Pioglitazone, a peroxisome proliferator-activated receptor gamma activator, ameliorates experimental autoimmune myocarditis by modulating Th1/Th2 balance

OBJECTIVES: The aim of this study is to investigate the effect of Peroxisome proliferator-activated receptor gamma (PPAR gamma) ligand on experimental autoimmune myocarditis (EAM). BACKGROUND: Rat EAM model resembles the giant cell myocarditis in human. Recent studies have suggested that Th1 cytokines are involved in the initiation and progression of EAM, whereas Th2 cytokines are associated with the remission. PPAR gamma, which is a member of nuclear hormone receptor superfamily, has been known to affect not only glucose homeostasis but also immune responses, by regulating the Th1/Th2 balance. METHODS: Lewis rats were immunized with cardiac myosin and divided into three groups: Group N, normal control rats (n = 16); Group E, EAM rats (n = 17); and Group P, EAM rats treated with a PPAR gamma activator pioglitazone (n = 20). RESULTS: Cardiac dysfunction and remodeling were inhibited in Group P compared to Group E. Heart weight/body weight ratio and the degree of inflammation and fibrosis were significantly lower in Group P than in Group E. The mRNA levels of macrophage inflammatory protein-1alpha (MIP-1alpha), which plays an important role in the recruitment of inflammatory cells in the early stage of EAM, were upregulated in the heart of Group E, but not in the heart of Group P. Furthermore, the treatment with pioglitazone decreased the expression levels of proinflamatory cytokine (TNF alpha and IL-1beta) genes and Th1 cytokine (IFN-gamma) genes, and increased the expression levels of Th2 cytokine (IL-4) gene. CONCLUSIONS: PPAR gamma ligands may have beneficial effects on myocarditis by inhibiting MIP-1alpha expression and modulating the Th1/Th2 balance.     

A CCR1 antagonist prevents the development of experimental autoimmune myocarditis in association with T cell inactivation

Chemokines play an important role in induction of chemotaxis of immune cells. CCR1 is a chemokine receptor expressed on neutrophils, monocytes, and T lymphocytes. The role of CCR1 in immunity is not well examined. We demonstrated the role of CCR1 on T lymphocytes and the effect of a CCR1 antagonist, BX471 in myocarditis. Lewis rats were immunized with cardiac myosin on day 0 to establish experimental autoimmune myocarditis. Rats were then administered BX471 subcutaneously every day (group BX0: n = 7) or from day 14 (group BX14: n = 7) and were killed on day 21. We confirmed expression of CCR1 in cells infiltrating the myocardium by immunohistochemistry and FACS analysis. The development of myocarditis was almost completely prevented in group BX0, and myocarditis-affected areas were significantly decreased in size in group BX14. Cardiac function was markedly improved. Ribonuclease protection assay showed that the CCR1 antagonist treatment suppressed mRNA expression for IL-6, IL-1beta, and TNF-alpha in the hearts. An antigen-specific T cell proliferation assay was performed with CD4-positive T cells isolated from control rats immunized with cardiac myosin. T cell proliferation was inhibited by the CCR1 antagonist. Additionally, we showed by Western blot that the CCR1 antagonist suppressed ERK1/2 and JNK activities in T cells stimulated with myosin and that IL-2 reversed this suppression. The CCR1 antagonist reduced the severity of EAM by inhibiting cytokine expression and inducing T cell inactivation. Thus, the CCR1 antagonist may provide a novel therapeutic strategy treatment of myocarditis.     

A cyclooxygenase-2 inhibitor alters Th1/Th2 cytokine balance and suppresses autoimmune myocarditis in rats

Acute myocarditis is a clinically serious disease; however, no effective treatment has been elucidated. Cyclooxygenase (COX)-2 is a key factor for progression of inflammation. Although inflammation is an essential pathological feature of acute myocarditis, the role of COX-2 in this process remains unclear. Thus, the purpose of this study was to clarify the role of COX-2 in acute myocarditis. We used a rat experimental autoimmune myocarditis (EAM) model and a specific COX-2 inhibitor in this study. Lewis rats were immunized on day 0 with porcine cardiac myosin to establish EAM. We administered the COX-2 inhibitor (meloxicam, 0.1 mg/kg per day) daily; the rats were killed on day 21. Echocardiograms, body and heart weight, heart rate, blood pressure, and histological and molecular examinations were performed. Cytokine expression in the hearts and cell proliferation against cardiac myosin were also analyzed. The COX-2 inhibition during the immune response (late) phase attenuated EAM development; however, the inhibition during the antigen priming (early) phase did not attenuate EAM. The COX-2 inhibitor altered Th1/Th2 cytokine balance and inhibited cell proliferation in vitro. The COX-2 inhibitor suppresses the development of EAM. COX-2 regulation is promising for treating acute myocarditis.     

Structural and electrical ventricular remodeling in rat acute myocarditis and subsequent heart failure

OBJECTIVE: We reported that experimental autoimmune myocarditis (EAM) rats showed dramatic changes in ventricular action potential and enhanced arrhythmogenicity in the acute phase, but mechanisms for this are still unclear. To investigate the mechanisms of cardiac remodeling in acute myocarditis and subsequent heart failure, physiological and molecular changes were evaluated along the time course of EAM. METHODS: Six-week-old Lewis rats were immunized with porcine cardiac myosin. On days 14, 21, 35 and 60 after immunization, histology, hemodynamics and electrophysiological parameters (i.e., effective refractory period (ERP), monophasic action potential duration (MAPD) and PVC inducibility) were evaluated and compared with control rats. After these studies, the expression levels of Kv(+) and L-Ca(2+) channels, ion transporters and BNP expressions in the left ventricle were examined by quantitative real time RT-PCR and Western blot analysis. RESULTS: EAM rats showed acute myocarditis with massive infiltration of the mononuclear cells on days 14 and 21. Subsequently, a chronic dilated cardiomyopathy (DCM)-like structural change was observed on day 60. Hemodynamic parameters were worse in EAM than controls. ERP and MAPD were longer in EAM than controls, with a peak on day 21, which was parallel to PVC inducibility. mRNA levels of Kv4.2, Kv1.5, KChIP2, frequenin and SERCA2a, and the protein levels of Kv4.2 and Kv1.5, were reduced, especially in the acute phase. CONCLUSIONS: The initial reduction of Ito-related molecules, such as the expression levels of Kv4.2, 1.5, frequenin and KChIP2, and the prolongation of MAPD are considered to be a key mechanism of ventricular remodeling and cause the characteristic clinical findings in EAM in the acute inflammatory phase and chronic DCM phase.     

Attenuation of experimental autoimmune myocarditis by blocking activated T cells through inducible costimulatory molecule pathway

OBJECTIVE: Inducible costimulator (ICOS) is a member of the CD28 family. Although inflammation is an essential pathological feature of myocarditis, the role of ICOS in myocarditis remains unclear. METHODS AND RESULTS: Lewis rats were immunized on day 0 with purified porcine cardiac myosin to establish experimental autoimmune myocarditis (EAM). Flow cytometry was used to examine expression of ICOS on myocardial infiltrating cells. Anti-ICOS antibody or ICOS-immunoglobulin (ICOSIg) was administered intravenously, and rats were killed on day 14 or 21 to study effects of ICOS/ICOS-ligand (ICOSL) pathway blockade during the antigen priming phase (days 0-14) or immune response phase (days 14-21), respectively. The heart weight to body weight ratio was determined, and histological examination and echocardiogram were performed to evaluate the severity of the disease. Cytokine expression in the heart and T cell proliferation against cardiac myosin were analyzed. Flow cytometry revealed that the majority of infiltrating cells, especially CD4-positive cells, expressed ICOS. Blockade of the ICOS/ICOSL pathway during the immune response phase attenuated EAM development. However, blockade of the ICOS/ICOSL pathway during the antigen priming phase did not attenuate and exacerbate EAM. Blockade of T cell activation through ICOS suppressed expression of cytokines including INF-gamma, IL-4, IL-6, IL-10, IL-1 beta, and TNF-alpha and inhibited T cell proliferation in vitro. CONCLUSIONS: Blockade of T cell activation through ICOS during the immune response phase regulates development of EAM, and therefore, ICOS may be an effective target for treating myocarditis.     

Peroxisome proliferation-activated receptor-gamma ligands ameliorate experimental autoimmune myocarditis

BACKGROUND: Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligands have been shown to ameliorate a variety of inflammatory conditions. The present study tested the hypothesis that PPAR-gamma ligands reduce experimental autoimmune myocarditis (EAM) associated with inhibition of the expansion and activation of T cells, as well as suppression of the expression of proinflammatory cytokines. METHODS AND RESULTS: EAM was induced in Lewis rats by immunization with porcine cardiac myosin. PPAR-gamma ligands, 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) 200 microg/kg/day i.p. and pioglitazone (PIO) 10 mg/kg/day orally, were administered for 3 weeks to rats with EAM. The results showed that enhanced PPAR-gamma expression was prominently stained in the nuclear and perinuclear regions of infiltrating inflammatory cells. Administration of PPAR-gamma ligands markedly reduced the severity of myocarditis, as shown by comparing the heart weight/body weight ratio, pericardial effusion scores, macroscopic scores and microscopic scores. PPAR-gamma ligands suppressed myocardial mRNA expression of inflammatory cytokines and the expression of interleukin (IL)-1beta protein in rats with EAM. In addition, 15d-PGJ(2) and PIO treatment suppressed the proliferative response and interferon-gamma production of T cell-enriched splenocytes from rats with EAM. Furthermore, the cytotoxic activity and myocardiogenic potential of these T cells were inhibited by 15d-PGJ(2) treatment. CONCLUSIONS: PPAR-gamma may play a role in the pathophysiology of EAM. PPAR-gamma ligands ameliorate the EAM associated with suppression of the expansion and activation of myocardiogenic T cells, as well as inhibition of the expression of proinflammatory cytokines. These results suggest that PPAR-gamma ligands such as 15d-PGJ(2) and PIO may have the potential to modulate human inflammatory heart diseases such as myocarditis.     

Tea catechins attenuate chronic ventricular remodeling after myocardial ischemia in rats

Tea catechins have many biological functions; these effects are induced by the suppression of several inflammatory factors. However, the effects of catechins on ventricular remodeling after myocardial ischemia have not been well investigated. To test the hypothesis that catechins can attenuate chronic ventricular remodeling after myocardial ischemia, we performed oral administration of catechins into rat myocardial ischemia models. We analyzed the mechanisms using physiological, pathological and molecular examinations. Although severe myocardial fibrosis with enhancement of inflammatory factors were observed in the non-treated ischemia group on day 28, catechins attenuated these changes with suppressed NF-kappaB and matrix metalloproteinases without systemic adverse effects. Catechins are potent for the suppression of chronic ventricular remodeling after myocardial ischemia because they are critically involved in the suppression of several inflammatory genes.     

Therapy with granulocyte colony-stimulating factor in the chronic stage, but not in the acute stage, improves experimental autoimmune myocarditis in rats via nitric oxide

We systematically investigated serial efficacy of granulocyte colony-stimulating factor (G-CSF) therapy upon experimental autoimmune myocarditis (EAM) in rats treated with and without the inhibition of nitric oxide (NO) with the analyses of tissue regeneration. G-CSF could mobilize multipotent progenitor cells of bone marrow into the peripheral blood and may improve ventricular function. A rat model of porcine myosin-induced EAM was used. After the immunization of myosin, G-CSF (10 μg/kg/day) or saline was injected intraperitoneally on days 0-21 in experiment 1 and on days 21-42 in experiment 2. Additional myosin-immunized rats were orally given 25 mg/kg/day of N^G-nitro-l-arginine methylester (l-NAME), an inhibitor of nitric oxide synthase (NOS), in each experiment (each group; n = 8-21). Serum cytokines and peripheral blood cell counts were measured in each group. In experiment 1, G-CSF treatment aggravated cardiac pathology associated with increased macrophage inflammatory protein-2 (MIP-2) and interleukin-6 (IL-6) levels and enhanced superoxide production. In experiment 2, G-CSF treatment reduced the severity of myocarditis with increased capillary density and improved left ventricular ejection fraction. In the rats with EAM treated with G-CSF associated with oral l-NAME treatment in experiment 2, the severity of myocarditis was not reduced. Myocardial c-kit⁺ cells were demonstrated only in G-CSF-treated group in experiment 2 but not in other groups. G-CSF has differential effects on EAM in rats associated with the modulation of cytokine network. The overwhelming superoxide production by G-CSF administration in the acute stage may worsen the disease. G-CSF therapy improved cardiac function via NO system in a rat model of myocarditis in the chronic stage, but not in the acute stage, possibly through the myocardial regeneration and acceleration of healing process.     

腺病毒载体介导的ICOSIg融合基因对实验性自身免疫性心肌炎作用的研究

目的探讨腺病毒载体介导的ICOSIg基因治疗对实验性自身免疫性心肌炎（EAM）的作用。方法将人ICOS胞外域与免疫球蛋白IgGFc段融合,构建ICOSIg融合基因的腺病毒表达载体P—Adeno—ICOSIg。PoeⅠ消化腺病毒载体后转染HEK293细胞,生产表达ICOSIg融合蛋白的腺病毒;构建增强型绿色荧光蛋白腺病毒作为对照。皮下注射猪心肌肌球蛋白诱导Lewis大鼠EAM模型,随机分为4组,分别于免疫当天（第0天）（A组,n=15）和第14天（B组,n=15）通过股静脉注射ICOSIg重组腺病毒,观察共刺激分子融合蛋白对T细胞激活阶段和炎症阶段的作用,C组（n=10）和D组（n=10）分别在第0天和第14天注射表达增强型绿色荧光蛋白（EGFP）的重组腺病毒。另设E组（n=10）未接受免疫的正常对照组。免疫第28天,超声心动图检测后处死动物。HE染色检测心肌炎症浸润程度。Westernblot检测心肌ICOS、ICOSL、B7—1及B7-2蛋白表达。实时定量RT—PCR定量心肌IL-2、IL-4和IFN-γ mRNA水平。结果第28天B组大鼠心功能指标（短轴缩短率：40．6±6．2比26．2±4．6,20．8±5．6,21．7±9．6）、心肌炎症程度较对照组显著改善。Western blot显示B组ICOS、ICOSL及B7—1蛋白表达显著下调,各组间B7-2蛋白表达差异未见统计学意义。B组大鼠心肌组织IFN-γ mRNA表达降低（19．8±7．9比66．6±4．5,79．6±5．9,80．9±5．1）,IL-4 mRNA表达增加（42．3±8．6比19．7±3．8,22．3±9．0,28．3±2．2）,IL-2表达各组间无显著差异,因此B组IFN-γ／IL-4比值显著低于A、C及D组（0．47±0．36比2．91±0．22,1．89±0．42,2．32±0．83）,表明Th细胞因子平衡向Th2方向偏离。结论炎症高峰期以ICOSIg阻断共刺激分子通路能够减轻EAM心肌组织炎症,改善大鼠心脏功能。其机制可能是下调心肌组织局部ICOS、ICOSL和B7—1表达及对炎症性细胞因子的抑制作用。     

Cardioprotective effects of sarcolemmal and mitochondrial K-ATP channel openers in an experimental model of autoimmune myocarditis. Role of the reduction in calcium overload during acute heart failure

It has been reported that K-ATP channel openers have a cardioprotective effect in acute ischemia as a pharmacological preconditioning effect. In the present study, the chronic effects of clinical K-ATP channel openers, ie, nicorandil (Nic) and mexiletine (Mex), on cardiac function were evaluated in a rat model of experimental autoimmune myocarditis (EAM). Nicorandil (3 or 10 mg/kg/day) or Mex (10 or 25 mg/kg/day) was administered to the EAM rats, and the effects were compared with those in untreated EAM rats (control EAM) and sham rats without EAM on day 21 (acute phase) or day 60 (chronic phase). In the acute phase, the control EAM rats exhibited a reduced left ventricular ejection fraction (LVEF) and prolonged monophasic action potential duration (MAPD). Neither drug had an affect on the LVEF or degree of myocarditis, but Mex 25 mg suppressed the MAPD prolongation. In the chronic phase, EAM+Nic and EAM+Mex 25 mg exhibited a higher LVEF than the control EAM. Although the control EAM exhibited sustained MAPD prolongation, the other groups showed recovery of the MAPD in the chronic phase. The mitochondorial redox state was lower in the control EAM than in the sham, and EAM+Nic exhibited a similar level of the redox state as the sham in the chronic phase. Nicorandil exhibited a cardioprotective effect through the protection of mitochondrial function. Mexiletine exhibited a cardioprotective effect possibly through a reduction in the calcium overload by shortening the MAPD in the acute phase.     

Allogeneic administration of fetal membrane-derived mesenchymal stem cells attenuates acute myocarditis in rats

We reported previously that the autologous administration of bone marrow-derived mesenchymal stem cells (BM-MSC) significantly attenuated myocardial dysfunction and injury in a rat model of acute myocarditis by stimulating angiogenesis and reducing inflammation. Because BM aspiration procedures are invasive and can yield low numbers of MSC after processing, we focused on fetal membranes (FMs) as an alternative source of MSC to provide a large number of cells. We investigated whether the allogeneic administration of FM-derived MSC (FM-MSC) attenuates myocardial injury and dysfunction in a rat myocarditis model. Experimental autoimmune myocarditis (EAM) was induced in male Lewis rats by injecting porcine cardiac myosin. Allogeneic FM-MSC obtained from major histocompatibility complex-mismatched ACI rats (5 × 10⁵ cells/animal) were injected intravenously into Lewis rats one week after myosin administration. At day 21, severe cardiac inflammation and deterioration of cardiac function were observed. The allogeneic administration of FM-MSC significantly attenuated inflammatory cell infiltration and monocyte chemoattractant protein 1 expression in the myocardium and improved cardiac function. In a T-lymphocyte proliferation assay, the proliferative response of splenic T lymphocytes was significantly lower in cells obtained from FM-MSC-treated EAM rats that reacted to myosin than in cells obtained from vehicle-treated rats with EAM. T-lymphocyte activation was significantly reduced by coculture with FM-MSC. The allogeneic administration of FM-MSC attenuated myocardial dysfunction and inflammation, and the host cell-mediated immune response was attenuated in a rat model of acute myocarditis. These results suggest that allogeneic administration of FM-MSC might provide a new therapeutic strategy for the treatment of acute myocarditis.     

14<Superscript>th</Superscript> INTERNATIONAL CONGRESS ON CARDIOVASCULAR PHARMACOTHERAPY

Objective Erythropoietin (EPO) has been shown to not only have cardioprotective effects but also attenuate autoimmune diseases. In the present study, we investigated the effect of EPO on cardiac inflammation and function, inflammatory cell infiltration, and cytokine expression in a rat model of experimental autoimmune myocarditis (EAM). Methods and results Male Lewis rats (6–8 weeks old) were immunized on day 0 with porcine cardiac myosin to establish EAM. The rats were subcutaneously administered either vehicle (saline) or human recombinant EPO (6,000 U/kg, 3 days/week) from day 0 to 20, and they were evaluated on day 21. In the EPO group, the inflammation area and heart weight/body weight ratio were significantly attenuated as compared with those in the vehicle group. Blood pressure and cardiac function were also improved in the EPO group. Immunohistochemistry revealed that EPO decreased the infiltration of macrophages and CD4 T cells, and degranulated mast cells in the myocardium. Real-time RT-PCR analysis demonstrated that inflammatory cytokine expression in the myocardium and lymphocytes was suppressed in the EPO group. However, in vitro experiments showed that EPO had no effect on antigen-induced proliferation and cytokine expression in lymphocytes. Conclusion EPO attenuates inflammatory cell infiltration and cytokine expression, and it improves cardiac function and reduces cardiac inflammation in EAM. This beneficial effect of EPO is unlikely to arise from a direct anti-inflammatory action on lymphocytes. These findings suggest the therapeutic potential of EPO for the treatment of myocarditis.     

Mycophenolate mofetil prevents the development of experimental autoimmune myocarditis

Experimental autoimmune myocarditis (EAM) is characterized by the appearance of multinucleated giant cells. EAM leads to severe myocardial damage and is a useful model of human giant cell myocarditis. We investigated whether mycophenolate mofetil (MMF), which is a potent immunosuppressant, prevents the development of myocarditis in a rat EAM model, and focused on the role of osteopontin (OPN) in the pathogenesis of this disorder. Adult Lewis rats were immunized with porcine cardiac myosin to establish EAM. The early MMF treatment completely prevented the development of EAM, and the late MMF treatment was also effective even against established EAM. Echocardiogram demonstrated that left ventricular function was also improved by the treatment with MMF. Real-time RT-PCR analysis showed that both early and late MMF treatments significantly inhibited myocarditis-induced OPN mRNA expression in the heart. Immunohistochemistry revealed that OPN expression was prominent in the myocardium on day 14, whereas expression was observed in the infiltrated macrophages on day 21. Mycophenolic acid (MPA) did inhibit agonist-induced OPN expression in cultured cardiomyocytes. These results show the therapeutic potential of MMF for autoimmune myocarditis and provide new insights into the pathogenesis of this disease.     

Tea catechins attenuate ventricular remodeling and graft arterial diseases in murine cardiac allografts

OBJECTIVE: Tea catechins have many biological functions; these effects are induced by the suppression of several inflammatory factors. However, effects of catechins on cardiac allograft rejection have not been well investigated. METHODS AND RESULTS: To test the hypothesis that catechins can attenuate ventricular remodeling and graft arterial diseases (GAD) in cardiac rejection, we orally administered catechins to murine cardiac recipients. We analyzed the mechanisms using immunohistochemistry, RNase protection, gel mobility shift, and cell proliferation assays. Although severe myocardial cell infiltration, fibrosis, and GAD with enhancement of inflammatory factors were observed in untreated class II mismatch allografts at day 60, catechins attenuated these changes with altered Th1/Th2 cytokine balance and suppressed NF-kappaB activation and cell proliferation. CONCLUSION: Catechins are potent agents for the suppression of chronic rejection because they are critically involved in the suppression of proinflammatory signaling pathways.     

Attenuation of autoimmune myocarditis in rats by mesenchymal stem cell transplantation through enhanced expression of hepatocyte growth factor

Mesenchymal stem cells (MSCs) have various effects, including angiogenic, myogenic, and paracrine actions. In this study, we determined whether MSC transplantation attenuates experimental autoimmune myocarditis (EAM). The mechanisms involved were also investigated. Male Lewis rats were immunized with myosin to establish EAM on day 0. MSCs, isolated from isogenic rats, were injected directly into the myocardium on day 14 (group MSC-2W), day 21 (group MSC-3W), or day 28 (group MSC-4W). In the MSC transplantation groups, cardiac systolic function detected by echocardiography was significantly improved, the EAM affected area determined by histological examination was significantly decreased, and capillary density was increased compared to that in the control groups. Expression of hepatocyte growth factor protein was enhanced by MSC transplantation. MSC transplantation inhibited myocardial expression of interleukin-2, -6, and -10 mRNAs. MSC transplantation reduces the severity of EAM by inducing neovascularization and inhibiting inflammatory cytokine production. Enhanced expression of hepatocyte growth factor was associated with these effects. Autoimmune myocarditis may be a good clinical target for MSC transplantation.     

特莫普利上调心肌硫氧化还原蛋白表达减轻心肌炎

目的 特莫普利是否能通过加强氧化还原调节机制减轻心肌炎。方法 Lewis大鼠自身免疫性心肌炎及离体培养的心肌细胞,免疫杂交检测特莫普利治疗前后硫氧化还原蛋白(TRX)表达,及其对心肌炎的影响。结果 特莫普利加强心肌细胞胞质定位的TRX表达,但对线粒体定位的TRX2表达则无明显改变。超氧化物歧化酶表达也无明显变化。特莫普利治疗从第1。21天,可减轻心肌炎的严重程度和降低氧化蛋白含量;但治疗从第15-21天,则无明显效果。本组心肌炎模型炎症大致从第15天开始,到21d达高峰,特莫普利从第1-21天治疗,可看做有2周的预治疗来上调心肌细胞TRX表达,通过加强TRX表达减轻心肌炎症。结论 TRX和经TRX修饰的氧化还原状态在自身免疫性心肌炎的发病中起重要作用,特莫普利治疗通过加强心肌TRX表达减轻心肌炎症。     

Hepatocyte growth factor ameliorates the progression of experimental autoimmune myocarditis: a potential role for induction of T helper 2 cytokines

Hepatocyte growth factor (HGF) plays a role in cell protection, antiapoptosis, antifibrosis, and angiogenesis. However, the role of HGF in the immune system is not well defined. We examined the influence of HGF on T cells and the effects of HGF therapy in acute myocarditis. Lewis rats were immunized on day 0 with cardiac myosin to establish experimental autoimmune myocarditis (EAM). Human HGF gene with hemagglutinating virus of the Japan-envelope vector was injected directly into the myocardium on day 0 or on day 14 (two groups of treated rats). Rats were killed on day 21. Expression of c-Met/HGF receptor in splenocytes and myocardial infiltrating cells was confirmed by immunohistochemical staining or FACS analysis. Myocarditis-affected areas were smaller in the treated rats than in control rats. Cardiac function in the treated rats was markedly improved. An antigen-specific T cell proliferation assay was done with CD4-positive T cells isolated from control rats stimulated with cardiac myosin. HGF suppressed T cell proliferation and production of IFN-gamma and increased production of IL-4 and IL-10 secreted from CD4-positive T cells in vitro. Additionally, TUNEL assay revealed that HGF reduced apoptosis in cardiomyocytes. HGF reduced the severity of EAM by inducing T helper 2 cytokines and suppressing apoptosis of cardiomyocytes. HGF has potential as a new therapy for myocarditis.     

Temocapril treatment ameliorates autoimmune myocarditis associated with enhanced cardiomyocyte thioredoxin expression

OBJECTIVE: Thioredoxin (TRX) is a redox regulatory protein that protects cells from various stresses. Angiotensin-converting enzyme (ACE) inhibitor was reported to enhance endogenous antioxidant enzyme activities. This study was carried out to investigate whether temocapril, a novel non-sulfhydryl-containing ACE inhibitor, reduces the severity of myocarditis via redox regulation mechanisms involving TRX. METHODS AND RESULTS: In normal rat myocytes in vitro and in vivo, Western blot showed that temocapril enhanced cytosolic redox regulatory protein TRX expression, but that neither mitochondrial TRX2 nor antioxidant enzymes, such as copper-zinc superoxide dismutase (Cu/Zn-SOD) or manganese superoxide dismutase (Mn-SOD) expression, was up-regulated by the preconditioning treatment. In rats with experimental autoimmune myocarditis (EAM), the severity of myocarditis and the protein carbonyl contents were less increased in temocapril treatment (10 mg/kg/day, orally) from day 1 to day 21, but not in temocapril treatment from day 15 to day 21. An immunohistochemical study showed that TRX stain was enhanced in infiltrating inflammatory cells and in damaged myocytes. Considering the characteristics of this model that myocardial inflammation begins around day 15 and increases until day 21, temocapril treatment for 3 weeks might be thought of as a preconditioning treatment. CONCLUSIONS: The results suggest that TRX and the redox state modified by TRX may play a crucial role in the pathophysiology of EAM. Temocapril ameliorates myocarditis associated with inducing TRX up-regulation in a preconditioning manner, although the mechanism of TRX up-regulation by temocapril remains to be elucidated.     

基质金属蛋白酶-9抑制剂治疗自身免疫性心肌炎大鼠模型的实验研究

目的 观察基质金属蛋白酶(MMP)-9抑制剂米诺环素对实验性自身免疫性心肌炎(EAM)Lewis大鼠模型的治疗效果,并探讨其机制.方法 Lewis大鼠60只,6～8周龄,体重150～170 g.双足底注射心肌C蛋白片段和完全弗氏辅佐剂的油状混合物,腹腔注射百日咳毒素建立自身免疫性心肌炎大鼠模型.根据药物干预的时期不同将大鼠分为早期干预组、中期干预组和晚期于预组3组,每组20只,不同的干预组又分为米诺环素治疗组(治疗组)和对照组两个亚组,每个亚组10只.早期干预组治疗组为腹腔注射50 mg/kg的米诺环素,1次/d,连续21 d,免疫注射后第1～21天.中期干预组治疗组为腹腔注射50 mg/kg的米诺环素,1次/d,连续21 d,免疫注射后第8～28天.晚期干预组治疗组为腹腔注射50 mg/kg的米诺环素,1次/d,连续21 d,免疫注射后第15～35天.各对照组均为在相同时间给予与治疗组相同体积的生理盐水腹腔注射.干预结束后,处死动物,心脏取材,进行系列检测.组织病理学石蜡切片苏木素-伊红(HE)染色检测心肌炎症分级,天狼猩红染色检测心肌胶原纤维含量,免疫组织化学染色检测心肌巨噬细胞和T细胞浸润,实时定量PCR检测MMP-2、MMP-9的mRNA表达水平,冰冻切片用于原位明胶酶谱法检测明胶酶活性.结果 早期干预组和中期干预组的治疗组大鼠心肌组织石蜡切片显示心肌组织炎症积分均显著低于其相应对照组[分别为1.51±0.36比3.03±1.35(P＜0.05)和2.11±0.82比3.75±0.29(P＜0.01)],免疫组织化学显示心肌巨噬细胞和T淋巴细胞浸润数目均显著低于其相应对照组,天狼猩红染色显示心肌间质纤维积分和纤维含量均明显低于其相应对照组[分别为1.51±0.35比2.75±0.29(P＜0.01)和1.61±0.42比2.50±0.41(P＜0.05)],实时定量PCR检测心肌MMP-2和MMP-9 mRNA表达低于其相应对照组,原位明胶酶法显示心肌明胶酶活性显著低于其相应对照组[分别为62 366±2131比162 367±5095(P＜0.01)和113 197±4809比184 256±5427(P＜0.01)].晚期干预组治疗组与对照组相比较心肌组织炎症积分、心肌巨噬细胞和T淋巴细胞浸润数目、心肌间质纤维化积分和纤维含量、心肌组织MMP-2和MMP-9 mRNA表达及心肌明胶酶活性差异均无统计学意义.结论 MMP-9抑制剂抑制EAM的早期病理发展,其机制可能与降低心肌炎症细胞浸润,延缓心肌间质纤维化,从转录水平降低明胶酶mRNA的表达,同时从蛋白水平降低明胶酶活性表达有关.

Abstract：

Objective To investigate the effects of matrix metalloproteinase-9 (MMP-9) inhibitor minocyclin hydrochloride in Lewis rats with experimental autoimmune myocarditis (EAM). Methods EAM was induced by injection of cardiac C protein emulsified in completed Freund adjuvant in double footpad and intra peritoneal injection of pertussis toxin on 6- to 8-week old Lewis rats. Sixty EAM Lewis rats were dividedinto 3 groups (early, middle and late intervention groups, n =20 each: 10 minocyclin treated and 10 control rats). In early intervention group, rats in treatment group received intraperitoneal injection of minocyclin hydrochloride from 1st to 21st day after immunization; in middle intervention group, rats were treated from 8th to 28th day after immunization and in late intervention group, rats were treated from 15th to 35th day after immunization (50 mg/kg body weight, once daily). Control rats received intraperitoneal injection of same volumetric physiological saline at corresponding time periods. At the end of intervention, rats were euthanatized and hearts were harvested. Paraffin sections were used for hematoxylin and eosin stain to determine the inflammatory score, for picrosirius stain to determine fibrosis score and collagen content, and for immunohistological stain to determine macrophages and T lymphocytes. Real time PCR was used to detect mRNA expression of myocardial MMP-2 and MMP-9. Cryostat sections were used for in situ zymography to detect protein activity of gelatinase. Results Inflammatory score in cardiac paraffin slides, number of cardiac macrophages and T lymphocytes, cardiac interstitial fibrosis score and content, expression of MMP-2, 9 mRNA and activity of gelatinase in treatment group were all significantly lower than in control group for early and middle intervention groups ( inflammatory score: early control group vs. treatment group: 3.03 ± 1.35 vs. 1.51 ±0. 36,P ＜0. 05, middle control group vs. treatment group: 3.75 ±0. 29 vs. 2. 11 ±0. 82,P ＜0. 01; cardiac interstitial fibrosis score, early control group vs. treatment group: 2. 75 ±0. 29 vs. 1.51 ± 0.35, P＜0.01, middle control group vs. treatment group: 2.50 ±0.41 vs. 1.61 ±0.42, P＜0.05;gelatinase, early control group vs. treatment group: 162 367 ±5095 vs. 62 366 ±2131, P ＜0. 01, middle control group vs. treatment group: 184 256 ±5427 vs. 113 197 ±4809, P ＜0. 01 ) while these parameters were similar between minocyclin-treated and control rats in late intervention group ( all P ＞ 0. 05 ).Conclusions MMP-9 plays an important role in the pathogenesis of autoimmune myocarditis. Inhibition of MMP-9 in early and middle stage could significantly attenuate inflammatory responses and myocardial fibrosis in this experimental EAM model.     

Attenuation of experimental autoimmune myocarditis by blocking T cell activation through 4-1BB pathway

4-1BB, a member of the tumor necrosis factor receptor (TNFR) family, binds the 4-1BB ligand (4-1BBL), works as a costimulatory molecule, and regulates T cell-mediated immune responses. Although inflammation is an essential pathological feature of myocarditis, the role of 4-1BB in experimental autoimmune myocarditis (EAM) remains unclear. Lewis rats were immunized on day 0 with purified porcine cardiac myosin to establish EAM. 4-1BB-immunoglobulin (4-1BBIg) was administered intraperitoneally (n = 6) a total of 9 times (3 times per week). Rats were killed on day 21 to study effects of 4-1BB/4-1BBL pathway blockade. For controls, isotype-matched human IgG was administered in other EAM rats (n = 6). Histologic and echocardiographic examination showed development of EAM attenuated by 4-1BBIg. Suppression of mRNA expression for IL-1α, IL-1β, IL-4, IL-6, and TNF-α was noted in the heart tissue treated with 4-1BBIg. Treatment with 4-1BBIg reduced production of Th1-type cytokines, and inhibited T cell proliferation in vitro. In the 4-1BB signaling pathway in splenocytes, 4-1BBIg suppressed JNK, p38, and IκB activity but not that of ERK1/2. Blockade of T cell activation through the 4-1BB/4-1BBL pathway regulates development of EAM; therefore, 4-1BB may be an effective target for treating myocarditis.