Regional analysis of inflammation and contractile function in reperfused acute myocardial infarction by in vivo 19F cardiovascular magnetic resonance in pigs.

Inflammatory cell infiltration is central to healing after acute myocardial infarction (AMI). The relation of regional inflammation to edema, infarct size (IS), microvascular obstruction (MVO), intramyocardial hemorrhage (IMH), and regional and global LV function is not clear. Here we noninvasively characterized regional inflammation and contractile function in reperfused AMI in pigs using fluorine (19F) cardiovascular magnetic resonance (CMR). Adult anesthetized pigs underwent left anterior descending coronary artery instrumentation with either 90 min occlusion (n = 17) or without occlusion (sham, n = 5). After 3 days, in surviving animals a perfluorooctyl bromide nanoemulsion was infused intravenously to label monocytes/macrophages. At day 6, in vivo 1H-CMR was performed with cine, T2 and T2* weighted imaging, T2 and T1 mapping, perfusion and late gadolinium enhancement followed by 19F-CMR. Pigs were sacrificed for subsequent ex vivo scans and histology. Edema extent was 35 ± 8% and IS was 22 ± 6% of LV mass. Six of ten surviving AMI animals displayed both MVO and IMH (3.3 ± 1.6% and 1.9 ± 0.8% of LV mass). The 19F signal, reflecting the presence and density of monocytes/macrophages, was consistently smaller than edema volume or IS and not apparent in remote areas. The 19F signal-to-noise ratio (SNR) > 8 in the infarct border zone was associated with impaired remote systolic wall thickening. A whole heart value of 19F integral (19F SNR × milliliter) > 200 was related to initial LV remodeling independently of edema, IS, MVO, and IMH. Thus, 19F-CMR quantitatively characterizes regional inflammation after AMI and its relation to edema, IS, MVO, IMH and regional and global LV function and remodeling.

Improved diagnosis of idiopathic giant cell myocarditis and cardiac sarcoidosis by myocardial gene expression profiling.

AIMS: Improvement of clinical diagnostics of idiopathic giant cell myocarditis (IGCM) and cardiac sarcoidosis (CS), two frequently fatal human myocardial diseases. Currently, IGCM and CS are diagnosed based on differential patterns of inflammatory cell infiltration and non-caseating granulomas in histological sections of endomyocardial biopsies (EMBs), after heart explantation or postmortem. We report on a method for improved differential diagnosis by myocardial gene expression profiling in EMBs. METHODS AND RESULTS: We examined gene expression profiles in EMBs from 10 patients with histopathologically proven IGCM, 10 with CS, 18 with active myocarditis (MCA), and 80 inflammation-free control subjects by quantitative RT-QPCR. We identified distinct differential profiles that allowed a clear discrimination of tissues harbouring giant cells (IGCS, CS) from those with MCA or inflammation-free controls. The expression levels of genes coding for cytokines or chemokines (CCL20, IFNB1, IL6, IL17D; P < 0.05), cellular receptors (ADIPOR2, CCR5, CCR6, TLR4, TLR8; P < 0.05), and proteins involved in the mitochondrial energy metabolism (CPT1, CYB, DHODH; P < 0.05) were deregulated in 2- to 300-fold, respectively. Bioinformatic analyses and correlation of the gene expression data with immunohistochemical findings provided novel information regarding the differential cellular and molecular pathomechanisms in IGCM, CS, and MCA. CONCLUSION: Myocardial gene expression profiling is a reliable method to predict the presence of multinuclear giant cells in the myocardium, even without a direct histological proof, in single small EMB sections, and thus to reduce the risk of sampling errors. This profiling also facilitates the discrimination between IGCM and CS, as two different clinical entities that require immediate and tailored differential therapy.

Adiponectin promotes coxsackievirus B3 myocarditis by suppression of acute anti-viral immune responses.

Adiponectin (APN) is an immunomodulatory adipocytokine that improves outcome in patients with virus-negative inflammatory cardiomyopathy and mice with autoimmune myocarditis. Here, we investigated whether APN modulates cardiac inflammation and injury in coxsackievirus B3 (CVB3) myocarditis. Myocarditis was induced by CVB3 infection of APN-KO and WT mice. APN reconstitution was performed by adenoviral gene transfer. Expression analyses were performed by qRT-PCR and immunoblot. Cardiac histology was analyzed by H&E-stain and immunohistochemistry. APN-KO mice exhibited diminished subacute myocarditis with reduced viral load, attenuated inflammatory infiltrates determined by NKp46, F4/80 and CD3/CD4/CD8 expression and reduced IFNß, IFNγ, TNFα, IL-1ß and IL-12 levels. Moreover, myocardial injury assessed by necrotic lesions and troponin I release was attenuated resulting in preserved left ventricular function. Those changes were reversed by APN reconstitution. APN had no influence on adhesion, uptake or replication of CVB3 in cardiac myocytes. In acute CVB3 myocarditis, cardiac viral load did not differ between APN-KO and WT mice. However, APN-KO mice displayed an enhanced acute immune response, i.e. increased expression of myocardial CD14, IFNß, IFNγ, IL-12, and TNFα resulting in increased cardiac infiltration with pro-inflammatory M1 macrophages and activated NK cells. Up-regulation of cardiac CD14 expression, type I and II IFNs and inflammatory cell accumulation in APN-KO mice was inhibited by APN reconstitution. Our observations indicate that APN promotes CVB3 myocarditis by suppression of toll-like receptor-dependent innate immune responses, polarization of anti-inflammatory M2 macrophages and reduction of number and activation of NK cells resulting in attenuated acute anti-viral immune responses.

Etiologies and predictors of mortality in an all-comer population of patients with non-ischemic heart failure.

BACKGROUND: Despite progress in diagnosis and therapy of heart failure (HF), etiology and risk stratification remain elusive in many patients. METHODS: The My Biopsy HF Study (German clinical trials register number: DRKS22178) is a retrospective monocentric study investigating an all-comer population of patients with unexplained HF based on a thorough workup including endomyocardial biopsy (EMB). RESULTS: 655 patients (70.9% men, median age 55 [45/66] years) with non-ischemic, non-valvular HF were included in the analyses. 489 patients were diagnosed with HF with reduced ejection fraction (HFrEF), 52 patients with HF with mildly reduced ejection fraction (HFmrEF) and 114 patients with HF with preserved ejection fraction (HFpEF). After a median follow-up of 4.6 (2.5/6.6) years, 94 deaths were enumerated (HFrEF: 68; HFmrEF: 8; HFpEF: 18), equating to mortality rates of 3.3% and 11.6% for patients with HFrEF, 7.7% and 15.4% for patients with HFmrEF and 5.3% and 11.4% for patients with HFpEF after 1 and 5 years, respectively. In EMB, we detected a variety of putative etiologies of HF, including incidental cardiac amyloidosis (CA, 5.8%). In multivariate logistic regression analysis adjusting for age, sex and comorbidities only CA, age and NYHA functional class III + IV remained independently associated with all-cause mortality (CA: HRperui 3.13, 95% CI 1.5-6.51; p = 0.002). CONCLUSIONS: In an all-comer population of patients presenting with HF of unknown etiology, incidental finding of CA stands out to be independently associated with all-cause mortality. Our findings suggest that prospective trials would be helpful to test the added value of a systematic and holistic work-up of HF of unknown etiology.

A distinct subgroup of cardiomyopathy patients characterized by transcriptionally active cardiotropic erythrovirus and altered cardiac gene expression.

Recent studies have detected erythrovirus genomes in the hearts of cardiomyopathy and cardiac transplant patients. Assessment of the functional status of viruses may provide clinically important information beyond detection of the viral genomes. Here, we report transcriptional activation of cardiotropic erythrovirus to be associated with strongly altered myocardial gene expression in a distinct subgroup of cardiomyopathy patients. Endomyocardial biopsies (EMBs) from 415 consecutive cardiac erythrovirus (B19V)-positive patients with clinically suspected cardiomyopathy were screened for virus-encoded VP1/VP2 mRNA indicating transcriptional activation of the virus, and correlated with cardiac host gene expression patterns in transcriptionally active versus latent infections, and in virus-free control hearts. Transcriptional activity was detected in baseline biopsies of only 66/415 patients (15.9 %) harbouring erythrovirus. At the molecular level, significant differences between cardiac B19V-positive patients with transcriptionally active versus latent virus were revealed by expression profiling of EMBs. Importantly, latent B19V infection was indistinguishable from controls. Genes involved encode proteins of antiviral immune response, B19V receptor complex, and mitochondrial energy metabolism. Thus, functional mapping of erythrovirus allows definition of a subgroup of B19V-infected cardiomyopathy patients characterized by virus-encoded VP1/VP2 transcripts and anomalous host myocardial transcriptomes. Cardiac B19V reactivation from latency, as reported here for the first time, is a key factor required for erythrovirus to induce altered cardiac gene expression in a subgroup of cardiomyopathy patients. Virus genome detection is insufficient to assess pathogenic potential, but additional transcriptional mapping should be incorporated into future pathogenetic and therapeutic studies both in cardiology and transplantation medicine.

Alternatively spliced tissue factor and full-length tissue factor protect cardiomyocytes against TNF-α-induced apoptosis.

Tissue Factor (TF) is expressed in various cell types of the heart, such as cardiomyocytes. In addition to its role in the initiation of blood coagulation, the TF:FVIIa complex protects cells from apoptosis. There are two isoforms of Tissue Factor (TF): "full length" (fl)TF--an integral membrane protein, and alternatively spliced (as)TF--a protein that lacks a transmembrane domain and can thus be secreted in a soluble form. Whether asTF or flTF affects apoptosis of cardiomyocytes is unknown. In this study, we examined whether asTF or flTF protects murine cardiomyocytes from TNF-α-induced apoptosis. We used murine cardiomyocytic HL-1 cells and primary murine embryonic cardiomyocytes that overexpressed either murine asTF or murine flTF, and stimulated them with TNF-α to initiate cell death. Apoptosis was assessed by annexin-V assay, propidium iodide assay, as well as activation of caspase-3 and -9. In addition, signaling via integrins, Akt, NFκB and Erk1/2, and gene-expression of Bcl-2 family members were analyzed. We here report that overexpression of asTF reduced phosphatidylserine exposure upon TNF-α-stimulation. asTF overexpression led to an increased expression and phosphorylation of Akt, as well as up-regulation of the anti-apoptotic protein Bcl-x(L). The anti-apoptotic effects of asTF overexpression were mediated via α(V)ß(3)/Akt/NFκB signaling and were dependent on Bcl-x(L) expression in HL-1 cells. The anti-apoptotic activity of asTF was also observed using primary cardiomyocytes. Analogous yet less pronounced anti-apoptotic sequelae were observed due to overexpression of flTF. Importantly, cardiomyocytes deficient in TF exhibited increased apoptosis compared to wild type cells. We propose that asTF and flTF protect cardiomyocytes against TNF-α-induced apoptosis via activation of specific signaling pathways, and up-regulation of anti-apoptotic members of the Bcl-2 protein family.

Transgenic overexpression of the adenine nucleotide translocase 1 protects cardiomyocytes against TGFß1-induced apoptosis by stabilization of the mitochondrial permeability transition pore.

AIMS: Since adenine nucleotide translocase 1 (ANT1) overexpression improved cardiac function in rats with activated renin-angiotensin system (RAS) and angiotensin II is known to enhance transforming growth factor ß (TGFß) signaling in cardiomyocytes, we assumed that ANT1 might modulate the classical TGFß/SMAD pathway. We therefore investigated whether the cardioprotective effect of ANT1 overexpression suppresses TGFß(1)-induced apoptosis, whether mitochondrial permeability transition pore (MPTP) regulation is involved, and SMAD signaling pathway is affected. METHODS AND RESULTS: Ventricular cardiomyocytes isolated from wild-type (WT) and ANT1 transgenic rats were treated with the apoptosis-inducing agent TGFß(1) (1 ng/ml). TGFß(1) treatment of WT cells enhanced the number of apoptotic cells by 31.8 ± 11.7% (p<0.01 vs. WT) measured by chromatin condensation. Apoptosis was blocked by 1µM cyclosporine A and by ANT1 overexpression. The protecting effect of ANT1 overexpression on TGFß(1)-induced apoptosis was verified by reduced caspase 3/7 activity and increased Bcl-2 expression. In addition, TGFß(1) decreased mitochondrial membrane potential as measured by JC-1 staining by 18.0 ± 3.7% in WT cardiomyocytes, but only by 7.2 ± 2.8% (p<0.05 vs. WT) in ANT1 cardiomyocytes. Cyclosporine A also attenuated the decline in mitochondrial membrane potential under TGFß(1) in WT cardiomyocytes. Determination of MPTP opening by Calcein assay in isolated cardiomyocytes and calcium retention assay in isolated mitochondria revealed a reduced open probability of MPTP after ANT1 overexpression. In addition to the effects of ANT1 on MPTP opening we investigated if ANT1 may interfere with the classical TGFß signaling pathway. Interestingly, ANT1-transgenic cardiomyocytes expressed less TGFß receptor II than WT cells. However, SMAD2 phosphorylation was already enhanced without TGFß(1) stimulation in these cells. Although no additional increase in SMAD2 phosphorylation was detectable after TGFß(1) treatment, SMAD signaling was still responsive to TGFß(1) indicated by an upregulation of SMAD7, a TGFß(1) target protein. CONCLUSION: Heart-specific overexpression of ANT1 leads to a reduced apoptotic response to TGFß(1) by preservation of the mitochondrial membrane potential, resistance to MPTP opening and altered TGFß signaling.

miRNA as activity markers in Parvo B19 associated heart disease.

Parvovirus B19 is a frequent virus detected in endomyocardial biopsies of patients with clinically suspected myocarditis or dilated cardiomyopathy (DCM). Viruses often cause a more symptomatic disease with increased tissue injury if they become reactivated. A disease-specific differential expression of microRNAs (miRNAs) has been described in the regulation of replicating viruses. Analyzing patients with latent and reactivated B19V infection, we found 29 differentially regulated miRNAs and, in order to test whether predicted genes are differentially expressed, selected mRNAs were tested by TaqMan-QPCR.

Mesenchymal stem cells improve murine acute coxsackievirus B3-induced myocarditis.

AIMS: Coxsackievirus B3 (CVB3)-induced myocarditis, initially considered a sole immune-mediated disease, also results from a direct CVB3-mediated injury of the cardiomyocytes. Mesenchymal stem cells (MSCs) have, besides immunomodulatory, also anti-apoptotic features. In view of clinical translation, we first analysed whether MSCs can be infected by CVB3. Next, we explored whether and how MSCs could reduce the direct CVB3-mediated cardiomyocyte injury and viral progeny release, in vitro, in the absence of immune cells. Finally, we investigated whether MSC application could improve murine acute CVB3-induced myocarditis. METHODS AND RESULTS: Phase contrast pictures and MTS viability assay demonstrated that MSCs did not suffer from CVB3 infection 4-12-24-48 h after CVB3 infection. Coxsackievirus B3 RNA copy number decreased in this time frame, suggesting that no CVB3 replication took place. Co-culture of MSCs with CVB3-infected HL-1 cardiomyocytes resulted in a reduction of CVB3-induced HL-1 apoptosis, oxidative stress, intracellular viral particle production, and viral progeny release in a nitric oxide (NO)-dependent manner. Moreover, MSCs required priming via interferon-γ (IFN-γ) to exert their protective effects. In vivo, MSC application improved the contractility and relaxation parameters in CVB3-induced myocarditis, which was paralleled with a reduction in cardiac apoptosis, cardiomyocyte damage, left ventricular tumour necrosis factor-α mRNA expression, and cardiac mononuclear cell activation. Mesenchymal stem cells reduced the CVB3-induced CD4- and CD8- T cell activation in an NO-dependent way and required IFN-γ priming. CONCLUSION: We conclude that MSCs improve murine acute CVB3-induced myocarditis via their anti-apoptotic and immunomodulatory properties, which occur in an NO-dependent manner and require priming via IFN-γ.

Increased C reactive protein, cardiac troponin I and GLS are associated with myocardial inflammation in patients with non-ischemic heart failure.

Inflammatory cardiomyopathy diagnosed by endomyocardial biopsy (EMB) is common in non-ischemic heart failure (HF) and might be associated with adverse outcome. We aimed to identify markers predicting myocardial inflammation in HF. We screened 517 patients with symptomatic non-ischemic HF who underwent EMB; 397 patients (median age 54 [IQR 43/64], 28.7% females) were included in this study. 230 patients were diagnosed with myocardial inflammation, defined as ≥ 7.0 CD3+ lymphocytes/mm2 and/or ≥ 35.0 Mac1 macrophages/mm2 and were compared to 167 inflammation negative patients. Patients with myocardial inflammation were more often smokers (52.4% vs. 39.8%, p = 0.013) and had higher C-reactive protein (CRP) levels (5.4 mg/dl vs. 3.7 mg/dl, p = 0.003). In logistic regression models CRP ≥ 8.15 mg/dl (OR 1.985 [95%CI 1.160-3.397]; p = 0.012) and Troponin I (TnI) ≥ 136.5 pg/ml (OR 3.011 [1.215-7.464]; p = 0.017) were independently associated with myocardial inflammation, whereas no association was found for elevated brain natriuretic peptide (OR 1.811 [0.873-3.757]; p = 0.111). In prognostic performance calculation the highest positive predictive value (90%) was detected for the combination of Global longitudinal strain (GLS) ≥ -13.95% and TnI ≥ 136.5 pg/ml (0.90 (0.74-0.96)). Elevated CRP, TnI and GLS in combination with TnI can be useful to detect myocardial inflammation. Smoking seems to predispose for myocardial inflammation.

Antibody-mediated enhancement of calcium permeability in cardiac myocytes.

Our study shows that antibodies, specific to the ADP/ATP carrier of the inner mitochondrial membrane, crossreact with the cell surface of cardiac myocytes, where the calcium channel seems to be the antigenic determinant. The antibodies enhanced the calcium current and suppressed its inactivation. Affinity-purified antibodies (IgG) exhibit an acute cytotoxic effect, which required extracellular calcium and was prevented by calcium channel blockers. Our findings suggest that antibody-mediated cytotoxicity results secondary to calcium overload caused by enhanced cellular calcium permeability, requiring no complement-dependent process.

Biglycan is required for adaptive remodeling after myocardial infarction.

BACKGROUND: After myocardial infarction (MI), extensive remodeling of extracellular matrix contributes to scar formation and preservation of hemodynamic function. On the other hand, adverse and excessive extracellular matrix remodeling leads to fibrosis and impaired function. The present study investigates the role of the small leucine-rich proteoglycan biglycan during cardiac extracellular matrix remodeling and cardiac hemodynamics after MI. METHODS AND RESULTS: Experimental MI was induced in wild-type (WT) and bgn(-/0) mice by permanent ligation of the left anterior descending coronary artery. Biglycan expression was strongly increased at 3, 7, and 14 days after MI in WT mice. bgn(-/0) mice showed increased mortality rates after MI as a result of frequent left ventricular (LV) ruptures. Furthermore, tensile strength of the LV derived from bgn(-/0) mice 21 days after MI was reduced as measured ex vivo. Collagen matrix organization was severely impaired in bgn(-/0) mice, as shown by birefringence analysis of Sirius red staining and electron microscopy of collagen fibrils. At 21 days after MI, LV hemodynamic parameters were assessed by pressure-volume measurements in vivo to obtain LV end-diastolic pressure, end-diastolic volume, and end-systolic volume. bgn(-/0) mice were characterized by aggravated LV dilation evidenced by increased LV end-diastolic volume (bgn(-/0), 111+/-4.2 microL versus WT, 96+/-4.4 microL; P<0.05) and LV end-diastolic pressure (bgn(-/0), 24+/-2.7 versus WT, 18+/-1.8 mm Hg; P<0.05) and severely impaired LV function (EF, bgn(-/0), 12+/-2% versus WT, 21+/-4%; P<0.05) 21 days after MI. CONCLUSIONS: Biglycan is required for stable collagen matrix formation of infarct scars and for preservation of cardiac hemodynamic function.

Long-term clopidogrel administration following severe coronary injury reduces proliferation and inflammation via inhibition of nuclear factor-kappaB and activator protein 1 activation in pigs.

BACKGROUND: The optimal duration of clopidogrel treatment following percutaneous coronary intervention (PCI) and the patient population that would benefit most are still unknown. In a porcine coronary injury model, we tested two different durations of clopidogrel treatment on severely or moderately injured arteries and examined the arterial response to injury. To understand the molecular mechanism, we also investigated the effects on transcription factors nuclear factor-kappaB (NF-kappaB) and activator protein 1 (AP-1). MATERIALS AND METHODS: In 24 cross-bred pigs, one coronary artery was only moderately injured by percutaneous transluminal coronary angioplasty (PTCA) and one coronary artery was severely injured by PTCA and subsequent beta-irradiation (Brachy group). Animals received 325 mg aspirin daily for 3 months and 75 mg clopidogrel daily for either 28 days [short-term (ST) clopidogrel group] or 3 months [long-term (LT) clopidogrel group]. RESULTS: After 3 months, the number of proliferating cells per cross-section differed significantly between ST and LT in both injury groups (PTCA(ST) 90.2 +/- 10.3 vs. PTCA(LT )19.2 +/- 4.7, P < 0.05; Brachy(ST) 35.8 +/- 8.4 vs. Brachy(LT) 7.5 +/- 2.0, P < 0.05). Similar results were seen for inflammatory cells (CD3(+) cells): PTCA(ST) 23.5 +/- 3.55 vs. PTCA(LT )4.67 +/- 0.92, P < 0.05; Brachy(ST) 83.17 +/- 11.17 vs. Brachy(LT) 20 +/- 4.82, P < 0.05). Long-term administration also reduced the activity of NF-kappaB and AP-1 by 62-64% and 42-58%, respectively. However, the effects of different durations of clopidogrel administration on artery dimensions were not statistically significant. CONCLUSIONS: Regarding inflammation and transcription factor activity at the PCI site, long-term clopidogrel administration is superior to short-term administration, especially in severely injured arteries. Transferring our results to the human situation, patients with more severely diseased arteries may benefit from a prolonged clopidogrel medication after PCI.

The German Transregional Collaborative Research Centre 'Inflammatory Cardiomyopathy--Molecular Pathogenesis and Therapy'. Methods and baseline results from a 3-centre clinical study.

OBJECTIVES: This report focuses on the design and methods of the 3-centre clinical study of the Transregional Collaborative Research Centre 'Inflammatory Cardiomyopathy--Molecular Pathogenesis and Therapy', which aims to establish a comprehensive research registry on the diagnostics, therapy and disease outcomes of patients with inflammatory cardiomyopathy (CMi). The study goals are to investigate specific disease sub-entities and to develop standardised strategies for diagnostics and treatment. METHODS: All consecutive patients with clinically suspected CMi, post-myocarditic cardiomyopathy and acute myocarditis are included in the research registry. Cardiopulmonary functional tests, clinical and patient data are obtained at baseline and subsequent readmission appointments and are linked to allow for prospective follow-up. Co-morbidities, quality of life, health- related behaviour and sociodemographic variables are ascertained using uniform self-administered questionnaires. PRESENT STATUS: By May 2008, 2,061 cases had been included in the research registry (1,300 data-sets completed). At registration, 335 patients were diagnosed with CMi. The mean age was 50 +/- 13 years and the mean ejection fraction was 39.9 +/- 15.8%. CONCLUSIONS: The broad range of the acquired molecular-biological, histological, immunohistological, clinical and patient data makes this the most comprehensive research registry on patients with CMi to date.

Rational of the use of aliskiren in hypertension and beyond.

Arterial hypertension is an independent risk factor for cardiovascular diseases and one of the major causes for mortality worldwide. Drugs, that control hypertension effectively are therefore needed to reduce hypertension induced morbidity and mortality. The inhibition of the renin-angiotensin-aldosterone-system (RAAS) is one target to control blood pressure in these patients. The new direct renin inhibitor aliskiren is one new substance on the market to inhibit the RAAS effectively by suppression of the plasma renin activity, which inhibits the RAAS at its rate-limiting step. Therefore, aliskiren in monotherapy and in combination might yield beneficial effects for the patients. Nevertheless, blood pressure lowering has to be combined with a reduction of target organ damage for all drug classes prescribed to patients with hypertension. Therefore, we review here the major effects of this new drug not only in regard to hypertension but also in regard to target organ damage reduction and possible changes in morbidity and mortality, which future trials will investigate.

Enhancement of endothelial nitric oxide synthase production reverses vascular dysfunction and inflammation in the hindlimbs of a rat model of diabetes.

AIMS/HYPOTHESIS: Reduced bioavailability of nitric oxide (NO) is a hallmark of diabetes mellitus-induced vascular complications. In the present study we investigated whether a pharmacological increase of endothelial NO synthase (eNOS) production can restore the impaired hindlimb flow in a rat model of severe diabetes. METHODS: A model of diabetes mellitus was induced in male Sprague-Dawley rats by a single injection of streptozotozin. Rats were treated chronically with the eNOS transcription enhancer AVE3085 (10 mg [kg body weight](-1) day(-1); p.o.) or vehicle for 48 days and compared with controls. Endothelial function and arterial BP were investigated in vivo using an autoperfused hindlimb model and TIP-catheter measurement, respectively. Protein production of eNOS, total and phosphorylated vasodilator-stimulated phosphoprotein (VASP) were assessed in their quadriceps muscle tissue, whereas cyclic GMP (cGMP) concentrations were assessed in blood plasma. RNA levels of intracellular and vascular cell adhesion molecules (ICAM-1 and VCAM-1) were measured by real-time PCR. RESULTS: Untreated diabetic rats showed significantly reduced quadriceps muscle contents of eNOS (-64%) and phosphorylated VASP (-26%) protein associated with impaired vascular function (maximum vasodilatation: -30%, p < 0.05) and enhanced production of ICAM-1 (+121%) and VCAM-1 (+156%). Chronic treatment with AVE3085 did not alter arterial BP or severe hyperglycaemia, but did lead to significantly increased production of eNOS (+95%), cGMP (+128%) and VASP phosphorylation (+65%) as well as to improved vascular function (+36%) associated with reduced production of ICAM-1 (-36%) and VCAM-1 (-58%). CONCLUSIONS/INTERPRETATION: In a rat model of severe diabetes, pharmacological enhancement of impaired eNOS production and NO-cGMP signalling by AVE3085 restores altered hindlimb blood flow and prevents vascular inflammation.

The cardiovascular influence of interleukin-1 beta on the expression of bradykinin B1 and B2 receptors.

The cytokine interleukin 1ss (IL-1ss) and the bradykinin receptors 1 (B1R) and 2 (B2R) are known to be upregulated in the ischemic heart. In the present study we investigated whether or not there is a causal link between these entities. Further we investigated whether or not pharmacological inhibition of IL-1ss release affects B1R and B2R regulation as well as left ventricular (LV) function in an in vivo rat model of myocardial infarction (MI). B1R and B2R mRNA levels were determined in cultured rat cardiomyocytes, aortic smooth muscle cells and cardiac fibroblasts (n=6 per group) under basal conditions, and after incubation of IL-1ss (40, 400 and 4000 pg/ml). Also, MI was induced in male Sprague-Dawley rats by ligation of the left descending coronary artery. Rats were treated with the interleukin converting enzyme inhibitor (ICEI) pralnacasan (50 mg/kg/day), or with a placebo. Three weeks after induction of MI, LV function was assessed using a 1.4 Millar TIP-catheter. Cardiac expressions of B1R and B2R mRNA were measured using ribonuclease-protection assays. Under basal conditions, both B1R and B2R were expressed in cardiomyocytes and smooth muscle cells, but not in cardiac fibroblasts. IL-1ss cultivation led only in cardiomyocytes to a significant upregulation of B1R mRNA. To a significant upregulation of B2R mRNA, it did not. In addition, ICEI treatment led in vivo to a significant downregulation of cardiac B1R mRNA, but not of B2R mRNA expression three weeks after induction of MI. Our data suggest that a causal link exists between cardiac IL-1ss content and B1R regulation after MI.

Genomic expression profiling of human inflammatory cardiomyopathy (DCMi) suggests novel therapeutic targets.

The clinical phenotype of human dilated cardiomyopathy (DCM) encompasses a broad spectrum of etiologically distinct disorders. As targeting of etiology-related pathogenic pathways may be more efficient than current standard heart failure treatment, we obtained the genomic expression profile of a DCM subtype characterized by cardiac inflammation to identify possible new therapeutic targets in humans. In this inflammatory cardiomyopathy (DCMi), a distinctive cardiac expression pattern not described in any previous study of cardiac disorders was observed. Two significantly altered gene networks of particular interest and possible interdependence centered around the cysteine-rich angiogenic inducer 61 (CYR61) and adiponectin (APN) gene. CYR61 overexpression, as in human DCMi hearts in situ, was similarly induced by inflammatory cytokines in vascular endothelial cells in vitro. APN was strongly downregulated in DCMi hearts and completely abolished cytokine-dependent CYR61 induction in vitro. Dysbalance between the CYR61 and APN networks may play a pathogenic role in DCMi and contain novel therapeutic targets. Multiple immune cell-associated genes were also deregulated (e.g., chemokine ligand 14, interleukin-17D, nuclear factors of activated T cells). In contrast to previous investigations in patients with advanced or end-stage DCM where etiology-related pathomechanisms are overwhelmed by unspecific processes, the deregulations detected in this study occurred at a far less severe and most probably fully reversible disease stage.

[State of diagnostics and therapy of inflammatory cardiomyopathie]. / Stand der Diagnostik und Therapie der inflammatorischen Kardiomyopathie.

Myocardial inflammation of cardiac muscle is caused by intramyocardial infiltration of immunological competent cells. Etiologically, the relevant factors are the direct or indirect influence of infectious pathogens, toxic, chemical or physical agents, allergic-hyperergic reactions and myocardial inflammatory events in the context of systemic diseases. Clinically significant infections include cardiotropic viruses, which are capable of causing acute or chronic damage to the myocardium under certain conditions. The wide variety of etiologies in inflammatory cardiomyopathy and its heterogeneous clinical presentations have impeded patients identification and consensus on the most appropriate diagnostic criteria and specific therapeutic strategies. Although the Dallas criteria have standardized the histological definition of active myocarditis, the recognition of true etiologies of acute and chronic stages of inflammatory cardiomyopathies require additional and more sensitive markers of tissue inflammation and molecular biological identification of responsible infectious agents. Actually, only biopsy-based diagnosis and characterization of patients allow identification of patients who may get benefit from specific immunosuppressive or anti-viral treatment strategies.

Treatment of coxsackievirus-B3-infected BALB/c mice with the soluble coxsackie adenovirus receptor CAR4/7 aggravates cardiac injury.

Coxsackie adenovirus receptor (CAR) is involved in immunological processes, and its soluble isoforms have antiviral effects on coxsackievirus B3 (CVB3) infection in vitro. We explored in this study the impact of CAR4/7, a soluble CAR isoform, on CVB3-induced myocarditis in BALB/c mice. BALB/c mice were treated daily with recombinant CAR4/7, beta-galactosidase (beta-Gal; as control protein) or buffer for 9 days. Half of each group was infected with CVB3 on day 3, and all mice were killed on day 9. Myocardial CVB3 titer, histology, and serology were analyzed. Treatment with CAR4/7 led to a significant reduction of myocardial CVB3 titer, whereas the application of beta-Gal had no detectable effect on the myocardial virus load. CAR4/7 application, however, resulted in increased myocardial inflammation and tissue damage in CVB3-infected hearts, whereas beta-Gal caused a degree of cardiac inflammation and injury similar to that in buffer-treated CVB3-infected control animals. CAR4/7 and beta-Gal treatment induced the production of antibodies against the respective antigens. CAR4/7-, but not beta-Gal-specific, virus-negative sera reacted against myocardial tissue and cellular membranous CAR, and significantly inhibited CVB3 infection in vitro. Thus, CAR4/7 suppressed CVB3 infection in vivo, supporting the concept of receptor analog in antiviral therapy. However, CAR4/7 treatment also leads to an aggravation of myocardial inflammation and injury most likely secondary to an autoimmune process.