Immunomodulation Therapy Using Tolerogenic Macrophages in a Rodent Model of Pulmonary Hypertension.

Inflammation plays a major role in the pathogenesis of pulmonary hypertension (PH). We sought to investigate the effects of a cell-based immunomodulation in a dysimmune model of PH. PH was induced in athymic nude rats using semaxinib (Su group, n = 6). Tolerogenic macrophages (toM) were generated from monocyte isolation and then injected either the day before semaxinib injection (Prevention group, n = 6) or 3 weeks after (Reversion group, n = 6). Six athymic nude rats were used as controls. In vivo trafficking of toM was investigated with bioluminescence imaging showing that toM were mainly located into the lungs until 48 h after injection. Right ventricular (RV) end-systolic pressure and RV systolic function were assessed at 4 weeks using echocardiography. Morphometric analysis and RNA sequencing of the lungs were realized at 4 weeks. Rats treated with toM (Prevention and Reversion groups) had a significantly lower RV end-systolic pressure at 4 weeks (respectively, 25 ± 8 and 30 ± 6 mmHg vs. 67 ± 9 mmHg, P < 0.001), while RV systolic dysfunction was observed in Su and Reversion groups. Mean medial wall thickness of small arterioles was lower in Prevention and Reversion groups compared with the Su group (respectively, 10.9% ± 0.8% and 16.4% ± 1.3% vs. 28.2% ± 2.1%, P < 0.001). Similarly, cardiomyocyte area was decreased in rats treated with toM (150 ± 18 and 160 ± 86 µm2 vs. 279 ± 50 µm2, P < 0.001). A trend toward upregulation of genes involved in pulmonary arterial hypertension pathobiology was found in Su rats, while KCNK3 was significantly downregulated (fold-change = 9.8, P < 0.001). Injection of toM was associated with a less severe phenotype of PH in rats exposed to angioproliferative stress. Preserved expression of KCNK3 may explain the protective effect of toM.

Myocardial gene expression profiles and cardiodepressant autoantibodies predict response of patients with dilated cardiomyopathy to immunoadsorption therapy.

AIMS: Immunoadsorption with subsequent immunoglobulin G substitution (IA/IgG) represents a novel therapeutic approach in the treatment of dilated cardiomyopathy (DCM) which leads to the improvement of left ventricular ejection fraction (LVEF). However, response to this therapeutic intervention shows wide inter-individual variability. In this pilot study, we tested the value of clinical, biochemical, and molecular parameters for the prediction of the response of patients with DCM to IA/IgG. METHODS AND RESULTS: Forty DCM patients underwent endomyocardial biopsies (EMBs) before IA/IgG. In eight patients with normal LVEF (controls), EMBs were obtained for clinical reasons. Clinical parameters, negative inotropic activity (NIA) of antibodies on isolated rat cardiomyocytes, and gene expression profiles of EMBs were analysed. Dilated cardiomyopathy patients displaying improvement of LVEF (≥20 relative and ≥5% absolute) 6 months after IA/IgG were considered responders. Compared with non-responders (n = 16), responders (n = 24) displayed shorter disease duration (P = 0.006), smaller LV internal diameter in diastole (P = 0.019), and stronger NIA of antibodies. Antibodies obtained from controls were devoid of NIA. Myocardial gene expression patterns were different in responders and non-responders for genes of oxidative phosphorylation, mitochondrial dysfunction, hypertrophy, and ubiquitin-proteasome pathway. The integration of scores of NIA and expression levels of four genes allowed robust discrimination of responders from non-responders at baseline (BL) [sensitivity of 100% (95% CI 85.8-100%); specificity up to 100% (95% CI 79.4-100%); cut-off value: -0.28] and was superior to scores derived from antibodies, gene expression, or clinical parameters only. CONCLUSION: Combined assessment of NIA of antibodies and gene expression patterns of DCM patients at BL predicts response to IA/IgG therapy and may enable appropriate selection of patients who benefit from this therapeutic intervention.

Infliximab treatment increases left ventricular ejection fraction in patients with rheumatoid arthritis: assessment of heart function by echocardiography, endothelin 1, interleukin 6, and NT-pro brain natriuretic peptide.

OBJECTIVE: To study the influence of anti-tumor necrosis factor-α (TNF-α) treatment on echocardiographic measures and concentrations of endothelin 1 (ET-1), interleukin 6 (IL-6), and amino-terminal fragment of pro-brain natriuretic peptide (NT-proBNP) in a cohort of 23 female patients with rheumatoid arthritis (RA). METHODS: We recruited 23 patients (mean age 51.3 ± 1.55 yrs) with RA resistant to treatment with disease-modifying antirheumatic drugs and average disease duration of 7.1 ± 1.0 years who had been selected to start treatment with the anti-TNF-α antagonist infliximab. Transthoracic echocardiographic examinations were performed before the first infusion and repeated after 1 year of treatment. Data for age, sex, RA disease activity by Disease Activity Score (DAS28) and echocardiographic data, NT-proBNP, IL-6, ET-1, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and other routine laboratory data were collected before treatment and after 1 year. RESULTS: Twelve months of treatment with infliximab resulted in reduction of RA activity (i.e., reduction of DAS and acute-phase reactants). There was increased left ventricle ejection fraction, from 58.5% before treatment to 63% after. Treatment with infliximab also resulted in significant reduction of ET-1 (1.26 fmol/ml before treatment vs 0.43 fmol/ml after), IL-6 (58.46 pg/ml vs 3.46 pg/ml), and NT-proBNP (43.06 fmol/ml vs 14.78 fmol/ml). These reductions were observed after just 4 months of treatment and remained significant until the termination of the study. CONCLUSION: In patients with RA, treatment with infliximab contributed significantly to increase in left ventricular ejection fraction. Improvement of cardiac function was shown by conventional echocardiography; there was reduction of biochemical markers of heart failure.

Apolipoprotein A-I mimetic peptide treatment inhibits inflammatory responses and improves survival in septic rats.

Systemic inflammation induces a multiple organ dysfunction syndrome that contributes to morbidity and mortality in septic patients. Since increasing plasma apolipoprotein A-I (apoA-I) and HDL may reduce the complications of sepsis, we tested the hypothesis that the apoA-I mimetic peptide 4F confers similar protective effects in rats undergoing cecal ligation and puncture (CLP) injury. Male Sprague-Dawley rats were randomized to undergo CLP or sham surgery. IL-6 levels were significantly elevated in plasma by 6 h after CLP surgery compared with shams. In subsequent studies, CLP rats were further subdivided to receive vehicle or 4F (10 mg/kg) by intraperitoneal injection, 6 h after sepsis induction. Sham-operated rats received saline. Echocardiographic studies showed a reduction in left ventricular end-diastolic volume, stroke volume, and cardiac output (CO) 24 h after CLP surgery. These changes were associated with reduced blood volume and left ventricular filling pressure. 4F treatment improved blood volume status, increased CO, and reduced plasma IL-6 in CLP rats. Total cholesterol (TC) and HDL were 79 +/- 5 and 61 +/- 4 mg/dl, respectively, in sham rats. TC was significantly reduced in CLP rats (54 +/- 3 mg/dl) due to a reduction in HDL (26 +/- 3 mg/dl). 4F administration to CLP rats attenuated the reduction in TC (69 +/- 4 mg/dl) and HDL (41 +/- 3 mg/dl) and prevented sepsis-induced changes in HDL protein composition. Increased plasma HDL in 4F-treated CLP rats was associated with an improvement in CO and reduced mortality. It is proposed that protective effects of 4F are related to its ability to prevent the sepsis-induced reduction in plasma HDL.

S100A8 and S100A9 mediate endotoxin-induced cardiomyocyte dysfunction via the receptor for advanced glycation end products.

Cardiovascular dysfunction as a result of sepsis is the leading cause of death in the critically ill. Cardiomyocytes respond to infectious pathogens with a Toll-like receptor-initiated proinflammatory response in conjunction with a decrease in contractility, although the downstream events linking Toll-like receptor activation and reduced cardiac contractility remain to be elucidated. Using microarray analysis of cardiac tissue exposed to systemic lipopolysaccharide (LPS), we discovered that 2 small calcium-regulating proteins (S100A8 and S100A9) are highly upregulated. HL-1 cardiomyocytes, isolated primary cardiomyocytes, and live mice were exposed to LPS, whereas beating HL-1 cells had S100A8 and S100A9 overexpressed and their calcium flux quantified. Using in vivo microbubble technology, we delivered S100A8 and S100A9 to normal mouse hearts; using the same technology, we inhibited S100A9 production in mouse hearts and subsequently exposed them to LPS. Coimmunoprecipitation of S100A8 and S100A9 identified interaction with RAGE (the receptor for advanced glycation end products), the cardiac function and postreceptor signaling of which were investigated. HL-1 cardiomyocytes, isolated primary cardiomyocytes, and whole hearts exposed to LPS have large increases in S100A8 and S100A9. Cardiac overexpression of S100A8 and S100A9 led to a RAGE-dependent decrease in calcium flux and, in the intact mouse, to a decreased cardiac ejection fraction, whereas knockdown of S100A9 attenuated LPS-induced cardiac dysfunction. Cardiomyocytes exposed to LPS express S100A8 and S100A9, leading to a RAGE-mediated decrease in cardiomyocyte contractility. This finding provides a novel mechanistic link between circulating pathogen-associated molecular products and subsequent cardiac dysfunction.

Antimyosin autoantibodies are associated with deterioration of systolic and diastolic left ventricular function in patients with chronic myocarditis.

OBJECTIVES: The study evaluates the clinical course and the development of systolic and diastolic left ventricular function in patients with chronic myocarditis with or without autoantibodies against cardiac myosin. BACKGROUND: Patients with myocarditis often show autoantibodies against cardiac myosin. The clinical and pathophysiologic significance of these antimyosin autoantibodies (AMAAB) is yet unknown. The results from studies comparing the clinical course and the development of left ventricular function in patients with chronic myocarditis with or without AMAAB are not yet available. METHODS: Thirty-three patients with biopsy proven chronic myocarditis underwent analysis of AMAAB, right and left heart catheterization and left ventriculography at baseline and after six months. Left ventricular volumes and ejection fraction as well as the time constant of left ventricular relaxation "tau" and the constant of myocardial stiffness "b" were determined at baseline and at follow-up. RESULTS: In 17 (52%) patients, AMAAB could be detected at baseline. After six months, AMAAB were still found in 13 (76%) initially antibody-positive patients. No initially antibody-negative (n = 16) patient developed AMAAB during follow-up. Clinical symptoms improved slightly in antibody-negative patients and remained stable in antibody-positive patients. Left ventricular ejection fraction developed significantly better in antibody-negative patients (+8.9 +/- 10.1%) compared with antibody-positive patients (-0.1 +/- 9.4%) (p < 0.012). Stroke volume (SV) and stroke volume index (SVI) also improved in antibody-negative patients (SV: +20 +/- 31 ml; SVI: +10 +/- 17 ml) compared with antibody-positive patients (SV: -14 +/- 43 ml; SVI: -8 +/- 22 ml) (SV: p < 0.015; SVI: p < 0.016). Left ventricular end-diastolic and end-systolic volumes and the time constant of left ventricular relaxation "tau" did not change significantly different in antibody-positive and antibody-negative patients. The constant of myocardial stiffness "b" improved significantly in antibody-negative patients (-6.1 +/- 10.8) compared with antibody-positive patients (+7.3 +/- 22.6) (p < 0.040). Analyzing only the persistently antibody-positive patients yielded essentially the same results. CONCLUSIONS: Antimyosin autoantibodies are associated with worse development of left ventricular systolic function and diastolic stiffness in patients with chronic myocarditis.

Steroid pretreatment improves graft recovery in a sheep orthotopic heart transplantation model.

BACKGROUND: Previous reports provide conflicting evidence concerning effects of steroids on recovery of cardiac function during procedures involving cardiopulmonary bypass. This study was designed to test the hypothesis that pretreatment of animals with steroids before heart transplantation improves graft hemodynamic function. METHODS: Four groups of sheep were studied: CON, nonsteroid-treated nontransplanted controls (n = 8); CON-S, steroid-treated nontransplanted controls (n = 5); TX, nonsteroid-treated transplanted animals (n = 5); and TX-S, steroid-treated transplanted animals (n = 5). Steroid-treated animals were given methylprednisolone 30 mg/kg immediately before surgery. Procedures for harvest and orthotopic transplantation were similar to those used clinically. Contractile function, left ventricular diameter, and cardiac output of control and transplanted hearts were measured for 6 hours. A 2 x 2 factorial repeated measures analysis of variance was used to determine statistical significance (p < 0.05). RESULTS: Steroid pretreatment produced significantly higher function in controls and transplanted animals compared with nonsteroid-treated animals. On average over 6 hours, significant steroid effects were observed for left ventricular peak systolic pressure, mm Hg (CON, 85 +/- 2; CON-S, 98 +/- 3; TX, 74 +/- 3; TX-S, 91 +/- 2); global stroke work, mJoule x cm(-2) (CON, 4.69 +/- 0.21; CON-S, 5.88 +/- 0.32; TX, 2.27 +/- 0.17; TX-S, 4.23 +/- 0.16); and peak rate of pressure relaxation (-dP/dt(max)), mm Hg/msec (CON, 1.23 +/- 0.05; CON-S, 1.44 +/- 0.08; TX, 0.60 +/- 0.03; TX-S, 2.04 +/- 0.13). Steroid pretreatment produced more stable recovery for transplanted animals. All five TX-S animals could be removed from inotropic support and had stable function for 6 hours. In contrast, 1 of 5 TX animals could not be removed from inotropic support, and 1 of 5 TX hearts failed 3 hours after transplant. Arterial blood PO2 values were significantly higher in steroid-treated animals than in nonsteroid treated animals. Blood systemic lactate, which was elevated after transplantation, returned to control level by 6 hours in the steroid-treated group but not in the nonsteroid-treated group. CONCLUSION: Steroid pretreatment of heart donors and recipients improved systolic and diastolic function and hemodynamic stability after transplantation. In addition, steroid pretreatment improved pulmonary gas exchange of control and transplanted animals.

Anti-tumor necrosis factor-alpha prevents decreased ventricular contractility in endotoxemic pigs.

It is not known how the decrease in left ventricular contractility following endotoxin exposure is mediated, or whether this decrease is preventable by antibodies to tumor necrosis factor-alpha (TNF alpha). Four groups of six anesthetized and instrumented pigs were pretreated with ovine polyclonal antibody to human TNF alpha (anti-TNF alpha), nonspecific IgG, or saline, and then treated with either endotoxin or saline. We measured hemodynamics and left ventricular pressures (Millar catheter) and volumes (conductance catheter). Left ventricular contractility was assessed using the slope (Emax) of the end-systolic pressure-volume relationship. Four hours after the start of endotoxin infusion in the nonspecific IgG pretreated group, Emax had decreased by 44 +/- 6% (p < 0.05), mean arterial pressure had decreased from 115 +/- 7 mm Hg to 70 +/- 10 mm Hg (p < 0.05), and cardiac output was rapidly decreasing after an initial increase (p < 0.05). Anti-TNF alpha significantly reduced the decrease in Emax (11 +/- 9%, p < 0.05), and the systemic hypotension (108 +/- 15 mm Hg to 99 +/- 6 mm Hg, p < 0.05), at 4 h, and prevented the late decrease in cardiac output. This suggests that TNF alpha is an important early mediator in sepsis leading to decreased left ventricular contractility.