional myosin-expressed miRNAs (miR-208b and miR-499). The aim of this study was to determine whether miR-208, miR-208b, and miR-499 are expressed with MHC mRNA in human dilated cardiomyopathy (DCM), and whether these levels are related to left ventricular (LV) function and to clinical outcomes. METHODS AND RESULTS Endomyocardial biopsy tissues were obtained from 82 patients with DCM and 21 subjects without LV dysfunction as controls. Levels of miR-208, miR-208b, and miR-499 were higher in DCM patients than in controls. Levels of alpha-MHC mRNA were lower in patients with DCM than in controls, whereas beta-MHC mRNA levels were higher in patients with DCM compared with controls. Levels of miR-208 were correlated with beta-MHC mRNA levels and myocardial collagen volume, whereas levels of miR-208b and miR-499 showed no correlation. After a mean follow-up of 517 days, an increase in miR-208 levels was shown to be a strong predictor of clinical outcomes (RR 3.4, 95% CI 1.1-11.2). CONCLUSIONS This study suggests that myocardial expression of miR-208 is associated with MHC mRNA expression and with poor clinical outcomes in patients with DCM. We conclude that miR-208 may therefore be involved in the progression of human DCM.

Effect of intensive lipid-lowering therapy on telomere erosion in endothelial progenitor cells obtained from patients with coronary artery disease

Telomere erosion of EPCs (endothelial progenitor cells) may be a key factor in endothelial cell senescence and is highly dependent on cellular oxidative damage. The aim of the present study was to investigate whether LLT (lipid-lowering therapy) with statins could attenuate EPC telomere erosion in patients with CAD (coronary artery disease). The study included 100 patients with stable CAD and 25 subjects without CAD as controls. CAD patients were randomized to 12 months of intensive LLT with atorvastatin or moderate LLT with pravastatin. EPCs were obtained from peripheral blood at baseline and after 12 months of statin therapy. Telomere length in EPCs was measured by FISH (fluorescence in situ hybridization) and oxidative DNA damage by flow cytometry of oxidized DNA bases. EPC telomere length was shorter in the CAD group than in the controls, and oxidative DNA damage to EPCs was higher in the CAD group compared with controls. After 12 months of therapy, changes in lipid profiles were greater in the intensive LLT group than in the moderate LLT group. Intensive LLT markedly increased EPC number and decreased oxidative DNA damage in EPCs (both P<0.05), with no change in telomere length. In contrast, moderate LLT did not change EPC counts or oxidative DNA damage, but showed telomere shortening (P<0.05). There was a weak negative correlation between changes in EPC number and LDL (low-density lipoprotein)-cholesterol levels after intensive LLT, whereas there was no correlation between them after moderate LLT. With in vitro culturing of EPCs subjected to oxidative stress, atorvastatin led to the prevention of EPC telomere shortening compared with pravastatin. In conclusion, the present study has demonstrated that intensive LLT may prevent EPC telomere erosion in patients with CAD, possibly contributing to the beneficial effects of intensive LLT in this disorder.

Cellular and molecular mechanisms of statins: an update on pleiotropic effects.

Coronary artery disease (CAD) is the leading cause of death worldwide. The efficacy and safety of statins (3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors) in primary and secondary prevention of CAD are confirmed in several large studies. It is well known that statins have some pleiotropic, anti-atherosclerotic effects. We review the molecular mechanisms underlying the beneficial effects of statins revealed in recently published studies. Endothelial cell injury is regarded as the classic stimulus for the development of atherosclerotic lesions. In addition, the inflammatory process plays an important role in the aetiology of atherosclerosis. In particular, chronic inflammation plays a key role in coronary artery plaque instability and subsequent occlusive thrombosis. Our previous reports and others have demonstrated beneficial effects of statins on endothelial dysfunction and chronic inflammation in CAD. A better understanding of the molecular mechanism underlying the effectiveness of statins against atherosclerosis may provide a novel therapeutic agent for the treatment of coronary atherosclerosis. The present review summarizes the cellular and molecular mechanism of statins against coronary atherosclerosis.

Expression of let-7i is associated with Toll-like receptor 4 signal in coronary artery disease: Effect of statins on let-7i and Toll-like receptor 4 signal

Toll-like receptor (TLR) 4 signal plays an important role in immunity in coronary artery disease (CAD). A recent report has demonstrated that one of the let-7 family microRNAs, let-7i, directly regulates Toll-like receptor 4 (TLR4) expression and contributes to immune response. The aim of this study was to determine whether let-7i is expressed with TLR4 in patients with CAD, and whether statins (atorvastatin or rosuvastatin) might affect these levels. To determine the effects of let-7i on TLR4 expression, human THP-1 cells transfected with let-7i were analyzed for TLR4 levels. This study included 98 patients with CAD and 48 subjects without CAD (non-CAD). Patients with CAD were randomized to 12 months of treatment with atorvastatin or rosuvastatin. Monocytes were obtained from peripheral blood at baseline and after 12 months of each type of therapy. Levels of let-7i and TLR4 were measured by real-time RT-PCR and FACS. Functional approaches to let-7i showed that transfection of let-7i into human THP-1 cells resulted in regulation of TLR4 expression. Levels of let-7i were lower in the CAD group than in the non-CAD group (0.98±0.42 vs. 4.65±1.21, P<0.01). There was a negative correlation between let-7i and TLR4 levels in patients with CAD (let-7i vs. TLR4 mRNA: r=-0.60, P<0.01; let-7i vs. TLR4 MFI: r=-0.32, P<0.01). The atorvastatin group had markedly increased let-7i levels and diminished TLR4 levels (all P<0.01), whereas the rosuvastatin group showed no change in these levels. This study suggests that atorvastatin down-regulates TLR4 signal via let-7i expression in CAD patients, possibly contributing to the beneficial effects of atorvastatin on let-7i-mediated TLR4 signal in this disorder.

Circulating Toll-like receptor 4-responsive microRNA panel in patients with coronary artery disease: results from prospective and randomized study of treatment with renin-angiotensin system blockade.

The extracellular miRNAs circulate in the bloodstream and may serve as novel diagnostic and therapeutic biomarkers. The aim of the present study was to investigate circulating Toll-like receptor 4 (TLR4)-responsive miRNA expression in patients with coronary artery disease (CAD) and to examine the effects of renin-angiotensin system (RAS) blockade and statins on miRNA levels. This study included 41 patients with CAD and 20 subjects without CAD (non-CAD). Plasma TLR4-responsive miRNA samples were analysed using a microarray assay for 1700 human miRNA. The candidate miRNAs were verified with real-time reverse transcription (RT)-PCR. Patients with CAD were randomized to 12 months of combined treatment with either telmisartan and atorvastatin [angiotensin II receptor blocker (ARB)] or enalapril and atorvastatin [angiotensin-converting enzyme inhibitor (ACEI)]. Plasma samples were obtained from peripheral blood at baseline and after 12 months. The microarray assay showed significant differences in seven TLR4-responsive miRNAs between the CAD and non-CAD groups (P<0.05). Real-time PCR verified that miR-31, miR-181a, miR-16 and miR-145 were significantly lower in the CAD group than in the non-CAD group (P<0.01). Levels of TLR4 protein were higher in the CAD group than in the non-CAD group (P<0.01) and were negatively correlated with levels of TLR4-responsive miRNAs. Receiver operating characteristic (ROC) curve analysis revealed that a panel of these four miRNAs was sensitive and specific enough to distinguish CAD from non-CAD [area under the curve (AUC)=0.93, 95% CI (confidence interval)=0.99-0.87]. Both ARB and ACEI groups showed increased TLR4-responsive miRNAs and diminished levels of TLR4 protein (P<0.05). Changes in miRNAs and TLR4 levels were greater in the ARB group than in the ACEI group (P<0.05). Circulating TLR4-responsive miRNAs including miR-31, miR-181a, miR-16 and miR-145 were significantly lower in patients with CAD compared with controls and these miRNAs may be involved in the pathogenesis of CAD.

Prospective, randomized, single-blind comparison of effects of 6 months of treatment with telmisartan versus enalapril on high-molecular-weight adiponectin concentrations in patients with coronary artery disease

BACKGROUND High-molecular-weight (HMW) adiponectin has important antiatherosclerotic properties. OBJECTIVES This study compared circulating HMW adiponectin concentrations and other parameters between patients with coronary artery disease (CAD) and participants without CAD. We investigated whether treatment with statins and either telmisartan or enalapril might affect HMW adiponectin and other parameters in patients with CAD. Finally, adiponectin concentrations were compared after 6 months of treatment between CAD patients with versus without cardiac events. METHODS Consecutive patients with stable CAD admitted to our hospital (Iwate Medical University School of Medicine, Iwate, Japan) for percutaneous coronary intervention (PCI) and stent implantation and with no previous treatment with renin-angiotensin system blockers or statins were recruited. Patients with CAD who met all eligibility criteria were randomly assigned using computer-generated numbers in a 1:1 ratio to receive telmisartan (40 mg/d) or enalapril (5 mg/d) for 6 months. In addition, all patients with CAD were treated with atorvastatin (10 mg/d). The patients without CAD received no treatment with telmisartan, enalapril, or atorvastatin. Plasma concentrations of total and HMW adiponectin were measured using a highly sensitive ELISA system before PCI or drug treatment (ie, baseline) and after 6 months of treatment. In addition, high-sensitivity C-reactive protein (hs-CRP) and homeostasis model assessment of insulin resistance (HOMA-IR) were measured. To evaluate cardiac events, follow-up coronary angiography was performed at least 6 months after PCI. RESULTS This study included 70 patients with stable CAD (mean [SD] age, 65.8 [10.9] years; male/female ratio, 55/15) and 25 participants with normal results on coronary angiography (non-CAD) (mean age, 63.5 [11.2] years; male/female ratio, 20/5). Baseline concentrations (mean [SD]) of HMW adiponectin were significantly lower in the CAD group than in the non-CAD group (2.0 [0.3] vs 9.2 [0.5] microg/mL; P < 0.01). The ratio of HMW to total adiponectin was also lower in the CAD group than in the non-CAD group (0.37 [0.02] vs 0.53 [0.02]; P < 0.01). Baseline concentrations of HMW adiponectin were negatively correlated with hs-CRP (r = -0.60) and HOMA-IR (r = -0.30) in patients with CAD. After 6 months of treatment, the telmisartan group showed significantly increased HMW adiponectin concentrations and HMW/total adiponectin ratio (HMW, 3.7 [0.7] vs 2.1 [0.5] microg/mL; P < 0.01 vs baseline; HMW/total, 0.44 [0.02] vs 0.39 [0.02]; P < 0.05 vs baseline), whereas HOMA-IR was significantly decreased (2.86 [1.93] vs 3.39 [1.77]; P < 0.05 vs baseline). HOMA-IR at follow-up was significantly lower in the telmisartan group than in the enalapril group (2.86 [1.93] vs 3.64 [1.45]; P < 0.05). In contrast, treatment with enalapril was not associated with any significant changes in total or HMW adiponectin concentrations, HMW/total adiponectin ratio, or HOMA-IR. Both the telmisartan and the enalapril groups showed significant decreases in hs-CRP after 6 months (P < 0.05 vs baseline). After 6 months of treatment with either telmisartan or enalapril, HMW adiponectin concentrations were 0.7 (0.2) microg/mL with cardiac events versus 3.2 (0.4) microg/mL without (P < 0.05); HMW/total concentrations were 0.25 (0.03) with cardiac events versus 0.43 (0.01) without (P < 0.01). In contrast, hs-CRP concentrations were higher in patients with cardiac events than in those without cardiac events (2.42 [0.52] vs 1.86 [0.45] log10 microg/dL; P < 0.01). CONCLUSION This study found that treatment with telmisartan and statins (but not enalapril and statins) was associated with a significant increase in HMW adiponectin concentrations and a decrease in insulin resistance in these patients with CAD.