Myocarditis following adeno-associated viral gene expression of human soluble TNF receptor (TNFRII-Fc) in baboon hearts.

Sequestration of tumor necrosis factor-alpha (TNFalpha) by TNF-receptor immunoglobulin G (IgG)-Fc fusion proteins can limit heart failure progression in rodent models. In this study we directly injected an adeno-associated viruses (AAV)-2 construct encoding a human TNF receptor II IgG-Fc fusion protein (AAV-TNFRII-Fc) into healthy baboon hearts and assessed virally encoded gene expression and clinical response. Adult baboons received direct cardiac injections of AAV-TNFRII-Fc ( approximately 5 x 10(12) viral/genomes/baboon) or an equivalent dose of AAV-2 empty capsids, and were analyzed after 5 or 12 weeks. Viral genomes were restricted to the myocardium, and routine analyses (blood cell counts, clinical chemistries) remained unremarkable. Echocardiograms were unchanged but electrocardiograms revealed marked ST- and T-wave changes consistent with myocarditis only in baboons receiving AAV-TNFRII-Fc. TNFRII serum levels peaked at approximately 3 times the baseline levels at 1-2 weeks postinjection and subsequently declined to baseline levels. TNFRII-Fc protein and transcripts were detected in the heart at harvest. After AAV injection, anti-AAV-2 antibody levels increased in all baboons, while anti-TNFRII-Fc could not be detected. Baboons that received AAV-TNFRII-Fc developed myocardial infiltrates including CD8+ cells. Thus, a cellular immune response to cardiac delivery of AAV encoding foreign proteins may be an important consideration for AAV-based cardiac gene therapy.

Polymorphisms in adenosine receptor genes are associated with infarct size in patients with ischemic cardiomyopathy.

The goal of this experiment was to identify the presence of genetic variants in the adenosine receptor genes and assess their relationship to infarct size in a population of patients with ischemic cardiomyopathy. Adenosine receptors play an important role in protecting the heart during ischemia and in mediating the effects of ischemic preconditioning. We sequenced DNA samples from 273 individuals with ischemic cardiomyopathy and from 203 normal controls to identify the presence of genetic variants in the adenosine receptor genes. Subsequently, we analyzed the relationship between the identified genetic variants and infarct size, left ventricular size, and left ventricular function. Three variants in the 3'-untranslated region of the A(1)-adenosine gene (nt 1689 C/A, nt 2206 Tdel, nt 2683del36) and an informative polymorphism in the coding region of the A3-adenosine gene (nt 1509 A/C I248L) were associated with changes in infarct size. These results suggest that genetic variants in the adenosine receptor genes may predict the heart's response to ischemia or injury and might also influence an individual's response to adenosine therapy.

Developing strategies to link basic cardiovascular sciences with clinical drug development: another opportunity for translational sciences.

Driven, at least in part, by the National Institutes of Health roadmap, an increasing number of studies has bridged the chasm between observations in the basic research laboratory and the clinical bedside. These studies have been an integral part in "translating" new discoveries into therapeutic initiatives. However, "translational medicine" has been used less frequently in the development of cardiovascular drugs or in predicting the potential cardiovascular toxicity of non-cardiac agents. Studies in animal models can provide important clues as to the potential cardiotoxicity of new therapeutic agents, as well as providing a template for the rational design of clinical trials. Three examples of drug development programs that might have been altered by clues available from laboratory studies include the development programs for the anti-cancer drug trastuzumab, the cyclooxygenase inhibitors, and the adenosine-receptor agonists and antagonists. Although mouse models may not always represent the physiology of humans, they provide important information that clinical scientists can utilize in designing safe programs for the evaluation of new pharmacologic agents.

Benefit and safety of enhanced external counterpulsation in treating coronary artery disease patients with a history of congestive heart failure.

Enhanced external counterpulsation (EECP) is used to noninvasively treat refractory angina patients, including those with a history of heart failure. The International EECP Patient Registry was used to examine the benefit and safety of EECP treatment, including a 6-month follow-up, in 1,957 patients, 548 with a history of heart failure. The heart failure cohort was older, with more females, a greater duration of coronary artery disease, more prior infarcts and revascularizations. Significantly fewer heart failure patients completed the course of EECP, and exacerbation of heart failure was more frequent, though overall major adverse cardiac events (MACE, i.e. death, myocardial infarction, revascularization) during treatment were not significantly different. The angina class improved in 68%, with comparable quality of life benefit, in the heart failure cohort. At 6 months, patients with congestive heart failure maintained their reduction in angina but were significantly more likely to have experienced a MACE end point.

Differential effects of overexpression of two forms of ephrin-A5 on neonatal rat cardiomyocytes.

Eph receptors constitute the largest family of receptor tyrosine kinases. Multiple transcripts of ephrin-A5, the cognate ligand of the EphA3 receptor, were found in neonatal rat cardiomyocytes. Two cDNA clones encoding the full-length ephrin-A5 (ephrin-A5 alpha) and a 27-amino acid deletion form (ephrin-A5 beta) were isolated. To examine the role of ephrin-A5 in cardiomyocytes, the cDNAs were inserted into adenoviral vectors, termed Ad.ephrin-A5 alpha and Ad.ephrin-A5 beta, respectively, and overexpressed in cardiomyocytes. The effect of ephrin-A5 on cardiomyocyte gene expression was investigated using a cDNA expression array and Western blot analysis. The results showed that both ephrin-A5 alpha and ephrin-A5 beta downregulated cyclin D2, cyclin-dependent kinase-4 proteins, and their cognate receptor EphA3, which were associated with reduced bromodeoxyuridine incorporation in cardiomyocytes. Whereas ephrin-A5 alpha and ephrin-A5 beta also induced differential gene expression, only ephrin-A5 beta significantly upregulated the transcription of brain natriuretic peptide and downregulated ras-related protein RAB2, protein kinase C inhibitor protein-1, clusterin, and insulin-like growth factor-binding protein. The results suggest that the two forms of ephrin-A5 share similar function while differ in regulating different sets of genes in cardiomyocytes.

Mitochondrial abnormalities in tumor necrosis factor-alpha-induced heart failure are associated with impaired DNA repair activity.

BACKGROUND: Recent studies suggest that mutations in cardiac mitochondrial DNA (mtDNA) may contribute to the development of dilated cardiomyopathy. The mechanisms that regulate those mutations, however, remain undefined. Thus, we studied cardiac mtDNA repair mechanisms, mtDNA damage, and mitochondrial structure and function in mice with heart failure secondary to overexpression of TNF-alpha (TNF1.6 mice). METHODS AND RESULTS: We studied mtDNA repair by measuring the uracil DNA glycosylase (mtUDG) and base excision repair activities. mtDNA damage was assessed by Southern blot of Fpg protein-digested mtDNA. Mitochondrial ultrastructural changes were examined by electron microscopy, and function by cytochrome c oxidase and succinate dehydrogenase activity assays. The results showed that both mtUDG and base excision repair activities were significantly reduced in TNF1.6 mouse heart. Fpg-sensitive sites were markedly increased in TNF1.6 mouse cardiac mtDNA, suggesting increased mtDNA damage. Mitochondrial function as demonstrated by cardiac cytochrome c oxidase activity was also markedly reduced. Cardiac ATP content was not changed, however, suggesting a shift from oxidative phosphorylation to glycolysis, as shown by increased LDH and ALT activities and lactate/pyruvate ratio. Ultrastructurally, the TNF1.6 mouse cardiac mitochondria became irregular in shape and smaller, and the cristae were decreased and appeared disorganized, with breaks. CONCLUSIONS: These results suggest that mtDNA mutations and mitochondrial structural and functional alterations in TNF-alpha-induced heart failure may be associated with reduced mtDNA repair activity, and the pathophysiological effects of TNF-alpha on the heart may be mediated, at least in part, through these changes in mitochondria.

Anti-tumor necrosis factor-alpha antibody limits heart failure in a transgenic model.

BACKGROUND: - Tumor necrosis factor (TNF)-alpha has been implicated in the pathophysiology of congestive heart failure. A strain of transgenic mice (TNF1.6) with cardiac-specific overexpression of TNF-alpha develop congestive heart failure. METHODS AND RESULTS: To determine the effect of anti-TNF-alpha therapy in this model, we studied 3 groups: TNF1.6 mice treated with saline, wild-type mice treated with saline, and TNF1.6 mice treated with TNF-alpha neutralizing antibody (cV1q) from 6 to 12 weeks of age. We used echocardiography to compare cardiac hypertrophy, function, and catecholamine response at 12 weeks of age versus baseline (6 weeks). cV1q treatment did not limit cardiac hypertrophy, but it significantly improved basal fractional shortening and responsiveness to beta-adrenergic stimulation, and it limited development of cardiac dilation. CONCLUSIONS: Blockade of TNF-alpha bioactivity by antibody therapy may both preserve cardiac function and partially reverse pathological changes in congestive heart failure.

Downregulation of matrix metalloproteinases and reduction in collagen damage in the failing human heart after support with left ventricular assist devices.

BACKGROUND: Left ventricular assist device (LVAD) support of the failing heart induces salutary changes in myocardial structure and function. Matrix metalloproteinases (MMPs) are increased in the failing heart and are induced by stretch in cardiac cells in vitro. We hypothesized that mechanical unloading may affect LV plasticity by regulating MMPs and their substrates. METHODS AND RESULTS: LV samples were collected from patients with dilated cardiomyopathy (DCM, n=14) or ischemic cardiomyopathy (ICM, n=16) at the time of implantation of the LVAD and again during cardiac transplantation. MMP-1, -3, and -9 were measured by ELISA, MMP-2 and -9 gelatinolytic activity by gelatin zymography, and tissue inhibitors of metalloproteinases (TIMPs) by Western blot. Total soluble and insoluble collagens were separated by pepsin solubilization, and the contents were determined by quantification of hydroxyproline. The undenatured soluble collagen was measured by Sircol collagen assay. The results showed that MMP-1 and -9 were decreased, whereas TIMP-1 and -3 were increased, but there was no change in MMP-2 and -3 and TIMP-2 and -4 after LVAD support. The undenatured collagen was increased, with the ratio of undenatured to total soluble collagens increased in ICM and that of insoluble to total soluble collagens increased in DCM after LVAD support. CONCLUSIONS: The reduced MMPs and increased TIMPs and ratios of undenatured to total soluble collagens and insoluble to total soluble collagens after LVAD support suggest that reduced MMP activity diminished damage to the matrix. These changes may contribute to the functional recovery and LV plasticity after LVAD support.

Matrix metalloproteinases in the progression of heart failure: potential therapeutic implications.

Matrix metalloproteinases (MMPs) are a family of functionally related zinc-containing enzymes that denature and degrade fibrillar collagens and other components of the extracellular matrix. Myocardial extracellular matrix remodelling and fibrosis regulated by MMPs are believed to be important contributors to the progression of heart failure. The role of MMPs in cardiac fibrosis and the progression of heart failure, along with the possibility of halting the progression of heart failure by modulating extracellular matrix remodelling are important issues under intense study. MMPs are increased in the failing hearts of both animal models and patients with heart failure. MMP inhibition may therefore modulate extracellular matrix remodelling and the progression of heart failure. It is a great advantage that various MMP inhibitors have been developed initially for the treatment of cancer, arthritis and other diseases believed to be associated with increased MMP activity. Several preclinical studies have shown that treatment of heart failure in animal models with MMP inhibitors results in less collagen matrix damage, favourable extracellular matrix remodelling, and improved cardiac structure and function. The results suggest that modulation of MMP activity can prevent myocardial dysfunction and the progression of heart failure through alterations in the remodelling process of extracellular matrix and the left ventricle. Although these promising results suggest potential benefits of MMP inhibition for human heart failure, no clinical data evaluating MMP inhibitors in heart failure have been reported. As the preclinical evidence continues to grow and the potential of MMP inhibition for the treatment of heart failure continues to unfold, MMP inhibition may prove to be an effective treatment for heart failure.

Effects of vesnarinone on peripheral circulating levels of cytokines and cytokine receptors in patients with heart failure: a report from the Vesnarinone Trial.

STUDY OBJECTIVES: Proinflammatory cytokines may contribute to disease progression in heart failure by virtue of the direct toxic effects that these molecules exert on the heart and the circulation. Accordingly, there is interest in developing therapeutic agents with anticytokine properties that might be used as adjunctive therapy to modulate proinflammatory cytokine levels in patients with heart failure. Previous experimental studies suggested that vesnarinone has potent anticytokine properties in vitro. Therefore, we examined the effects of vesnarinone on circulating levels of cytokines and cytokine receptors in a large-scale, multicenter, clinical trial of patients with moderate-to-advanced heart failure: the Vesnarinone Trial (VEST). METHODS: Circulating levels of tumor necrosis factor (TNF)-alpha, soluble TNF-receptor type 1, soluble TNF-receptor type 2, as well as interleukin (IL)-6 and soluble IL-6 receptor (sIL-6R) were measured on plasma samples by enzyme-linked immunosorbent assay at baseline and at 24 weeks in patients who were receiving placebo (n = 352), 30 mg of vesnarinone (n = 367), and 60 mg of vesnarinone (n = 327). RESULTS: Treatment with 30 mg and 60 mg of vesnarinone had no effect on circulating levels of cytokines or cytokine receptors in patients with advanced heart failure over a 24-week period. CONCLUSIONS: In contrast to the potent anticytokine effects observed with vesnarinone in experimental studies in vitro, the results of this clinical study suggest that vesnarinone does not have any measurable anticytokine effects in vivo in patients with moderate-to-advanced heart failure.

Overexpression of tumor necrosis factor- alpha activates both anti- and pro-apoptotic pathways in the myocardium.

We have previously reported that mice with cardiac-specific overexpression of tumor necrosis factor (TNF)- alpha develop myocardial inflammation, cardiac hypertrophy, and dilated cardiomyopathy. TNF- alpha is reported to induce apoptosis in cultured cardiac myocytes. To investigate the role of apoptosis in this transgenic model, wild-type controls (WT) and transgenic mice (TG) at the age of 1, 8, and 40 weeks were analyzed. Increased incidence of apoptosis in TG was indicated by DNA laddering. TUNEL assays revealed that the frequencies of apoptotic cells were increased in the TG myocardium at all ages. However, as revealed by histochemical and immunofluorescent methods, most of the apoptotic cells appeared to be non-myocytes even in the mice with overt congestive heart failure. To elucidate the signaling pathways responsible for TNF- alpha induced apoptosis, expression of apoptosis-related genes were evaluated by multi-probe RNase protection assays. Transcripts for death-domain-related proteins, including TNFR1, Fas, FADD, TRADD, and RIP, were constitutively expressed in WT and upregulated in the TG myocardium. Expression of caspase-1 through -8 was also enhanced in TG. While both anti- and pro-apoptotic Bcl-2 family genes were constitutively expressed in WT, TNF- alpha overexpression strongly induced anti-apoptotic A1 in the myocardium. Furthermore, TNF- alpha overexpression activated NF- kappa B, a mediator of anti-apoptotic pathways, in the myocardium. Thus, overexpression of TNF- alpha activated both anti- and pro-apoptotic pathways in the myocardium, resulting in an increase of apoptosis, primarily in non-myocytes. These results suggest that TNF- alpha by itself is not sufficient to induce apoptosis in cardiac myocytes in vivo.

UV light synergistically enhances the cardiotoxic effects of interleukin 1beta through peroxynitrite formation.

BACKGROUND: Proinflammatory cytokines play an important role in chronic cardiac diseases. METHODS AND RESULTS: Neonatal rat cardiomyocytes were exposed to interleukin (IL)-1beta (2 ng/mL) for 4 days. We assessed contractility through videomicroscopy and calcium transients with the Ca(2+)-sensitive dye fura-2. In IL-1beta-treated cells, the UV excitation (380 nm) necessary to induce dye fluorescence effected a rapid cessation of Ca(2+) transients and contraction, accompanied by calcium overload originating from an intracellular compartment. This occurred in the absence of fura-2 but required UV illumination. Incubation with 10 mmol/L N-acetylcysteine prevented this response, suggesting a free radical-mediated event. However, exposure to IL-1beta either increased or did not change the activity of the free radical scavengers superoxide dismutase, catalase, and glutathione peroxidase. In contrast, lipid peroxidation increased by 600% (P < or =.0001) in the IL-1beta plus UV-treated cells, an effect eliminated by L-NMMA. L-NMMA also completely abolished the UV-mediated cytotoxicity. We used immunohistochemistry to localize nitrotyrosine accumulation in the myocytes cotreated with IL-1beta and UV, an effect that was also blocked by L-NMMA. CONCLUSIONS: We hypothesize that the toxic radical peroxynitrite, arising from nitric oxide and superoxide anion, may be responsible for tetany and acute cardiomyocyte death. These results demonstrate the potential role of peroxynitrite in cardiotoxicity, which may be important in cardiac diseases associated with proinflammatory cytokines.

The International EECP Patient Registry (IEPR): design, methods, baseline characteristics, and acute results.

BACKGROUND: In 1998, the International EECP Patient Registry (IEPR) was organized to document patient characteristics, safety, and efficacy during the treatment period, and long-term outcomes. All centers with EECP facilities were invited to join the voluntary Registry. The Registry population comprises all patients starting EECP therapy for treatment of angina pectoris in participating centers. HYPOTHESIS: The study was undertaken to determine whether EECP is a safe and effective treatment for patients with angina pectoris regardless of their suitability for revascularization by more conventional techniques. METHODS: After 18 months of operation, 43 clinical centers representing over half of clinical sites using the EECP system contributed cases. The data reported here were collected before the first EECP treatment and upon completion of final treatment. EECP can be used for patients ineligible for either coronary artery bypass graft (CABG) or percutaneous coronary intervention (PCI), as well as for those who prefer noninvasive treatment to avoid or delay revascularization. In this report, patients considered to be candidates for revascularization are compared with those not considered suitable. RESULTS: Of the 978 patients analyzed, 70% had Canadian Cardiovascular Society Classification class III or IV angina before starting treatment, and 62% used nitroglycerin. Most (81%) had been previously revascularized, and 69% were considered unsuited for either PCI or CABG at the time of starting EECP. A full treatment course (usually 35 h) was completed in 86%, of whom 81% reported improvement of at least one angina class immediately after the last treatment. CONCLUSION: In a broad patient population, EECP has been shown to be a safe and effective treatment.

Controlled trial of intravenous immune globulin in recent-onset dilated cardiomyopathy.

BACKGROUND: This prospective placebo-controlled trial was designed to determine whether intravenous immune globulin (IVIG) improves left ventricular ejection fraction (LVEF) in adults with recent onset of idiopathic dilated cardiomyopathy or myocarditis. METHODS AND RESULTS: Sixty-two patients (37 men, 25 women; mean age +/-SD 43.0+/-12.3 years) with recent onset (/=0.10 from study entry, and 20 (36%) of 56 normalized their ejection fraction (>/=0.50). The transplant-free survival rate was 92% at 1 year and 88% at 2 years. CONCLUSIONS: These results suggest that for patients with recent-onset dilated cardiomyopathy, IVIG does not augment the improvement in LVEF. However, in this overall cohort, LVEF improved significantly during follow-up, and the short-term prognosis remains favorable.

Cytokines and cytokine receptors in advanced heart failure: an analysis of the cytokine database from the Vesnarinone trial (VEST).

BACKGROUND: Previous reports have shown that elevated circulating levels of cytokines and/or cytokine receptors predict adverse outcomes in patients with heart failure. However, these studies were limited by small numbers of patients and/or they were performed in a single center. In addition, these studies did not have sufficient size to address the influence of age, race, sex, and cause of heart failure on the circulating levels of these inflammatory mediators in patients with heart failure. METHODS AND RESULTS: We analyzed circulating levels of cytokines (tumor necrosis factor [TNF] and interleukin-6) and their cognate receptors in 1200 consecutive patients who were enrolled in a multicenter clinical trial of patients with advanced heart failure. This analysis constitutes the largest analysis of cytokines and cytokine receptors to date. Analysis of the patients receiving placebo showed that increasing circulating levels of TNF, interleukin-6, and the soluble TNF receptors were associated with increased mortality. In men, there was a linear increase in circulating levels of TNF with advancing age. Women < or = 50 years of age had relatively low levels of TNF, but TNF levels were disproportionately higher in women >50 years of age. No differences existed in cytokines and/or cytokine receptors in whites versus nonwhites, and circulating levels of cytokines and cytokine receptors were significantly greater in patients with ischemic heart disease. CONCLUSIONS: Cytokines and cytokine receptors are independent predictors of mortality in patients with advanced heart failure. Moreover, circulating levels of cytokines are modified by age, sex, and cause of heart failure.

Effects of soluble TNF receptor treatment on lipopolysaccharide-induced myocardial cytokine expression.

Tumor necrosis factor (TNF)-alpha plays a key role in the pathogenesis of septic shock syndrome, and myocardial TNF-alpha expression may contribute to this pathophysiology. We examined the myocardial expression of TNF-alpha-related cytokines and chemokines in mice exposed to lipopolysaccharide (LPS) and tested the effects of anti-TNF therapy on myocardial cytokine expression. Cytokine mRNA levels were measured by RNase protection assay, and protein levels in the plasma and myocardium were assessed by enzyme-linked immunosorbent assays. LPS (4 microg/g body wt ip) induced marked cytokine expression, including TNF-alpha, interleukin (IL)-1beta, IL-6, and monocyte chemotactic protein (MCP)-1, in both the plasma and myocardium. Pretreatment with adenovirus-mediated TNF receptor fusion protein (AdTNFR1; 10(9) plaque-forming units iv) decreased plasma cytokine levels. In contrast, whereas myocardial IL-1beta expression was also suppressed, expression of IL-6 and MCP-1 was not inhibited by AdTNFR1. In summary, anti-TNF treatment differentially altered the cytokine expression in the plasma and myocardium during endotoxemia. Inability to block myocardial expression of IL-6 and MCP-1 suggests a possible mechanism for the failure of anti-TNF therapies in the treatment of endotoxin shock.