Qiutang Zeng

miR-146a in PBMCs modulates Th1 function in patients with acute coronary syndrome

The upregulation of Th1 cells has been suggested to have an essential function in the development of atherosclerosis (AS). Recent studies indicate that miR‐146a is a microRNA specifically and highly expressed in Th1‐driven autoimmune disease. The aim of the study was to investigate the possible mechanisms of the miR‐146a in the onset of acute coronary syndrome (ACS). The results showed that the expression of miR‐146a in peripheral blood mononuclear cells (PBMCs) was significantly increased in patients with ACS. We showed that overexpression of miR‐146a in PBMCs could significantly upregulate the function of Th1 cells. Furthermore, we showed that miR‐146a treatment could modulate the Th1 differentiation through posttranscriptional enhancing the T‐bet pathway in PBMCs. In addition, this study also provided evidence that miR‐146a treatment in vitro could induce the protein expression of TNF‐α, MCP‐1, NF‐κB p65, which are key pro‐inflammatory cytokines and critical transcription factor in AS. In contrast, miR‐146a inhibitor could attenuate these phenomena significantly. The results support the concept that miR‐146a may be a novel regulatory factor in Th1 differentiation and a new therapeutic target for AS and ACS.

Interleukin-37 ameliorates myocardial ischaemia/reperfusion injury in mice

Innate immune and inflammatory responses are involved in myocardial ischaemia/reperfusion (I/R) injury. Interleukin (IL)‐37 is a newly identified member of the IL‐1 family, and functions as a fundamental inhibitor of innate immunity and inflammation. However, its role in myocardial I/R injury remains unknown. I/R or sham operations were performed on male C57BL/6J mice. I/R mice received an injection of recombinant human IL‐37 or vehicle, immediately before reperfusion. Compared with vehicle treatment, mice treated with IL‐37 showed an obvious amelioration of the I/R injury, as demonstrated by reduced infarct size, decreased cardiac troponin T level and improved cardiac function. This protective effect was associated with the ability of IL‐37 to suppress production of proinflammatory cytokines, chemokines and neutrophil infiltration, which together contributed to a decrease in cardiomyocyte apoptosis and reactive oxygen species (ROS) generation. In addition, we found that IL‐37 inhibited the up‐regulation of Toll‐like receptor (TLR)‐4 expression and nuclear factor kappa B (NF‐kB) activation after I/R, while increasing the anti‐inflammatory IL‐10 level. Moreover, the administration of anti‐IL‐10R antibody abolished the protective effects of IL‐37 in I/R injury. In‐vitro experiments further demonstrated that IL‐37 protected cardiomyocytes from apoptosis under I/R condition, and suppressed the migration ability of neutrophils towards the chemokine LIX. In conclusion, IL‐37 plays a protective role against mouse myocardial I/R injury, offering a promising therapeutic medium for myocardial I/R injury.

Downregulation of T Helper Cell Type 3 in Patients with Acute Coronary Syndrome

BACKGROUND AND AIMSnThere is an imbalance between Th1 and Th2 in the development and progression of atherosclerosis and in patients with acute coronary syndrome (ACS) including acute myocardial infarction (AMI) and unstable angina. T helper cell type 3 (Th3), which primarily secretes transforming growth factor beta-1 (TGF-beta1), has been shown to inhibit both Th1 and Th2 cells. The present study was designed to investigate whether Th3 cells are involved in plaque destabilization and the onset of ACS.nnnMETHODSnNinety one patients who underwent diagnostic catheterization were classified into four groups (AMI group, unstable angina group, stable angina group and chest pain syndrome group). The cell frequencies of Th1, Th2 and Th3 were detected using flow cytometry, and the concentrations of their related cytokines IFN-gamma, IL-4 and TGF-beta1 were studied by ELISA.nnnRESULTSnApart from the imbalance between Th1 and Th2, results revealed a significant decrease in peripheral Th3 number and levels of TGF-beta1 in patients with ACS as compared with those in patients with stable angina and chest pain syndrome (p<0.01).nnnCONCLUSIONSnDownregulation of Th3 cells in patients with ACS may play a potential role in plaque destabilization and the onset of ACS.

Metformin inhibits nuclear factor κB activation and decreases serum high-sensitivity C-reactive protein level in experimental atherogenesis of rabbits

Previous studies demonstrated that metformin has obvious antiatherogenic properties, but the exact mechanism remains unclear. Therefore, we established an atherosclerotic rabbit model in order to investigate the potential effects of metformin on transcription factor nuclear factor κB (NF-κB) and serum high-sensitivity C-reactive protein (hs-CRP) level, which had been regarded as proatherogenic factors. New Zealand rabbits were randomly divided into three groups: a control group (n = 8), an atherosclerotic group (AS group, n = 8), and a metformin treatment group (Met group, n = 8). The experimental atherosclerotic rabbit model was successfully established at the end of the 8th week. From the 9th week, rabbits in the Met group were administered with 150 mg/kg metformin daily by gavage. Blood samples were collected at days 0 and 8, and at 16 weeks to detect the level of blood lipid and serum glucose. At the end of the experiment, blood samples were withdrawn for determining serum hs-CRP. Aortic samples were harvested for histomorphometric analysis. Immunohistochemistry and Western blotting were used to detect the expression of NF-κB subunit p65 in nuclear extracts and phosphorylation of inhibitor of nuclear factor κB (IκB) in cytoplasmic extracts. An experimental atherosclerotic rabbit model was successfully established. The expression of nuclear NF-κB subunit p65 and cytoplasmic phosphorylation of IκB protein in the vessel wall was enhanced (P < 0.01, respectively) in the AS group, and serum hs-CRP level was significantly increased in the AS group compared with the control group (3.90 ± 0.25 mg/l versus 1.36 ± 0.14 mg/l, P < 0.01). Treatment with metformin significantly attenuated the progression of aortic atherosclerosis. In the Met group, there was a marked reduction in nuclear NF-κB subunit p65 and cytoplasmic phosphorylation of IκB protein expression (P < 0.01). Serum hs-CRP concentration was also significantly decreased (3.20 ± 0.20 mg/l versus 3.90 ± 0.25 mg/l, P < 0.05). Metformin inhibits the phosphorylation of IκB and the activation of NF-κB in the vessel wall of experimental atherogenesis of rabbits, as well as decreasing the serum level of hs-CRP, thus suggesting that metformin has vascular anti-inflammatory properties, which may be one of its antiatherogenic mechanisms.

Total cholesterol content of erythrocyte membranes is associated with the severity of coronary artery disease and the therapeutic effect of rosuvastatin

Abstract Introduction. Numerous studies suggest that total cholesterol content of erythrocyte membranes (CEM) might play a critical role in atherosclerotic plaque progression and instability. However, the exact role of CEM in atherosclerosis remains obscure. Our study was designed to investigate the association between CEM and the severity of coronary artery disease (CAD), and to assess the effect of rosuvastatin on CEM levels. Methods. CEM levels were assessed in 136 participants, including acute coronary syndrome (ACS) (non-ST-segment elevation ACS (NSTEACS) and ST-segment elevation myocardial infarction (STEMI)), stable angina pectoris (SAP), and controls. The Gensini score was used to estimate the severity of CAD. Additionally, 54 patients with CAD were medicated with rosuvastatin, 5 or 10 mg once daily, and then checked at 6 months. Results. The highest level of CEM was found in the STEMI group, followed by the NSTEACS, the SAP, and the control groups. Gensini score in group IV (CEM > 141.6 μg/mg) was markedly higher compared with group I (CEM ≤77.6 μg/mg). Gensini scores in group II (77.6 < CEM ≤111.1 μg/mg) and group III (111.1 < CEM ≤141.6 μg/mg) were also higher than in group I (all P < 0.001). Furthermore, a positive correlation was found between CEM levels and Gensini score (r = 0.714, P < 0.001). CEM levels were dose-dependently reduced by rosuvastatin therapy. Conclusions. CEM levels are positively associated with the severity of CAD, meaning that CEM might contribute to the development of CAD. Importantly, rosuvastatin could decrease CEM levels in patients with CAD and might effectively help to attenuate the progression of CAD.

Establishment of nasal tolerance to heat shock protein-60 alleviates atherosclerosis by inducing TGF-β-dependent regulatory T cells

SummaryMounting evidence supports that a newly identified regulatory T cell (Treg), CD4+LAP+ Treg, is associated with oral tolerance induction and following inhibition of atherosclerosis, but little is described about whether nasal tolerance to antigen likewise induces the novel Tregs production and the relevant antiatherosclerotic benefit. We investigated the effect of nasal administration of heat shock protein-60 (HSP60) on atherogenesis. HSP60 or phosphate buffer solution (PBS) was nasally administered to six-week-old male ApoE−/− mice. At the 10th week after the nasal administration, there was a significant decrease in atherosclerotic plaque areas of aortic roots in the HSP60-treated mice as compared with those in the PBS-treated mice. Atherosclerosis suppression was accompanied with a significant increase in CD4+LAP+ and CD4+CD25+Foxp3+ Tregs and a concurrently increased production of TGF-β in the HSP60-treated mice. The protective effect of HSP60 was offset by injection of anti-TGF-β antibody. It is concluded that nasal administration of HSP60 can inhibit atherosclerotic formation through immune tolerance which is established by Tregs depending on the induction of anti-inflammatory cytokine TGF-β. Immune tolerance induced by nasal administration of HSP60 may provide an alternative therapeutic method for atherosclerosis.Mounting evidence supports that a newly identified regulatory T cell (Treg), CD4+LAP+ Treg, is associated with oral tolerance induction and following inhibition of atherosclerosis, but little is described about whether nasal tolerance to antigen likewise induces the novel Tregs production and the relevant antiatherosclerotic benefit. We investigated the effect of nasal administration of heat shock protein-60 (HSP60) on atherogenesis. HSP60 or phosphate buffer solution (PBS) was nasally administered to six-week-old male ApoE−/− mice. At the 10th week after the nasal administration, there was a significant decrease in atherosclerotic plaque areas of aortic roots in the HSP60-treated mice as compared with those in the PBS-treated mice. Atherosclerosis suppression was accompanied with a significant increase in CD4+LAP+ and CD4+CD25+Foxp3+ Tregs and a concurrently increased production of TGF-β in the HSP60-treated mice. The protective effect of HSP60 was offset by injection of anti-TGF-β antibody. It is concluded that nasal administration of HSP60 can inhibit atherosclerotic formation through immune tolerance which is established by Tregs depending on the induction of anti-inflammatory cytokine TGF-β. Immune tolerance induced by nasal administration of HSP60 may provide an alternative therapeutic method for atherosclerosis.

Overexpression of CXCL16 promotes a vulnerable plaque phenotype in Apolipoprotein E–Knockout Mice

BACKGROUNDnCXCL16 has been shown to be involved in atherosclerotic lesion development, but its role in preexisting lesions is still unclear. This study aims to assess the effect of CXCL16 on the stability of preexisting lesions.nnnMETHODSnWe firstly measured plasma CXCL16 level in Apolipoprotein E-Knockout (ApoE KO) mice with either high-cholesterol diet (HCD) or normal diet (ND) by enzyme-linked immunosorbent assay (ELISA). Then, silastic collars were placed around the carotid arteries in HCD-ApoE KO mice to accelerate atherosclerotic lesions. Five weeks later, CXCL16 was overexpressed by intravenous injection of lentivirus carrying CXCL16 transgene. Two weeks after infection, lesions were stained with hematoxylin and eosin (HE) and with oil red O. Biomarkers in the lesions, such as MMPs, CCL2, VCAM-1 and TNF-α were measured by real-time polymerase chain reaction (RT-PCR), which indicate the instability of plaques.nnnRESULTSnThe level of CXCL16 in plasma was higher in HCD-ApoE KO mice as compared to ND-ApoE KO mice. Circulating CXCL16 overexpression does not affect the size of preexisting plaques, but it leads to vulnerable plaque morphology and increases the expression of markers of plaque destabilization.nnnCONCLUSIONnSystemic CXCL16 becomes much higher in atherosclerosis, and it could be a potential atherogenic biomarker. Overexpression of CXCL16 promotes the evolution of preexisting lesions to vulnerable plaques in ApoE KO mice.

Inhibition of IFN Regulatory Factor-1 Down-Regulate Th1 Cell Function in Patients with Acute Coronary Syndrome

BackgroundThe crucial role of T helper (Th) cells and chronic inflammation in atherosclerosis and coronary artery disease is no longer controversial. Evidence has revealed that Th cell type 1 (Th1) is closely associated with the pathogenesis of acute coronary syndrome (ACS). But the mechanisms involved in the generation of Th1 cells have not been fully elucidated. IFN regulatory factor (IRF)-1 is a pleiotropic transcription factor involved in innate immunity and chronic inflammation disease. The study was undertaken to investigate the potential effect of IRF-1 on the Th1 cell function in patients with ACS in vitro.MethodsPatients with clinical presentation of chest pain, stable angina, unstable angina, and acute myocardial infarction were enrolled in this study. Circulating CD4+ T cells were enriched and analyzed for mRNA and protein expression of IRF-1. Silencing IRF-1 gene with small interfering RNA in CD4+ T cells from patients with ACS was performed to explore the possible mechanisms involved in ACS.ResultsThe results demonstrated that the expression of IRF-1 in CD4+ T cells was significantly increased in patients with ACS and positively correlated with plasma Th1 cytokine profile. Inhibition of IRF-1 in CD4+ T cells from patients with ACS prevented the induction of the frequencies and cytokines expression of Th1 cells. In addition, this study also revealed that IRF-1 modulate Th1 differentiation through establishing IL-12 responsiveness by acting on IL-12 receptor β1.ConclusionThe present data demonstrate that inhibition of IRF-1 obviously decrease the function of Th1 cells and may be a novel participator in the progress of ACS.

Genome-Wide Analysis of DNA Methylation and Acute Coronary Syndrome.

Rationale: Acute coronary syndrome (ACS) is a leading cause of death worldwide. Immune functions play a vital role in ACS development; however, whether epigenetic modulation contributes to the regulation of blood immune cells in this disease has not been investigated. Objective: We conducted an epigenome-wide analysis with circulating immune cells to identify differentially methylated genes in ACS. Methods and Results: We examined genome-wide methylation of whole blood in 102 ACS patients and 101 controls using HumanMethylation450 array, and externally replicated significant discoveries in 100 patients and 102 controls. For the replicated loci, we further analyzed their association with ACS in 6 purified leukocyte subsets, their correlation with the expressions of annotated genes, and their association with cardiovascular traits/risk factors. We found novel and reproducible association of ACS with blood methylation at 47 cytosine-phosphoguanine sites (discovery: false discovery rate <0.005; replication: Bonferroni corrected P<0.05). The association of methylation levels at these cytosine-phosphoguanine sites with ACS was further validated in at least 1 of the 6 leukocyte subsets, with predominant contributions from CD8+ T cells, CD4+ T cells, and B cells. Blood methylation of 26 replicated cytosine-phosphoguanine sites showed significant correlation with expressions of annotated genes (including IL6R, FASLG, and CCL18; P<5.9×10−4), and differential gene expression in case versus controls corroborated the observed differential methylation. The replicated loci suggested a role in ACS-relevant functions including chemotaxis, coronary thrombosis, and T-cell–mediated cytotoxicity. Functional analysis using the top ACS-associated methylation loci in purified T and B cells revealed vital pathways related to atherogenic signaling and adaptive immune response. Furthermore, we observed a significant enrichment of the replicated cytosine-phosphoguanine sites associated with smoking and low-density lipoprotein cholesterol (Penrichment⩽1×10−5). Conclusions: Our study identified novel blood methylation alterations associated with ACS and provided potential clinical biomarkers and therapeutic targets. Our results may suggest that immune signaling and cellular functions might be regulated at an epigenetic level in ACS.

Effect of minocycline postconditioning and ischemic postconditioning on myocardial ischemia-reperfusion injury in atherosclerosis rabbits

This study examined the protective effect of ischemic postconditioning (IPoC) and minocycline postconditioning (MT) on myocardial ischemia-reperfusion (I/R) injury in atherosclerosis (AS) animals and the possible mechanism. Forty male healthy rabbits were injected with bovine serum albumin following feeding on a high fat diet for 6 weeks to establish AS model. AS rabbits were randomly divided into 3 groups: (1) I/R group, the rabbits were subjected to myocardial ischemia for 35 min and then reperfusion for 12 h; (2) IPoC group, the myocardial ischemia lasted for 35 min, and then reperfusion for 20 s and ischemia for 20 s [a total of 3 cycles (R20s/I20s×3)], and then reperfusion was sustained for 12 h; (3) MT group, minocycline was intravenously injected 10 min before reperfusion. The blood lipids, malondialdehyde (MDA), superoxide dismutase (SOD), soluble cell adhesion molecule (sICAM), myeloperoxidase (MPO), and cardiac troponin T (cTnT) were biochemically determined. The myocardial infarction size (IS) and apoptosis index (AI) were measured by pathological examination. The expression of bcl-2 and caspase-3 was detected in the myocardial tissue by using reverse transcription-polymerase chain reaction (RT-PCR). The results showed that the AS models were successfully established. The myocardial IS, the plasma levels of MDA, sICAM, MPO and cTnT, and the enzymatic activity of MPO were significantly decreased, and the plasma SOD activity was significantly increased in IPoC group and MT group as compared with I/R group (P<0.05 for all). The myocardial AI and the caspase-3 mRNA expression were lower and the bcl-2 mRNA expression was higher in IPoC and MT groups than those in I/R group (all P<0.05). It is concluded that the IPoC and MT can effectively reduce the I/R injury in the AS rabbits, and the mechanisms involved anti-oxidation, anti-inflammation, up-regulation of bcl-2 expression and down-regulation of caspase-3 expression. Minocycline can be used as an effective pharmacologic postconditioning drug to protect myocardia from I/R injury.SummaryThis study examined the protective effect of ischemic postconditioning (IPoC) and minocycline postconditioning (MT) on myocardial ischemia-reperfusion (I/R) injury in atherosclerosis (AS) animals and the possible mechanism. Forty male healthy rabbits were injected with bovine serum albumin following feeding on a high fat diet for 6 weeks to establish AS model. AS rabbits were randomly divided into 3 groups: (1) I/R group, the rabbits were subjected to myocardial ischemia for 35 min and then reperfusion for 12 h; (2) IPoC group, the myocardial ischemia lasted for 35 min, and then reperfusion for 20 s and ischemia for 20 s [a total of 3 cycles (R20s/I20s×3)], and then reperfusion was sustained for 12 h; (3) MT group, minocycline was intravenously injected 10 min before reperfusion. The blood lipids, malondialdehyde (MDA), superoxide dismutase (SOD), soluble cell adhesion molecule (sICAM), myeloperoxidase (MPO), and cardiac troponin T (cTnT) were biochemically determined. The myocardial infarction size (IS) and apoptosis index (AI) were measured by pathological examination. The expression of bcl-2 and caspase-3 was detected in the myocardial tissue by using reverse transcription-polymerase chain reaction (RT-PCR). The results showed that the AS models were successfully established. The myocardial IS, the plasma levels of MDA, sICAM, MPO and cTnT, and the enzymatic activity of MPO were significantly decreased, and the plasma SOD activity was significantly increased in IPoC group and MT group as compared with I/R group (P<0.05 for all). The myocardial AI and the caspase-3 mRNA expression were lower and the bcl-2 mRNA expression was higher in IPoC and MT groups than those in I/R group (all P<0.05). It is concluded that the IPoC and MT can effectively reduce the I/R injury in the AS rabbits, and the mechanisms involved anti-oxidation, anti-inflammation, up-regulation of bcl-2 expression and down-regulation of caspase-3 expression. Minocycline can be used as an effective pharmacologic postconditioning drug to protect myocardia from I/R injury.