acei mi146 TRAF

Clinical Science (2010)119,395–405(Printed in Great Britain)doi:10.1042/CS20100003

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

T oll-like receptor 4levels

Yuji TAKAHASHI,Mamoru SATOH,Yoshitaka MINAMI,Tsuyoshi TABUCHI,Tomonori ITOH and Motoyuki NAKAMURA

Division of Cardiology,Department of Internal Medicine and Memorial Heart Center,Iwate Medical University School of Medicine,Uchimaru 19-1,Morioka 020-8505,Iwate,Japan

A B S T R A C T

The TLR4(T oll-like receptor 4)signal plays an important role in immunity in CAD (coronary artery disease).miR-146a/b (where miR is microRNA)regulates the TLR4downstream 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 TLR4signal in CAD patients,and whether combined treatment with a statin and RAS inhibition might affect these levels.A total of 66patients with CAD and 33subjects without CAD (non-CAD)were enrolled.Patients with CAD were randomized to 12months 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 12months.Levels of miR-146a/b ,IRAK1mRNA,TRAF6mRNA and TLR4mRNA/TLR4protein were signi?cantly higher in the CAD group than in the non-CAD group (all P <0.01).Levels of miR-146a/b were positively correlated with IRAK1mRNA and TRAF6mRNA levels.After 12months 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 TLR4mRNA/TLR4protein 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 TLR4signal in CAD patients,possibly contributing to the anti-atherogenic effects of ARBs and statins in this disorder.

Key words:angiotensin-converting enzyme inhibitor (ACEI),angiotensin II receptor blocker (ARB),atherosclerosis,interleukin-1-receptor-associated kinase 1(IRAK1),microRNA (miRNA),Toll-like receptor 4(TLR4),tumour-necrosis-factor-receptor-associated factor 6(TRAF6).

Abbreviations:ACEI,angiotensin-converting enzyme inhibitors;ACS,acute coronary syndrome;AngII,angiotensin II;ARB,AngII receptor blocker;BMI,body mass index;BP ,blood pressure;CAD,coronary artery disease;CI,con?dence interval;FBS,fetal bovine serum;GAPDH,glyceraldehyde-3-phosphate dehydrogenase;HbA 1c ,glycated haemoglobin;HDL,high-density lipoprotein;hsCRP ,high-sensitivity C-reactive protein;IL-1,interleukin-1;IRAK1,IL-1-receptor-associated kinase 1;LDL,low-density lipoprotein;MFI,mean ?uorescence intensity;miRNA (miR),microRNA;NF-κB,nuclear factor κB;PBMC,peripheral blood mononuclear cell;PCI,percutaneous coronary intervention;RAS,renin–angiotensin system;t-BHP ,t-butyl hydroperoxide;TLR,Toll-like receptor;TNF,tumour necrosis factor;TRAF6,TNF-receptor-associated factor.Correspondence:Dr Mamoru Satoh (email m_satoh@imu.ncvc.go.jp).

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C l i n i c a l S c i e n c e

TLRs(Toll-like receptors)have been identi?ed as key recognition components of pathogen-associated molecular patterns controlling innate immune responses in mammals[1].Our previous reports have demonstrated that activation of the TLR4signal is involved in the downstream release of in?ammatory cytokines in circulating monocytes obtained from patients with CAD (coronary artery disease)[2,3].It has therefore been suggested that a potential pathophysiological link exists between the TLR4signal and immune response in coronary atherosclerosis.

It has been reported that miRNAs/miRs(microRNAs) are the most abundant family of small non-coding RNAs and regulate mRNA translation of target genes through the RNA interference pathway[4,5]. Taganov et al.[6]have demonstrated a role for one human miRNA family,miR-146,in controlling TLR4and its downstream cytokine signalling in a human monocytic cell line.In particular,miR-146a/b regulates the NF-κB(nuclear factorκB)-dependent genes,such as IRAK1[IL-1(interleukin-1)-receptor-associated kinase1]and TRAF6[TNF(tumour necrosis factor)-receptor-associated factor6],through a negative-feedback regulation loop[6].Recent reports have indicated elevated miR-146expression in patients with chronic in?ammatory diseases,such as rheumatoid arthritis and psoriasis,which have been shown to have increased levels of in?ammatory cytokines[7,8].It has become evident that coronary atherosclerosis is an in?ammatory disease involving an immune response during its initiation and progression[9].However,it is unclear whether monocytic expression of miR-146a/b is related to the TLR4signal in patients with CAD.

Clinical studies have reported that RAS(renin–angiotensin system)blockades including ARBs [AngII(angiotensin II)receptor blockers]and ACEIs (angiotensin-converting enzyme inhibitors),have an anti-atherosclerotic effect in patients with cardiovascular risk factors or CAD[10,11].A recent report found that ARB treatment reduced TLR4expression in PBMCs (peripheral blood mononuclear cells)obtained from patients with CAD[12].It has also been reported that statins(3-hydroxy-3-methylglutaryl-CoA reductase inhibitors)have pleiotropic effects including anti-in?ammatory properties[13].Moreover,an experimental study has shown that RAS inhibitors and statins have a combined effect on in?ammatory pro?les,such as in?ammatory cytokines and atherosclerosis[14]. However,it remains uncertain whether combined treatment with RAS inhibitors and statins affects the TLR4signalling pathway in patients with CAD.

In the present study,our aim was to determine whether miR-146a/b is expressed with its target genes(IRAK1and TRAF6)and TLR4,and whether it could be modi?ed by combined treatment with RAS inhibition and a statin by comparing the effects of an ARB with those of an ACEI in patients with CAD.

MATERIALS AND METHODS

Study population

A total of66patients with stable CAD were admitted to our hospital for PCI(percutaneous coronary intervention)and stent implantation,and none had previous exposure to treatment with RAS inhibitors and statins.All of the enrolled patients underwent initial diagnostic coronary angiography.CAD was diagnosed on the basis of(i)a history of typical chest pain on effort, (ii)documented exercise-induced myocardial ischaemia, (iii)angiographically proven CAD,and(iv)the absence of ACS(acute coronary syndrome)for3months before blood sampling.Patients were excluded from the study if they had clinical signs of acute infection,severe renal failure(serum levels of creatine>3mg/dl),secondary hypertension or rheumatoid disease,or if they were suspected of having a malignant or primary wasting disorder.

The non-CAD group comprised33subjects with suspected CAD on the basis of symptoms and/or minor ECG changes.Subsequent coronary angiography and close clinical examination failed to show any evidence of CAD,and these subjects were thus designated as non-CAD.In addition,blood samples were obtained from ?ve healthy volunteers for in vitro study(?ve males;age, 39.3+?9.5years).

Approval for the study protocol was obtained from the ethics committee of the Iwate Medical University School of Medicine(H17-73).All patients agreed to participate in the study and written informed consent was obtained in each case.

Study design

The present study was designed as a prospective randomized investigator-blinded study.The investigators were blinded to the allocation of each subject’s treatment group and thus did not know whether patients were receiving the study medication.Patient with CAD who met all of the eligibility criteria were randomly assigned using computer-generated random numbers in a1:1 ratio to receive telmisartan(40mg/day)and atorvastatin (10mg/day)(ARB group)or enalapril(5mg/day)and atorvastatin(10mg/day)(ACEI group)for a period of 12months.

Blood sampling

Fasting peripheral blood was collected from patients with CAD in the morning after an overnight fast before PCI and treatment with the RAS inhibitors and the statin for baseline data and again after12months of treatment

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with RAS inhibitors and the statin.Fasting peripheral blood was also collected from the non-CAD group in the morning after an overnight fast.

Clinical follow-up study

Follow-up coronary angiography was carried out at least 12months after PCI in all patients with CAD.Cardiac events were de?ned as target lesion revascularization(the lesion segment with5mm margins from each end),target or other vessel revascularization,cardiac death and ACS.

Cell preparation

PBMCs were isolated from peripheral blood samples obtained from all subjects by Ficoll–Paque density gradi-ent centrifugation.To exclude platelet contamination, isolated PBMCs were washed and centrifuged twice with PBS containing5mM EDTA for15min at400g at 20?C[15].Isolated PBMCs were left to adhere to a plastic dish(120min at37?C)and then detached from the dish by incubation in ice-cold PBS.After washing three times with PBS,the cells were resuspended at a?nal concentration of1×106cells/ml in RPMI1640(Sigma–Aldrich).

Real-time PCR for miR-146a/b,IRAK1 mRNA,TRAF6mRNA and TLR4mRNA

Total RNA including the small RNA fraction was extracted from isolated PBMCs using a mirVana TM Paris miRNA isolation kit(Ambion),according to the manufacturer’s instructions.

Extracted RNA from isolated PBMCs and human total RNA as a control(Applied Biosystems)were initially reverse-transcribed using a High Capacity cDNA Archive kit(Applied Biosystems)and were then ampli?ed in a10μl PCR mixture and primer set for ampli?cation of human miR-146a(assay ID000468),miR-146b(assay ID001097)and U6 (assay ID001093)using TaqMan?microRNA assays, according to the manufacturer’s recommended protocol (Applied Biosystems).Levels of IRAK1(assay ID Hs00355570_m1),TRAF6(assay ID Hs00377558_m1), TLR4(assay ID Hs01061963_m1)and GAPDH (glyceraldehyde-3-phosphate dehydrogenase;assay ID Hs99999905)mRNA were ampli?ed using TaqMan?Gene Expression assays(Applied Biosystems).The ampli?cation steps consisted of denaturation at95?C, followed by40cycles of denaturation at95?C for15s and then annealing at60?C for1min.All reactions were carried out on the7500real-time PCR system (Applied Biosystem)using the TaqMan?Universal PCR master mix and assays on demand(Applied Biosystems). Relative quanti?cation was carried out using the threshold cycle(C t)method for recurrent compared with primary with U6or GAPDH as an endogenous control, and fold changes were calculated for each gene[16].Replicates with a C t>40were excluded.The assay was run in triplicate for each case to allow for assessment of technical variability.To account for PCR ampli?cation of contaminating genomic DNA,a control without reverse transcription was included.To improve the accuracy of the real-time PCR for quanti?cation, ampli?cations were performed in triplicate for each RNA sample.

Flow cytometric analysis

The amount of TLR4and CD14on the cell surface of PBMCs was measured by FACS.Isolated PBMCs were incubated with FITC-conjugated mouse anti-(human TLR4)antibodies(Santa Cruz Biotechnology)and PerCP-conjugated CD14antibody(Becton Dickinson). Isotype-matched irrelevant control IgG was used as a control(Becton Dickinson).TLR4levels in CD14-positive cells were measured by a FACScan?ow cytometer(Becton Dickinson)and are shown as the MFI (mean?uorescence intensity).

Laboratory measurements

Laboratory data were measured by standard biochem-istry methods in our hospital laboratory.All analyses were performed in the same run from samples that were frozen and stored at?80?C immediately after centrifu-gation.hsCRP(high-sensitivity C-reactive protein)was quanti?ed using a latex-enhanced immunonephelometric assay(detection range,0.035–2.200mg/dl;CardioPhase hs-CRP;Dade Behring).

Transfection with miR-146a/b mimic

and inhibitor

Human THP-1cells,an undifferentiated promonocytic cell line(A.T.C.C.,Manassas,VA,U.S.A.)were maintained by twice weekly passage in ATCC-RPMI 1460medium,10%(v/v)FBS(fetal bovine serum; Gibco BRL),100units/ml penicillin and100μg/ml streptomycin at37?C with5%CO2,and then cultured at a density of1×106cells/ml.For miR-146a/b mimic and precursor transfections,THP-1cells were transfected with20ng/ml pre-miR TM miR-146a/b precursor molecules(Ambion)and20ng/ml anti-miR TM miR-146a/b inhibitors(Ambion)using the NeoFx transfection agent(Ambion).Those cells were used for experiments24h after transfection and were exposed at each passage to0.1μM t-BHP(t-butyl hydroperoxide; Sigma–Aldrich)for an additional48h.Levels of miR-146a/b,IRAK1mRNA and TRAF6mRNA were measured using the methods described above.

Cell culture with oxidant treatment Atorvastatin(0.5μM;P?zer),telmisartan(30μM; Sigma–Aldrich)and enalapril(100mM;Sigma–Aldrich) were dissolved in DMSO(Gibco BRL;DMSO?nal

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concentration,0.05%).To induce oxidative stress, cultured PBMCs from?ve healthy volunteers were grown in RPMI1640medium(Sigma–Aldrich)with10% (v/v)heat-inactivated FBS(Gibco BRL),100units/ml penicillin,100μg/ml streptomycin and each drug or drug vehicle(0.05%DMSO)and were exposed at each passage to0.1μM t-BHP(Sigma–Aldrich)or PBS.After48h of culture,levels of miR-146a/b,IRAK1mRNA,TRAF6 mRNA and TLR4mRNA were measured as described above.

To assess cell viability,we used Trypan Blue(Gibco BRL)exclusion staining of cultured cells;cell viability was over90%using this method in all experiments.

Statistical analysis

All values are presented as means+?S.D.Kolmogorov–Smirnov analysis was performed to assess the data distribution.An unpaired Student’s t test was performed for normally distributed data,and the non-parametric Mann–Whitney U test was performed where this was not appropriate.Statistical analysis of categorical variables was also carried out usingχ2analysis and Fisher’s exact analysis.After12months of combined treatment with RAS inhibitors and statin,comparisons between the two groups(ARB group and ACEI group) were analysed using a two-way repeated measures ANOVA for normally distributed variables and using the Kruskal–Wallis test for non-normally distributed variables.When applicable,signi?cant differences were analysed further with Dunnett post-hoc tests.Pearson’s correlation coef?cients were used to examine the relationship between miR-146a/b,TLR4,IRAK1and TRAF6.Multivariate association between miR-146a/b, TLR4,IRAK1and TRAF6levels and clinical outcomes was evaluated with logistic regression models.Relative risks are reported with logistic regression models adjusted for factors that are independently associated with the outcome variable.A value of P<0.05was considered statistically signi?cant.

RESULTS

Baseline and clinical characteristics Baseline characteristics of the study populations are shown in Table1.There were no signi?cant differences in age,percentage of males,BMI(body mass index)or number of subjects with a past history of dyslipidaemia between the CAD and non-CAD groups.There were signi?cant differences in other clinical parameters between the two groups(P<0.05).

Clinical characteristics in ARB and ACEI groups

As shown in Table2,there was no signi?cant differences in baseline characteristics between the ARB Table1Baseline and clinical characteristics of study population

*P<0.05compared with the non-CAD group.N/A,not available.

CAD group Non-CAD group Characteristic(n=66)(n=33) Age(years)66.2+?9.564.2+?10.3 Male gender(n)52(79%)26(79%) BMI(kg/m2)25.1+?2.724.9+?3.0 Hypertension(n)42(64%)*13(39%) Diabetes(n)15(23%)*3(9%) Dyslipidaemia(n)24(36%)10(30%) Previous CAD(n)18(27%)*0 Smoking(n)17(26%)*5(15%) Angiographic degree of CAD(n)

Single-vessel disease38(58%)*0

Multi-vessel disease28(42%)*0 Systolic BP(mmHg)141.1+?13.1*133.8+?13.8 Diastolic BP(mmHg)81.4+?12.2*75.4+?12.3 Fasting glucose(mg/dl)123.5+?17.8*106.3+?13.9 HbA1c(%) 5.5+?0.4* 5.3+?0.4 HDL-cholesterol(mg/dl)48.8+?13.3*58.5+?15.9 LDL-cholesterol(mg/dl)114.7+?31.0*88.3+?22.6 hsCRP(mg/dl)0.421+?0.246*0.113+?0.140 Medication(n)

Aspirin66(100%)N/A Ticlopidine hydrochloride26(39%)N/A Clopidogrel sulfate40(61%)N/A

β-Blockers15(23%)N/A Calcium antagonists30(45%)N/A Nitrates28(42%)N/A

and ACEI groups.After12months of treatment,BP (blood pressure)and laboratory data[LDL(low-density lipoprotein)-cholesterol and hsCRP]were decreased in both the ARB and ACEI groups.HDL(high-density lipoprotein)-cholesterol levels were increased in both groups.

Levels of miR-146a/b,IRAK1mRNA,TRAF6 mRNA,TLR4mRNA and TLR4MFI

There was no difference in average C t of U6between the CAD and non-CAD patients(21.3+?2.1compared with 21.4+?2.0arbitrary units;P value was not signi?cant). Levels of miR-146a/b were higher in the CAD group than in the non-CAD group(P<0.01;Figure1A).

Levels of IRAK1and TRAF6mRNA were higher in the CAD group compared with the non-CAD group (both P<0.01;Figures1B).Levels of TLR4mRNA and TLR4MFI were higher in the CAD group than in the non-CAD group(both P<0.01;Figures1C and 1D).The average C t of GAPDH did not differ between the two groups(23.2+?2.7compared with23.1+?2.5

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Table2Baseline clinical characteristics and changes in these characteristics after12months in the ARB and ACEI groups

*P<0.05compared with baseline.

Characteristic ARB group(n=33)ACEI group(n=33) Age(years)65.5+?9.966.9+?9.4

Male gender(n)26(79%)26(79%)

BMI(kg/m2)24.3+?3.225.3+?2.7 Hypertension(n)20(61%)22(67%) Diabetes(n)7(21%)8(24%) Dyslipidaemia(n)12(36%)12(36%) Previous CAD(n)9(27%)9(27%) Smoking(n)9(27%)8(24%) Angiographic degree of CAD(n)

Single-vessel disease18(55%)20(60%)

Multi-vessel disease15(45%)13(40%) Systolic BP(mmHg)

Baseline141.3+?11.8141.0+?14.6 Follow-up123.2+?11.4*126.2+?15.5* Diastolic BP(mmHg)

Baseline82.6+?11.180.1+?13.2 Follow-up70.8+?10.1*70.2+?11.7* Fasting glucose(mg/dl)121.6+?20.1125.3+?15.3 HbA1c(%) 5.4+?0.3 5.5+?0.4

HDL-cholesterol(mg/dl)

Baseline48.7+?13.748.8+?13.0 Follow-up56.6+?16.5*53.5+?16.3* LDL-cholesterol(mg/dl)

Baseline116.7+?32.4112.6+?30.0 Follow-up81.5+?18.0*79.0+?21.4* hsCRP(mg/dl)

Baseline0.432+?0.2930.411+?0.193 Follow-up0.093+?0.093*0.114+?0.160* Medication(n)

Aspirin33(100%)33(100%) Ticlopidine hydrochloride13(40%)13(40%) Clopidogrel sulfate20(60%)20(60%)

β-Blockers7(21%)8(24%) Calcium antagonists16(48%)14(42%) Nitrates13(40%)15(45%)

arbitrary units in the CAD and non-CAD groups respectively).

Levels of miR-146a/b were positively correlated with IRAK1and TRAF6mRNA levels in both the CAD and non-CAD groups(miR-146a and IRAK1mRNA: r=0.72,P<0.01;miR-146a and TRAF6mRNA: r=0.79,P<0.01;miR-146b and IRAK1mRNA: r=0.76,P<0.01;miR-146b and TRAF6mRNA: r=0.82,P<0.01)(Figure2).Levels of miR-146a/b were slightly positively correlated with TLR4mRNA levels(miR-146a and TLR4mRNA:r=0.66,P<0.01; miR-146b and TLR4mRNA:r=0.67,P<0.01).Cultured cells transfected with the

miR-146a/b mimic and inhibitor

THP-1cells were transfected with the miR-146a/b mimic and inhibitor followed by challenge with t-BHP for48h.Levels of miR-146a/b expression were higher in the miR-146a/b mimic-transfected cells than the mock-transfected cells(Figure3A).On the other hand, miR-146a/b levels were lower in the miR-146a/b inhibitor-transfected cells than the mock-transfected cells (Figure3B).To determine the effects of gain-of-function or loss-of-function of miR-146a/b on IRAK1and TRAF6 expression,samples from transfected cells were analysed for levels of IRAK1and TRAF6mRNA.THP-1cells transfected with the miR-146a/b mimic had a decrease in IRAK1and TRAF6mRNA levels compared with mock-transfected cells(Figure3C).THP-1cells transfected with the miR-146a/b inhibitor had an increase in IRAK1 and TRAF6mRNA compared with mock-transfected cells(Figure3D).

In vitro study with RAS inhibitor

and statin treatment

Levels of miR-146a/b,IRAK1mRNA,TRAF6mRNA and TLR4mRNA increased in t-BHP-stimulated PBMCs compared with those treated with PBS(Figure4). These levels were decreased in t-BHP-stimulated PBMCs treated with the RAS inhibitor(telmisartan or enalapril) and atorvastatin compared with those treated with the vehicle.In addition,these levels were decreased in t-BHP-stimulated PBMCs treated with telmisartan and atorvastatin compared with those treated with enalapril and atorvastatin(Figure4).

Effect of RAS blockade and statin on

miR-146a/b,IRAK1mRNA,TRAF6mRNA and TLR4mRNA levels

Figures4and5show changes in miR-146a/b,IRAK1 mRNA,TRAF6mRNA and TLR4mRNA levels after combined treatment with RAS inhibitors and statin. There was no signi?cant difference in baseline levels of miR-146a/b,IRAK1mRNA,TRAF6mRNA and TLR4mRNA between the ARB and ACEI groups (Figures5and6).For both ARB and ACEI,combined treatment resulted in decreased miR-146a/b,IRAK1 mRNA,TRAF6mRNA and TLR4mRNA levels after 12months(all P<0.01;Figures5and6).Decreases in miR-146a/b levels,IRAK1mRNA and TRAF6mRNA after treatment were greater in the ARB group than in the ACEI group(Figure5).In addition,decreases in TLR4 mRNA and TLR4MFI after treatment were greater in the ARB group than in the ACEI group(Figures6D and6E).

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Figure 1Comparison of miR-146a/b (A),IRAK1and TRAF6mRNA (B),TLR4mRNA (C)and TLR4MFI (D)levels between the CAD (n =66)and non-CAD (n =33)groups

Levels of miR-146a/b ,IRAK1mRNA,TRAF6mRNA,TLR4mRNA and TLR4MFI were higher in the CAD group than in the non-CAD group.*P <0.01compared with the non-CAD

group.

Figure 2Correlation between miR-146a/b and IRAK1and TRAF6mRNA levels in CAD and non-CAD groups

There was a positive correlation between miR-146a/b and both IRAK1and TRAF6mRNA levels.Correlations are:(A)r =0.72,P <0.01;(B)r =0.79,P <0.01;(C)r =0.76,P <0.01;and (D)r =0.85,P <0.01.

Clinical follow-up study

At the time of follow-up,cardiac events were observed in 13out of 66patients with CAD at 12months after PCI.Cardiac events consisted of target lesion revascularization in 11subjects,and target or other vessel revascularization in three subjects (both target lesion and other vessel revascularizations were shown in one patient).When the CAD group was divided into tertiles according to miR-146a and miR-146b levels,and IRAK1mRNA,TRAF6mRNA or TLR4mRNA levels at baseline,high levels of miR-146a and TLR4mRNA were independent predictors of cardiac events {miR-146a :relative risk =6.32[95%CI (con?dence interval)=1.13–35.19],P =0.04;TLR4mRNA:relative

risk =9.44(95%CI = 1.10–88.10),P =0.04}after

adjusting for various clinical parameters [age,gender,culprit lesion (left anterior descending coronary artery),past history of hypertension,diabetes mellitus,previous CAD or smoking,BP ,fasting glucose,HbA 1c (glycated haemoglobin),LDL-cholesterol and hsCRP].

DISCUSSION

Among the family of TLRs,TLR4is considered to be an important player in the initiation and progression of atherosclerotic disease [17].Our previous studies and other reports have shown a relationship between

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Figure 3Levels of miR-146a/b ,IRAK1mRNA and TRAF6mRNA in THP-1cells transfected with an miR-146a/b mimic and inhibitor

(A)Levels of miR-146a/b were higher in the miR-146a/b mimic-transfected cells than in mock-transfected cells.(B)Levels of miR-146a/b were lower in the miR-146a/b inhibitor-transfected cells than in mock-transfected cells.(C)IRAK1and TRAF6mRNA levels were lower in the miR-146a/b mimic-transfected cells than in mock-transfected cells.(D)IRAK1and TRAF6mRNA levels were higher in miR-146a/b inhibitor-transfected cells than in mock-transfected cells.*P <0.01compared with mock

transfection.

Figure 4Effect of RAS blockade (telmisartan or enalapril)and atorvastatin on levels of miR-146a/b (A),IRAK1and TRAF6mRNA (B),and TLR4mRNA and TLR4MFI (C)in cultured PBMCs stimulated with t-BHP or PBS

All values in t-BHP-stimulated PBMCs treated with vehicle (0.05%DMSO)are de?ned as 100%.Percentage changes in miR-146a/b ,IRAK1mRNA,TRAF6mRNA,TLR4mRNA and TLR4MFI levels were lower in t-BHP-stimulated PBMCs treated with an RAS inhibitor (telmisartan or enalapril)and atorvastatin than in those treated with drug vehicle.In addition,percentage changes in these levels were lower in t-BHP-stimulated monoyctes treated with telmisartan and atorvastatin compared with those treated with enalapril and atorvastatin.*P <0.05compared with t-BHP-stimulated PBMCs treated with vehicle.#P <0.05compared with t -BHP-stimulated PBMCs treated with enalapril and atorvastatin.

an activated TLR4signal and its downstream pathway in circulating monocytes obtained from patients with CAD [2,3,18].In addition,expression of TLR4in in?ltrating macrophages within the coronary arteries may be an important factor underlying coronary plaque

destabilization and rupture in CAD [19].In agreement with these reports,the present study has shown an increase in TLR4levels (both mRNA and protein levels)in the CAD group when compared with the non-CAD group.A mouse model has reported that loss

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Figure 5Effect of treatment with an ARB and statin or an ACEI and statin on miR-146a/b ,IRAK1mRNA and TRAF6mRNA in the CAD group

Levels of miR-146a (A),miR-146b (B),IRAK1mRNA (C)and TRAF6mRNA (D)at the time of follow-up (post)were markedly decreased in both the ARB and ACEI groups compared with at baseline (Pre)(P <0.01).Decreases in miR-146a/b ,IRAK1mRNA and TRAF6mRNA after treatment were greater in the ARB group than in the ACEI group (P <0.05).

of TLR4reduced the severity of atherosclerosis and altered atherosclerotic plaque [20].It has been suggested that a potential pathophysiological link exists between activation of the TLR4signal and the progression of coronary atherosclerosis.

It has been shown previously that the miR-146family (miR-146a/b )regulates downstream TLR4signalling (IRAK1and TRAF6)through a negative-feedback regulation loop [6].The human genome contains miR-146a and miR-146b genes on chromosomes 5and 10respectively [6,21,22].Promoter analysis of miR-146has demonstrated that both miR-146a and miR-146b function as negative regulators of adaptor molecules of IRAK1and TRAF6.This suggests that IRAK1and TRAF6are targeted for post-transcriptional control by both miR-146a and miR-146b [6].IRAK1and TRAF6activate NF-κB and AP-1(activating protein-1)transcription factors,and then up-regulate the TLR4-mediated immune response [23].An in vitro study using human monocytic cell line stimulated with LPS (lipopolysaccharide)has shown that elevation of miR-146expression occurred in an NF-κB-dependent manner [6].Therefore miR-146a/b has been shown to be induced by pro-in?ammatory stimuli such as TLR4,IL-1and TNF-α[24].A novel ?nding of the present study was

that levels of miR-146a/b and its target genes (both

IRAK1and TRAF6)were higher in PBMCs obtained from the CAD group than in the non-CAD group.It has been reported that the key kinases downstream of TLR4,including IRAK1and TRAF6,are activated in PBMCs from patients with CAD as well as activation of TLR4[25,26].A recent study has shown that the expression of miR-146a in PBMCs was signi?cantly increased in patients with ACS [27].In addition,levels of miR-146a/b were positively correlated with levels of IRAK1,TRAF6and TLR4mRNA expression.Our gain-of-function and loss-of-function approaches to miR-146a/b have shown that transfection of miR-146a/b into THP-1monocytes resulted in a down-regulation of IRAK1and TRAF6mRNA expression,but it remains uncertain whether endogenous IRAK1or TRAF6mRNA levels were in?uenced by miR-146a/b expression in humans.This discrepancy between our in vivo and in vitro studies may suggest that the impairment of IRAK1/TRAF6regulation by miR-146a/b could contribute to the prolonged activation of TLR4and its downstream signalling in patients with CAD.It is therefore speculated that this impairment of IRAK1/TRAF6regulation by miR-146a/b may play a role in the pathogenesis of CAD.Further studies will be

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Figure 6Effect of treatment with an ARB and statin or an ACEI and statin on TLR4mRNA and TLR4MFI levels in the CAD group

(A)CD14-positive cell population (R1)was gated by using PerCP against cell granularity (SSC;side scatter).Using the distribution,levels of cell-surface ?uorescence can be measured.The ordinate relates to the relevant cell number,whereas ?uorescence intensity can be seen on the abscissa,representing TLR4levels in the ARB group (B)or the ACEI group (C).A shift to the left demonstrates a decrease in the amount of TLR4staining in a cell.Speci?c MFI was measured in each case.Levels of TLR4mRNA (D)and TLR4MFI (E)at the time of follow-up (Post)were markedly decreased in both the ARB and ACEI groups compared with baseline (Pre)(P <0.01).Decreases in TLR4mRNA and TLR4MFI after treatment were greater in the ARB group than in the ACEI group (P <0.05).needed to clarify the mechanism by which miR-146a/b dysregulates IRAK1/TRAF6expression in patients with CAD.

Another important ?nding of the present study was that combined treatment with RAS inhibitors and a statin reduced monocytic levels of miR-146a/b ,IRAK1mRNA,TRAF6mRNA and TLR4mRNA in patients with CAD.RAS inhibitors,including ARBs and ACEIs,exert potent anti-atherosclerotic effects,which are mediated not only by their anti-hypertensive properties,but also by their anti-in?ammatory properties [28].An in vitro study has reported that AngII up-regulated TLR4-dependent signalling and then induced an in?ammatory response [28].The present study has also suggested that RAS inhibitors block the AngII-induced TLR4signalling pathway [29].RAS inhibitors may therefore decrease monocytic activation of TLR4through inhibition of AngII and then decrease miR-146a/b levels due to down-regulation of the TLR4signal.In particular,decreases in miR-146a/b ,IRAK1,TRAF6and TLR4levels after treatment were greater in the ARB group than in the ACEI group.It has been reported that treatment with an ARB reduced NF-κB activity in human PBMCs,whereas treatment with an ACEI had no effect

on NF-κB activity [30].It has been speculated that treatment with an ARB may directly down-regulate NF-κB activity and repress miR-146a/b levels in patients with CAD.It has also been reported that oxidized LDL activated the RAS and increased TLR4expression in cultured endothelial cells [31].An in vitro study using PBMCs treated with statins has shown that they decreased several TLR4transcription factors,such as IRAK1and TRAF6,and then inhibited the TLR4signal [32].Our present in vitro study has shown that telmisartan and atorvastatin had a greater effect than enalapril and atorvastatin on miR-146a/b ,IRAK1and TRAF6.An Apoe (apolipoprotein E-knockout mouse model has reported that combined treatment with an ARB and a statin decreased in?ammatory mediators compared with combined treatment with an ACEI and statin or treatment with ARB,ACEI or statin alone [14].From these observations,combined treatment with telmisartan and a statin may down-regulate IRAK1,TRAF6and TLR4expression,and then repress miR-146a/b resulting from the down-regulation of the TLR4signalling pathway in CAD.

Our 12-month follow-up study showed that high levels of miR-146a and TLR4mRNA at baseline are

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potent independent predictors of cardiac events through regression analysis.Our present study and those of others have reported a marked TLR4expression in human atherosclerotic plaques[19,33],and indicated that oxidative stress up-regulated TLR4expression in macrophages[34],suggesting an association between TLR4,in?ammatory and immune responses,and coronary atherosclerosis.Therefore activation of the TLR4signal may induce miR-146a/b expression as a negative regulator,and then induce progression of coronary atherosclerosis in patients with CAD.

A limitation of the present study is the small number of CAD and non-CAD groups.A further limitation is the relatively low dose of enalapril(5mg/day) compared with other reports from Western countries; this dose was chosen because it is the most commonly prescribed dose of enalapril in Japan(the maximum approved dose of enalapril is10mg daily)[35].However, there was no difference between the AR

B and ACEI groups with regard to BP after12months of therapy, which suggests that enalapril(5mg/day)and telmisartan (40mg/day)have the same anti-hypertensive effect.It was an ethical criterion of the study that the statin had to be administered to all patients with CAD because LDL-cholesterol levels in the majority of these patients were over100mg/dl.AHA(American Heart Association)/ACC(American College of Cardiology) guidelines for secondary prevention in patients with CAD state that the goal of treatment is an LDL-cholesterol level<100mg/dl[36].In addition,the present study could not elucidate the mechanism by which combined treatment with an ARB and statin affects miR-146a/b expression in patients with CAD.Further studies will therefore be needed to determine a causal relationship between these therapeutic agents in CAD.

Conclusions

The present study has suggested that dysregulation of miR-146a/b expression may contribute to the prolonged activation of TLR4and its downstream signalling in PBMCs obtained from patients with CAD.In addition, combined treatment with an ARB and statin decrease miR-146a/b levels,its target genes(IRAK1and TRAF6) and TLR4in patients with CAD,possibly contributing to the synergic effects of this therapeutic combination in this disorder.

AUTHOR CONTRIBUTION

Yuji Takahashi conceived and designed the study, interpreted the data,and revised the manuscript;Mamoru Satoh conceived and designed the study,and drafted the manuscript;Yoshitaka Minami analysed and interpreted the data;Tsuyoshi Tabuchi conceived and designed the study,and interpreted the data;Tomonori Itoh drafted the manuscript;and Motoyuki Nakamura revised the manuscript.

FUNDING

This study was supported by a Grant-in-Aid for General Scienti?c Research from the Japanese Ministry of Education,Science,Sports and Culture[grant number 20590886],and Keiryokai Research Foundation[grant number98].

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Received5January2010/4May2010;accepted4June2010

Published as Immediate Publication4June2010,doi:10.1042/CS20100003

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