Yucheng Zhong

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.

MicroRNA-208a Dysregulates Apoptosis Genes Expression and Promotes Cardiomyocyte Apoptosis during Ischemia and Its Silencing Improves Cardiac Function after Myocardial Infarction

Aims. miR-208a is associated with adverse outcomes in several cardiac pathologies known to have increased apoptosis, including myocardial infarction (MI). We investigated if miR-208a has proapoptotic effects on ischemic cardiomyocytes and if its silencing has therapeutic benefits in MI. Methods and Results. The effect of miR-208a on apoptosis during ischemia was studied in cultured neonatal mice myocytes transfected with agomir or antagomir. Differential gene expression was assessed using microarrays. MI was induced in male C57BL/6 mice randomly assigned to antagomir (n = 6) or control group (n = 7), while sham group (n = 7) had sham operation done. Antagomir group received miR208a antagomir, while control and sham group mice received vehicle only. At 7 and 28 days, echocardiography was done and thereafter hearts were harvested for analysis of apoptosis by TUNEL method, fibrosis using Massons trichrome, and hypertrophy using hematoxylin and eosin. miR-208a altered apoptosis genes expression and increased apoptosis in ischemic cardiomyocytes. Therapeutic inhibition of miR-208a decreased cardiac fibrosis, hypertrophy, and apoptosis and significantly improved cardiac function 28 days after MI. Conclusion. miR-208a alters apoptosis genes expression and promotes apoptosis in ischemic cardiomyocytes, and its silencing attenuates apoptosis, fibrosis, and hypertrophy after MI, with significant improvement in cardiac function.

Thymic Stromal Lymphopoietin Attenuates the Development of Atherosclerosis in ApoE−/− Mice

Background Thymic stromal lymphopoietin (TSLP) is a cytokine with multiple effects on the body. For one thing, TSLP induces Th2 immunoreaction and facilitates allergic reaction; for another, it promotes the differentiation of naturally occurring CD4+CD25+Foxp3+ regulatory T cells (nTregs) and maintains immune tolerance. However, the exact role of TSLP in atherosclerosis remains unknown. Methods and Results In vitro, we examined the phenotype of TSLP‐conditioned bone marrow dendritic cells (TSLP‐DCs) of apolipoprotein E–deficient (ApoE−/−) mice and their capacity to induce the differentiation of Tregs. Our results indicated that TSLP‐DCs obtained the characteristics of tolerogenic dendritic cells and increased a generation of CD4+ latency‐associated peptide (LAP)+ Tregs and nTregs when cocultured with naive T cells. In addition, the functional relevance of TSLP and TSLP‐DCs in the development of atherosclerosis was also determined. Interestingly, we found that TSLP was almost absent in cardiovascular tissue of ApoE−/− mice, and TSLP administration increased the levels of antioxidized low‐density lipoprotein IgM and IgG1, but decreased the levels of IgG2a in plasma. Furthermore, mice treated with TSLP and TSLP‐DCs developed significantly fewer (32.6% and 28.2%, respectively) atherosclerotic plaques in the aortic root compared with controls, along with increased numbers of CD4+LAP+ Tregs and nTregs in the spleen and decreased inflammation in the aorta, which could be abrogated by anti‐TGF‐β antibody. Conclusions Our results revealed a protective role for TSLP in atherosclerosis that is possibly mediated by reestablishing a tolerogenic immune response, which may represent a novel possibility for treatment or prevention of atherosclerosis.

Intranasal immunization with heat shock protein 60 induces CD4(+) CD25(+) GARP(+) and type 1 regulatory T cells and inhibits early atherosclerosis.

Atherosclerosis is an autoimmune inflammatory disease involving both innate and adaptive immune mechanisms. Immune tolerance induction may have therapeutic potential for the suppression of atherosclerosis. Current interest is directed towards mucosal tolerance induction, especially nasal tolerance. Previous studies have shown that heat shock protein 60 (HSP60) is recognized as an important autoantigen in atherosclerosis, and nasal or oral HSP60 can induce tolerance and ameliorate atherosclerosis by inducing several subsets of regulatory T cells (Tregs) such as latency‐associated peptide (LAP)+ and forkhead box transcription factor 3 (FoxP3)+ Tregs. However, little is known regarding the detailed mechanisms of nasal tolerance. Here, we again investigated the impact of nasal HSP60 on atherosclerosis and the mechanisms underlying the anti‐atherosclerosis responses. We found that nasal HSP60 caused a significant 33·6% reduction in plaque size at the aortic root in the early stages of atherosclerosis (P < 0·001). Notably, a significant increase in activated CD4+CD25+ glycoprotein A repetitions predominant (GARP)+ Tregs, type 1 Tregs (Tr1 cells), and CD4+CD25+FoxP3+ Tregs, as well as a marked decrease in the numbers of type 1 and 17 T helper cells was detected in the spleens and cervical lymph nodes of HSP60‐treated mice. Moreover, nasal HSP60 increases the production of transforming growth factor (TGF)‐β and interleukin (IL)‐10 and decreases the secretion of IFN‐γ and IL‐17. Interestingly, the atheroprotective role of nasal HSP60 treatment was abrogated partly by the neutralization of IL‐10. Our findings show that nasal administration of HSP60 can attenuate atherosclerotic formation by inducing GARP+ Tregs, Tr1 cells and FoxP3+ Tregs, and that these Tregs maintain immune homeostasis by secreting IL‐10 and TGF‐β.

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.

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

BACKGROUND CXCL16 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. METHODS We 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. RESULTS The 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. CONCLUSION Systemic 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.

Elevated Plasma IL-38 Concentrations in Patients with Acute ST-Segment Elevation Myocardial Infarction and Their Dynamics after Reperfusion Treatment

Objective. Recent studies suggest that IL-38 is associated with autoimmune diseases. Furthermore, IL-38 is expressed in human atheromatous plaque. However, the plasma levels of IL-38 in patients with ST-segment elevation myocardial infarction (STEMI) have not yet to be investigated. Methods. On admission, at 24 h, at 48 h, and at 7 days, plasma IL-38, C-reactive protein (CRP), cardiac troponin I (cTNI), and N-terminal of the prohormone brain natriuretic peptide (NT-proBNP) levels were measured and IL-38 gene in peripheral blood mononuclear cells (PBMCs) was detected in STEMI patients. Results. The results showed that plasma IL-38 levels and IL-38 gene expression in PBMCs were significantly increased in STEMI patients compared with control group and were time dependent, peaked at 24 h. In addition, plasma IL-38 levels were dramatically reduced in patients with reperfusion treatment compared with control group. Similar results were also demonstrated with CRP, cTNI, and NT-proBNP levels. Furthermore, IL-38 levels were found to be positively correlated with CRP, cTNI, and NT-proBNP and be weakly negatively correlated with left ventricular ejection fraction (LVEF) in STEMI patients. Conclusions. The results indicate that circulating IL-38 is a potentially novel biomarker for patients with STEMI and IL-38 might be a new target for MI study.

Heme Oxygenase-1 Restores Impaired GARP+CD4+CD25+ Regulatory T Cells from Patients with Acute Coronary Syndrome by Upregulating LAP and GARP Expression on Activated T Lymphocytes

Background: Accumulating evidence shows that the pathological autoreactive immune response is responsible for plaque rupture and the subsequent onset of acute coronary syndrome (ACS). Naturally occurring CD4+CD25+regulatory T cells (nTregs) are indispensable in suppressing the pathological autoreactive immune response and maintaining immune homeostasis. However, the number and the suppressive function of glycoprotein-A repetitions predominant (GARP) + CD4+ CD25+ activated nTregs were impaired in patients with ACS. Recent evidence suggests that heme oxygenase-1 (HO-1) can regulate the adaptive immune response by promoting the expression of Foxp3. We therefore hypothesized that HO-1 may enhance the function of GARP+ CD4+ CD25+Tregs in patients with ACS and thus regulate immune imbalance. Methods: T lymphocytes were isolated from healthy volunteers (control, n=30) and patients with stable angina (SA, n=40) or ACS (n=51). Half of these cells were treated with an HO-1 inducer (hemin) for 48 h, and the other half were incubated with complete RPMI-1640 medium. The frequencies of T-helper 1 (Th1), Th2, Th17 and latency-associated peptide (LAP) +CD4+ T cells and the expression of Foxp3 and GARP by CD4+CD25+T cells were then assessed by measuring flow cytometry after stimulation in vitro. The suppressive function of activated Tregs was measured by thymidine uptake. The levels of transforming growth factor-1 (TGF-β1) in the plasma were measured using enzyme-linked immunosorbent assay (ELISA). The expression levels of the genes encoding these proteins were analyzed by real-time polymerase chain reaction. Results: Patients with ACS exhibited an impaired number and suppressive function of GARP+ CD4+ CD25+Tregs and a mixed Th1/Th17-dominant T cell response when compared with the SA and control groups. The expression of LAP in T cells was also lower in patients with ACS compared to patients with SA and the control individuals. Treatment with an HO-1 inducer enhanced the biological activity of GARP+ CD4+ CD25+Tregs and resulted in increased expression of LAP and GARP by activated T cells. Conclusions: The reduced number and impaired suppressive function of GARP+ CD4+ CD25+Tregs result in excess effector T cell proliferation, leading to plaque instability and the onset of ACS. HO-1 can effectively restore impaired GARP+ CD4+ CD25+Tregs from patients with ACS by promoting LAP and GARP expression on activated T cells.

Impaired Circulating CD4+LAP+ Regulatory T Cells in Patients with Acute Coronary Syndrome and Its Mechanistic Study

Objective CD4+ latency-associated peptide (LAP)+ regulatory T cells (Tregs) are a newly discovered T cell subset in humans and the role of these cells in patients with acute coronary syndrome (ACS) has not been explored. We designed to investigate whether circulating frequency and function of CD4+LAP+ Tregs are defective in ACS. Methods One hundred eleven ACS patients (acute myocardial infarction and unstable angina) and 117 control patients were enrolled in the study. The control patients consisted of chronic stable angina (CSA) and chest pain syndrome (CPS). The frequencies of circulating CD4+LAP+ Tregs and the expression of the transmembrane protein glycoprotein-A repetitions predominant (GARP) on CD4+ T cells were determined by flow cytometry. The function of CD4+LAP+ Tregs was detected using thymidine uptake. Serum interleukin-10 (IL-10) and transforming growth factor-β protein (TGF-β) levels were detected using ELISA and expression of GARP mRNA in peripheral blood mononuclear cells (PBMCs) was measured by real time-polymerase chain reaction. Results We found ACS patients had a significantly lower frequency of circulating CD4+LAP+ Tregs, and the function of these cells was reduced compared to controls. The expression of GARP in CD4+ T cells and the serum levels of TGF-β in ACS patients were lower than those of control patients. The serum levels of IL-10 were similar between the two cohorts. Conclusions A novel regulatory T cell subset, defined as CD4+LAP+ T cells is defective in ACS patients.

Impairment of Circulating CD4 + CD25 + GARP + Regulatory T Cells in Patients with Acute Coronary Syndrome

Background: Atherosclerosis (AS) is an inflammatory and immune disease. Regulatory T cells (Tregs) suppress the activation of T cells and have been shown to play a protective role during the pathogenesis of AS. However, specific markers for Tregs are lacking. Recently, glycoprotein A repetitions predominant (GARP) was discovered as a specific marker of activated Tregs, and we therefore utilized GARP as a specific surface marker for Tregs in the current study. Methods: To assess whether GARP+ Tregs are downregulated in patients with acute coronary syndrome (ACS), we examined CD4+CD25+GARP+ T cell frequencies as well as their associated cytokines and suppressive function. Additionally, we compared GARP expression to that of FOXP3, which may be more sensitive as a marker of activated Tregs in patients with ACS. Results: Patients with ACS demonstrated a significant decrease in circulating CD4+CD25+GARP+ Tregs. Moreover, the suppressive function of Tregs and levels of related cytokines were also impaired in ACS patients compared to those with stable angina (SA) or normal coronary artery (NCA). Additionally, after TCR stimulation, peripheral blood mononuclear cells (PBMCs) from patients with ACS exhibited a decrease in CD4+CD25+GARP+ Tregs. Conclusions: These fnding indicate that circulating CD4+CD25+GARP+ Tregs are impaired in patients withACS. Thus, targeting GARP may promote the protective function of Tregs in ACS.