Pioglitazone modulates the balance of effector and regulatory T cells in apolipoprotein E deficient mice.

BACKGROUND AND AIMS: Pioglitazone (PIO) affects T cell-mediated immunity through actions of peroxisome proliferator activated receptor γ (PPARγ). Effector and regulatory T cells control the development of atherosclerosis, a chronic inflammatory disease affecting the arterial blood vessels. The aim of this study was to examine whether PIO ameliorates atherosclerosis by altering the balance of effector and regulatory T cells. METHODS AND RESULTS: To explore the effect of PIO on early and advanced atherosclerosis, apolipoprotein E deficient (ApoE-/-) mice were fed western diet and received PIO (20 mg/kg/day) by gastric gavage at 6 or 14 weeks of age, respectively for 8 weeks. Data showed PIO markedly inhibited early fatty streak formation. Further, although the advanced fibrofatty plaque sizes were not significantly reduced, the numbers of smooth muscle cells within lesions were increased and higher collagen concentrations were produced. In general, macrophage expression in lesions was decreased. Additionally, the expression of Foxp3(+) cells was increased in lesions and spleens in mice at all PIO treatment stages, whereas the CD4(+)IFN-γ(+)/CD4(+)IL-4(+) cell ratios were reduced. CONCLUSION: PIO inhibited early atherosclerotic lesion formation and increased the stability of advanced atherosclerotic plaques in ApoE-/- mice, which was associated with altering the balance of effector and regulatory T cells.

Olmesartan, a novel angiotensin II type 1 receptor antagonist, reduces severity of atherosclerosis in apolipoprotein E deficient mice associated with reducing superoxide production.

BACKGROUND AND AIM: Oxidative stress may play an important role in the development of atherosclerosis. Some angiotensin II type 1 (AT(1)) receptor antagonists have the capacity of reducing oxidative stress in addition to the hemodynamic actions. Accordingly, we assessed the hypothesis that olmesartan, a novel AT(1) receptor antagonist, reduced the severity of atherosclerosis in apolipoprotein (apo) E-deficient mice associated with reducing oxidative stress. METHODS AND RESULTS: Atherosclerosis was induced in apo E-deficient mice fed a high fat diet. Mice were intraperitoneally treated with an injection of olmesartan (1mg/kg/day) daily over 8 weeks, and were compared with the untreated controls. Blood pressure was not changed significantly by the olmesartan treatment. Fatty streak plaque developed in apo E-deficient mice, and was suppressed in mice that received olmesartan. In addition, olmesartan reduced not only superoxide production but the overload of oxidative stress in aortic walls. There were no significant differences in serum lipid levels between olmesartan-treated and -untreated groups. In vitro study showed that both olmesartan and its active metabolite RNH-6270, an enantiomer of olmesartan, suppressed interferon-γ, macrophage inflammatory protein-2, and thioredoxin (a marker of oxidative stress) concentrations in cultured cells. CONCLUSION: Olmesartan may suppress atherosclerosis via reducing not only superoxide production but also the overload of oxidative stress in this animal model.

Cardioprotective effects of peroxisome proliferator activated receptor gamma activators on acute myocarditis: anti-inflammatory actions associated with nuclear factor kappaB blockade.

OBJECTIVE: To test the hypothesis that activation of peroxisome proliferator activated receptor gamma (PPAR-gamma) reduces experimental autoimmune myocarditis (EAM) associated with inhibitor kappaB (IkappaB) alpha induction, blockade of nuclear factor kappaB (NF-kappaB), and inhibition of inflammatory cytokine expression. METHODS: EAM was induced in Lewis rats by immunisation with porcine cardiac myosin. PPAR-gamma activators 15-deoxy-Delta(12,14)-prostaglandin J2 (15d-PGJ2) and pioglitazone (PIO) were administered to rats with EAM. RESULTS: Enhanced PPAR-gamma expression was prominently stained in the nuclear and perinuclear regions of infiltrating inflammatory cells. Administration of 15d-PGJ2 and PIO greatly reduced the severity of myocarditis and suppressed myocardial mRNA and protein expression of inflammatory cytokines in rats with EAM. In addition, treatment with PPAR-gamma activators enhanced IkappaB concentrations in the cytoplasmic fractions and nuclear fractions from inflammatory myocardium. Concurrently, NF-kappaB was greatly activated in myocarditis; this activation was blocked in the 15d-PGJ2 treated and PIO treated groups. CONCLUSIONS: PPAR-gamma may have a role in the pathophysiology of EAM. Because an increase in IkappaB expression and inhibition of translocation of the NF-kappaB subunit p65 to the nucleus in inflammatory cells correlated with the protective effects of PPAR-gamma activators, these results suggest that PPAR-gamma activators act sequentially through PPAR-gamma activation, IkappaB induction, blockade of NF-kappaB activation, and inhibition of inflammatory cytokine expression. These results suggest that PPAR-gamma activators such as 15d-PGJ2 and PIO may have the potential to modulate human inflammatory heart diseases such as myocarditis.

Immunoglobulin treatment prevents congestive heart failure in murine encephalomyocarditis viral myocarditis associated with reduction of inflammatory cytokines.

We have previously shown that immunoglobulin therapy suppressed murine coxsackievirus B3 myocarditis. In the present study, we examined the effects of immunoglobulin upon murine myocarditis induced by encephalomyocarditis virus, which is not pathogenic to humans. Antiviral activity of immunoglobulin (Venilon) against encephalomyocarditis virus could not be detected in vitro. The production of cytokines was decreased in virus-infected macrophages by the treatment of immunoglobulin in vitro. Immunoglobulin (1 g/kg/day) was administered intraperitoneally to the virus-infected C3H/He mice daily for 2 weeks, beginning simultaneously with virus inoculation in experiment I and on day 14 after virus inoculation in experiment II. In experiment I, survival rate did not differ significantly between immunoglobulin-treated and untreated groups. In experiment II, survival rate was higher in immunoglobulin compared with control groups. Immunoglobulin administration suppressed the development of myocardial necrosis with T-lymphocyte infiltrates in mice not only in the acute viremic but in the chronic aviremic stages concomitantly associated with the reduction of inflammatory cytokines, i.e., tumor necrosis factor-alpha, interferon-gamma, macrophage inflammatory protein-2, and interleukin-6. Taken together, immunoglobulin therapy could have the potential to prevent congestive heart failure.

T cell-mediated immune response enhances the severity of myocarditis in secondary cardiotropic virus infection in mice.

In this report, we showed that a previous enterovirus exposure in ordinary mice with normal T cell function, but not in T cell-deficient mice, can influence development of myocardial inflammation with a second virus exposure. Inoculation of 4-week-old male BALB/c-nu/+ (euthymic and normal T cell function) mice with amyocarditic Coxsackie virus B1 (CB1), followed by inoculation 28 days later with myocarditic variant of Coxsackie virus B3 (CB3-m) resulted in more intense myocardial inflammation and injury than was seen in BALB/c-nu/+ inoculated with CB1, followed by inoculation with non-enterovirus, i.e., encephalomyocarditis virus (EMC) or influenza A virus and in age-matched BALB/c-nu/+ mice secondary inoculated with CB3-m alone. In contrast, this phenomenon of the enhancement of the severity of myocarditis by a secondary CB3-m inoculation was not seen in BALB/c-nu/nu (athymic and T cell- deficient) mice. Interestingly, inoculation of BALB/c-nu/+ mice with CB1, followed by inoculation 28 days later with another amyocarditic variant of Coxsackie virus B3 (CB3-o), resulted in more severe myocarditis than was seen in age-matched BALB/c-nu/+ mice secondary inoculated CB3-o alone. Myocardial-activated T cells and elevated serum interleukin-6 were involved in the exacerbation of the disease during the reinfection. T cell-mediated immune responses to a conserved antigenic epitope among the enteroviruses may be involved in the exacerbation of myocardial inflammatory disease during a second enterovirus infection.

Fc receptor-mediated inhibitory effect of immunoglobulin therapy on autoimmune giant cell myocarditis: concomitant suppression of the expression of dendritic cells.

In the present study, the mechanisms and importance of the Fc portion of immunoglobulin in experimental giant cell myocarditis were examined. Giant cell myocarditis was induced in rats by immunization of porcine cardiac myosin. Human intact immunoglobulin (1 g. kg(-1). d(-1)) or F(ab')(2) fragments of human immunoglobulin (1 g. kg(-1). d(-1)) were administered intraperitoneally daily on days 1 to 21. Intact immunoglobulin administration significantly ameliorated myocarditis, but F(ab')(2) fragments did not. The ribonuclease protection assay revealed that therapy with intact immunoglobulin, but not F(ab')(2) fragments, suppressed the mRNA expressions of inflammatory and proinflammatory cytokines. Immunohistochemical analysis showed that therapy with intact immunoglobulin, but not F(ab')(2) fragments, suppressed dendritic cell (DC) expression during both the early and the subsequent fulminant phases. Moreover, the early treatment of intact immunoglobulin until the 11th day or 14th day, when the expression of DCs was completely suppressed, ameliorated myocarditis. However, the late treatment of intact immunoglobulin beginning on day 15, when the expression of DCs had already been completed, failed to ameliorate the condition. An in vitro study showed that intact immunoglobulin, but not F(ab')(2) fragments, suppressed the lipopolysaccharide-induced interleukin-1beta production associated with the downregulation of CD32 antigen (Fcgamma receptor II) expression. Thus, intact immunoglobulin therapy markedly suppressed myocarditis as a result of Fc receptor-mediated anti-inflammatory action, and the suppression of the disease was associated with the suppression of DCs, ie, the suppression of the initial antigen-priming process in experimental giant cell myocarditis.

Serum thioredoxin (TRX) levels in patients with heart failure.

An increase in oxidative stress is thought to be involved in the progression of heart disease, but the serum level of thioredoxin (TRX), which regulates the cellular redox state, has not been investigated in patients with heart diseases. The present study determined serum TRX levels with a sandwich enzyme-linked immunosorbent assay in a total of 39 patients with dilated cardiomyopathy (DCM) (n=5), acute coronary syndrome (ACS) (n=7) or stable angina (n=18), including effort angina (n=7) and vasospastic angina (n=11), and in control subjects (n=7). The serum TRX level in patients with New York Heart Association (NYHA) functional classes III and IV (n=8, 33.3+/-8.6 ng/ml) was significantly higher than in the control subjects (n=7, 14.0+/-4.6 ng/ml). In addition, the serum TRX levels correlated positively with the severity of NYHA class, and negatively with the left ventricular ejection fraction. The serum TRX levels were elevated in patients with ACS and DCM compared with the controls. These results indicate a possible association between TRX concentration and the severity of heart failure.

Difference in thioredoxin expression in viral myocarditis in inbred strains of mice.

Redox regulating mechanisms may be involved in the pathogenesis of viral myocarditis and thioredoxin (TRX) is a small multifunctional protein that contains a redox active sequence. The present study investigated the histopathology and characteristics of TRX expression in acute coxsackievirus B3 myocarditis in inbred strains of mice (severe myocarditis in DBA/2 mice, moderate myocarditis in BALB/c mice and mild myocarditis in C57BL/6 mice). Thioredoxin was upregulated and its expression correlated with the severity of the disease. In addition, 8-hydroxy-2'-deoxyguanosine, which is an established marker for oxidative stress, was concominantly positive in damaged myocytes. Thus, TRX may be specifically induced by the acute inflammatory stimuli in murine viral myocarditis, and the severity and development of acute viral myocarditis may be regulated by the cellular redox state.

Enhanced production of macrophage inflammatory protein 2 (MIP-2) by in vitro and in vivo infections with encephalomyocarditis virus and modulation of myocarditis with an antibody against MIP-2.

Interleukin-8 (IL-8) is a chemotactic cytokine for neutrophils and lymphocytes. Macrophage inflammatory protein 2 (MIP-2) is a murine counterpart of IL-8. The present study was performed to determine whether MIP-2 aggravates murine myocarditis. We examined (i) the MIP-2-producing activity of encephalomyocarditis (EMC) virus-infected cultured macrophages, (ii) serial plasma MIP-2 levels in EMC virus-induced mice by enzyme-linked immunosorbent assay, and (iii) the effects of antimouse MIP-2 monoclonal antibody (MAb) in vivo upon myocarditis. The production of MIP-2 increased in an infection dose- and time-dependent manner in virus-infected RAW 264. 7 macrophages. Five-week-old C(3)H/He mice were inoculated with EMC virus. Plasma MIP-2 levels were significantly elevated in mice on days 7 and 14 postinfection. Mice were injected subcutaneously with anti-MIP-2 MAb at 10 microg/day (group 2) or 100 microg/day (group 3) on days 0 to 5 and were observed until day 21. Uninfected control mice (group 1) were prepared. The survival rate was higher in the anti-MIP-2-treated group (group 3), but not in group 2, than in the control group. Histopathological analysis revealed that cellular infiltration and myocardial necrosis with macrophage and T-cell accumulation were less prominent in the anti-MIP-2 MAb-treated group, but not in group 2, compared to the level in the controls. MIP-2 is an important naturally occurring inflammatory cytokine in myocarditis, and anti-MIP-2 MAb treatment may prevent the inflammatory response.

Protection against murine coxsackievirus B3 myocarditis by T cell vaccination.

T cell vaccination regulates autoimmunity by the modification of helper and suppressor T cells. The present study was performed to examine whether T cell vaccination can prevent viral myocarditis in vivo. We used coxsackievirus B3 myocarditis in mice as an animal model with the analysis of lymphokine-activated killer cell activity. Vaccination of the mice with T lymphocytes significantly prolonged survival and improved cardiac histology of murine myocarditis. The effects of T cell vaccination were most evident when T cells sensitized with the same virus were used. Vaccination of the mice with T cells from other strains of mice showed lesser protective effects. Clearance of myocardial virus was not affected by this treatment. The efficacy of T cell vaccination was confirmed in vitro by the decrease of the lymphokine-activated killer cell activity against EL-4 tumor cells and cultured myocytes. T cell vaccination of mice prolonged survival and improved myocardial lesions of animals inoculated with coxsackievirus B3.

Immunoglobulin treatment ameliorates murine myocarditis associated with reduction of neurohumoral activity and improvement of extracellular matrix change.

OBJECTIVES: We examined effects of immunoglobulin on murine myocarditis induced by encephalomyocarditis virus, not pathogenic to humans, and analyzed the plasma cytokine and catecholamine levels and the changes of the extracellular matrix with or without the treatment. BACKGROUND: We have previously shown that immunoglobulin therapy suppressed murine coxsackievirus B3 myocarditis by an antiviral effect. However, it is not yet determined whether beneficial effects of immunoglobulin for myocarditis are due to antiviral effects or to other unknown effects. METHODS: Antiviral activity of human immunoglobulin (Polyglobin-N) against encephalomyocarditis virus was determined in vitro. Immunoglobulin (1 g/kg/day) was administered intraperitoneally to the virus-infected mice daily for two weeks, beginning simultaneously with virus inoculation in experiment I and on day 14 after virus inoculation in experiment II. RESULTS: Antiviral activity of immunoglobulin could not be detected in the assay of a plaque-reduction method in vitro. The in vivo study showed that immunoglobulin administration ameliorated both myocardial necrosis with interstitial fibrin deposition in experiment I and interstitial fibrosis with the improvement of ventricular remodeling in experiment II by the reduction of plasma catecholamines, interferon-alpha, and soluble intercellular adhesion molecule-1. CONCLUSIONS: Immunoglobulin therapy could suppress myocarditis associated with the improvement of extracellular matrix changes by the reduction of neurohumoral activity.

Role of MIP-2 in coxsackievirus B3 myocarditis.

Interleukin-8 (IL-8) is a chemotactic cytokine for neutrophils and lymphocytes. Macrophage inflammatory protein-2 (MIP-2) is a murine counterpart of IL-8. The present study was performed to determine the role of MIP-2 in murine myocarditis. We examined (1) the MIP-2 producing activity of Coxsackievirus B3 (CB3)-infected cultured macrophages, (2) serial plasma MIP-2 levels in CB3-induced mice by enzyme-linked immunosorbent assay (ELISA), and (3) the effects of anti-mouse MIP-2 monoclonal antibody (mAb) in vivo upon myocarditis. The production of MIP-2 increased in an infection dose- and time-dependent manner in virus-infected RAW 264.7 macrophages. Three-week-old C(3)H/He mice were inoculated with CB3. Plasma MIP-2 levels were significantly elevated in mice on days 7, 10 and 14 post-infection. Mice were injected subcutaneously with anti-MIP-2 mAb at 10 microg/day (Group 2) or 100 microg/day (Group 3) on days 0-7, and were observed until day 14. Uninfected control mice (Group 1) were injected with saline. Survival rate was higher in the anti-MIP-2-treated group (Group 3), but not in Group 2, than in the control group. Histopathological analysis revealed that cellular infiltration and myocardial necrosis with macrophage and T cell accumulation were less prominent in the anti-MIP-2 mAb-treated groups as compared to the controls. MIP-2 is an important naturally occurring inflammatory cytokine in CB3 myocarditis, and anti-MIP-2 mAb treatment may prevent the inflammatory response.

Strain difference in rats with experimental giant cell myocarditis.

Immunogenetic mechanisms may be involved in the pathogenesis of myocarditis and dilated cardiomyopathy. The present study investigated the incidence, histopathology and histocompatibility characteristics of experimental giant cell myocarditis in various strains of rats. Experimental giant cell myocarditis was induced by immunization with porcine cardiac myosin in Lewis (RT-1(l)), Dahl (DIR/Eis) (RT-1(l)), Fisher (RT-1(lv 1)) rats, but not in Dahl (DIS/Eis) (RT-1(l)) or Brown Norway (RT-1(n)). Myocarditis was most severe in the Lewis rats and their heart weight/body weight ratio was significantly higher than that of control rats immunized with Freund's complete adjuvant alone. In conclusion, this study provides evidence that the expression and severity of experimental giant cell myocarditis may be determined mainly by genetic factors, including both major histocompatibility complex genes as well as other genes, which may be controlled by an immune mechanism.

Upregulation of thioredoxin (TRX) expression in giant cell myocarditis in rats.

To examine the possible involvement of a redox regulating mechanism in the pathogenesis of immune-mediated myocarditis, myocarditis was induced by immunization of porcine cardiac myosin in rats and immunohistochemistry and Western blot for thioredoxin (TRX) were performed. Immunohistochemistry for 8-hydroxy-2'-deoxyguanosine (8-OHdG) and nuclear factor kappa-B (NF-kappaB) was also performed. TRX was upregulated in the acute stage, but not in the chronic stage, and the expression was correlated with the severity of the disease. Damaged myocytes were strongly immunostained for 8-OHdG and NF-kappaB. Thus, TRX may be specifically induced by acute inflammatory stimuli, and the development of acute immune-mediated myocarditis may be regulated by the cellular redox state via TRX.

Significance of pericardial cytokines in giant cell myocarditis in rats--pathological comparison to viral myocarditis in mice.

To investigate the precise disease progression in myocarditis, Lewis rats were injected with porcine cardiac myosin, and C3H/He mice were inoculated with coxsackievirus B3. Both were killed serially, and the hearts were stained with hematoxylin-eosin to compare their pathological characteristics. In viral myocarditis, viral replication in the myocardium resulted in myocardial necrosis with inflammation, and the lesions were distributed transmurally, as previously reported. On the other hand, in giant cell myocarditis, inflammatory lesions appeared at first around the capillaries in the epicardium, and thereafter spread transmurally. Pericardial effusion was noticed in all the rats with myocarditis in the fulminant stage. Levels of interleukin (IL) -1beta and IL-6 in the pericardial effusion were elevated compared with the serum cytokines at the peak of inflammation. However, interferon-gamma in both the pericardial effusion and serum was not elevated. The cause of the myocardial lesions that developed in rats with giant cell myocarditis may be some active inflammatory process via the pericardial effusion.

A nuclear complex containing PPARalpha/RXRalpha is markedly downregulated in the hypertrophied rat left ventricular myocardium with normal systolic function.

The expression of genes encoding fatty acid utilization enzymes is coordinately downregulated during the development of cardiac hypertrophy and failure. However, molecular mechanisms that mediate this downregulation are unknown. Peroxisome proliferator-activated receptor (PPAR) response elements (PPREs) have been identified in promoters of many genes involved in fatty acid utilization, where they function as positive regulatory elements. PPARs bind to PPREs as heterodimers with retinoid X receptors (RXRs). Primary cardiac myocytes from neonatal rats were transfected with a reporter construct driven by the C promoter of rat acyl-coenzyme A synthetase (ACS) gene. Stimulation with phenylephrine, a potent inducer of hypertrophy, markedly downregulated the activity of this promoter. By use of electrophoretic mobility-shift assays (EMSAs) using PPRE in the rat ACS promoter as a probe, we found a sequence-specific protein-DNA complex in the nuclear extract from adult rat left ventricular (LV) myocardium. Supershift experiments revealed that this complex was immunoreactive for PPARalpha and RXRalpha. We compared the activity of this complex in LV nuclear extracts from Dahl salt-sensitive rats (DSs) with hypertension and control age-matched Dahl salt-resistant rats (DRs). Even at the stage of concentric LV hypertrophy with normal systolic function, the activity of the band was markedly diminished in DSs compared with DRs. However, immunoblot analyses showed no difference in LV expression levels of PPARalpha or RXRalpha between DSs and DRs. These findings indicate that a nuclear complex of PPARalpha/RXRalpha is present in adult rat LV and is markedly downregulated in the hypertrophied LV from DS rats, which may account for the loss of transcriptional activation. The downregulation of this complex precedes LV systolic dysfunction and is mediated at the posttranslational levels.

HLA antigens in patients with variant angina in Japan.

There have been few reports on examining the susceptibility of variant angina. Accordingly, the major histocompatibility complexes (HLA-A, -B, -C, -DR) of unrelated Japanese patients with variant angina were examined. There were no significant differences in the frequency of HLA-A,-B, -C, and -DR antigens between patients and controls (n = 100). Although endothelial dysfunction with pathological abnormalities is suggested to be one of the etiological factors in vasospasm, immunogenetic abnormalities linked to HLA system might not play a role in the pathogenesis of variant angina.

Interstitial fibrin-fibronectin deposition with T cell infiltrates precedes fibrosis in murine viral myocarditis.

This study was carried out to investigate interstitial fibrin and fibronectin deposition and subsequent myocardial connective tissue abnormalities in BALB/c-nu/+ (euthymic and normal T cell function) and BALB/c-nu/nu (athymic and T cell-deficient) mice. Both types of mice were inoculated with encephalomyocarditis virus and sacrificed periodically. Sections of the hearts were stained with haematoxylin-eosin, trichrome, lymphocyte subsets, silver impregnation, and fibrin or fibronectin. In addition, myocardial collagen concentration was measured. Interstitial fibrin and fibronectin appeared in parallel with inflammatory T lymphocytes and myocardial necrosis in the BALB/c-nu/+ mice. The changes increased until 14 days, subsequently decreasing with time. Interstitial fibrosis and abnormal reticulin fibres were absent until 7 days postinfection, and then increased with time until 60 days. In BALB/c-nu/nu mice, in contrast, although myocardial necrosis and fibrin-fibronectin deposition associated with immature T lymphocytes were evident on days 7 and 14, subsequent myocardial fibrosis and reticulin fibre abnormalities were minimal on days 30 and 60. In BALB/c-nu/+ mice, myocardial collagen concentration increased on day 30, but it did not in BALB/c-nu/nu mice. Thus, interstitial fibrin-fibronectin deposition resulting from virus-induced and T lymphocyte-mediated myocyte necrosis precedes the subsequent development of interstitial fibrosis and abnormal reticulin architectures in this model of murine myocarditis.

Extracellular matrix remodeling in coxsackievirus B3 myocarditis.

The connective tissue abnormality in relation to T lymphocytes was investigated in murine myocarditis. Inbred BALB/c-nu/+ (euthymic and normal T cell function) and BALB/c-nu/nu (athymic and T cell deficient) mice were inoculated with coxsackievirus B3 (CB3). Hearts were stained with hematoxylin-eosin, Malloryazan, and silver impregnation, for reticulin fiber abnormalities, and T lymphocyte subsets. In BALB/c-nu/+ mice, active myocardial necrosis appeared parallel with T lymphocyte infiltrates, that is, it was absent on day 0, increased until 14 days, and then decreased with time. In contrast, the abnormal reticulin fiber architecture and interstitial fibrosis increased with time until 60 days, when ventricular remodeling was noted. In the hearts of BALB/c-nu/nu mice, although minimal myocardial necrosis associated with infiltrating immature T lymphocytes was noted earlier, subsequent interstitial fibrosis and reticulin fiber abnormalities were not documented later. The abnormal reticulin fiber architecture seen in BALB/c-nu/+ mice may contribute to the extracellular matrix remodeling in murine CB3 myocarditis in which dilated cardiomyopathy develops later.