Tomonari Akatsu

Elevated circulating levels of heat shock protein 70 are related to systemic inflammatory reaction through monocyte Toll signal in patients with heart failure after acute myocardial infarction

Recent studies have shown that heat shock protein (HSP) 70 may serve as a “damage signal” to the immune system and could be the endogenous ligand for Toll‐like receptor (TLR) 4 mediating synthesis of inflammatory cytokines.

Myocardial osteopontin expression is associated with collagen fibrillogenesis in human dilated cardiomyopathy.

Osteopontin (OPN), an extracellular matrix (ECM) protein, plays an important role in myocardial remodeling by promoting collagen synthesis and accumulation in experimental animal models.

Expression of Toll-like receptor 4 is associated with enteroviral replication in human myocarditis

Previous studies have demonstrated that inflammatory cytokine expression associated with enteroviral (EV) infection may play an important role in human myocarditis. However, the mechanism of the host immune response against viral pathogens has not been fully understood. The aim of the present study was to determine whether Toll-like receptor 4 (TLR4) and EV RNA are present in human myocarditis. Endomyocardial biopsy samples were obtained from 44 patients with myocarditis and five controls. Levels of plus- and minus-strand EV RNAs and TLR4 mRNA were measured by real-time reverse transcriptase-PCR. Immunohistochemical analysis was performed to identify the cellular source of TLR4 and the EV capsid protein VP1. EV RNA was present in 21 patients with myocarditis and these patients were defined as having either active viral replication ( n =15) or latent viral persistence ( n =6). Neither strand of EV RNA was detected in controls. TLR4 mRNA expression levels were higher in myocarditis patients than in controls (TLR4/glyceraldehyde-3-phosphate dehydrogenase ratio 1.48+/-0.17 compared with 0.08+/-0.06, P <0.001). A positive correlation was found between EV RNA and TLR4 levels (plus-strand vs TLR4: r =0.66, P <0.001; minus-strand vs TLR4: r =0.48, P <0.001). TLR4 immunostaining was observed in infiltrating cells and myocytes in patients with myocarditis. The EV capsid protein VP1 was also found in myocytes. The myocarditis group with EV replication and high levels of TLR4 showed significantly lower systolic function. The present study has shown that increased expression of TLR4 is associated with EV replication and that these RNA levels are related to cardiac dysfunction in human myocarditis.

Toll-like receptor 4 is expressed with enteroviral replication in myocardium from patients with dilated cardiomyopathy

Expressions of innate immune response proteins, most notably proinflammatory cytokines, against enteroviral (EV) infection have been documented in the heart of human dilated cardiomyopathy (DCM). Toll-like receptor 4 (TLR4) activates signaling pathways leading to the expression of proinflammatory cytokines implicated the etiology of DCM. We sought to determine whether EV replication activates TLR4-dependent immune response in myocardium obtained from patients with DCM. Endomyocardial biopsy tissues were obtained from 56 patients with DCM and 10 controls. Levels of plus- and minus-strand EV RNA and TLR4 mRNA were measured by real-time RT-PCR. Immunohistochemical analysis was performed to identify the cellular source of EV capsid protein VP1 and TLR4. Both plus- and minus-strand EV RNA were detected in 19 DCM patients (34%). Neither strand of EV RNA was detected in controls. TLR4 mRNA levels were higher in DCM patients than in controls (P<0.001). A positive correlation was found between TLR4 levels and each strand type of EV RNA in EV RNA-positive patients (plus-strand vs TLR4: r=0.69, P<0.001; minus-strand vs TLR4: r=0.65, P=0.002). VP1/TLR4 double staining showed extensive colocalization of VP1 and TLR4 proteins in cytoplasm of cardiac myocytes in myocardium obtained from DCM patients. EV RNA-positive patients showed lower systolic function and larger ventricular volume compared with EV RNA-negative patients left ventricular ejection fraction (LVEF): P=0.002; left ventricular end-systolic diameter (LVESD): P=0.004). The DCM subgroup with high TLR4 levels showed lower LVEF and larger LVESD than the subgroup with TLR4 levels (both P<0.001). This study suggests that myocardial expression of TLR4 associates with EV replication in human DCM. EV RNA and TLR4 mRNA levels may correlate with LV dysfunction in DCM. The expression of TLR4 against EV replication may be involved in the pathogenesis of DCM.

C‐reactive protein co‐expresses with tumor necrosis factor‐α in the myocardium in human dilated cardiomyopathy

C‐reactive protein (CRP) has recently been reported to be present in cardiac tissue and to stimulate the production of proinflammatory cytokines. Cardiac expression of tumor necrosis factor‐α (TNF‐α) plays an important role in the pathogenesis of dilated cardiomyopathy (DCM).

Increased mRNA expression of tumour necrosis factor-α and its converting enzyme in circulating leucocytes of patients with acute myocardial infarction

Tumour necrosis factor-alpha (TNF-alpha) plays an important role in myocardial damage in acute myocardial infarction (AMI). It has recently been discovered that TNF-alpha-converting enzyme (TACE) cleaves precursor TNF-alpha into its mature form. However, it remains unknown whether TNF-alpha expression is related to TACE expression in circulating leucocytes in AMI. Blood samples were obtained from 37 patients with AMI within 24 h of onset and eight healthy controls. Plasma TNF-alpha levels were measured by ELISA. Total mRNA was then extracted from circulating leucocytes, and the expression levels of TACE and TNF-alpha mRNAs were determined by reverse transcriptase-PCR. Plasma TNF-alpha levels were significantly higher in patients with Killips classes III and IV AMIs (17.1+/-5.0 pg/ml, n =11) than in those with Killips classes I and II AMIs (13.7+/-4.2 pg/ml, n =26), or controls (13.0+/-1.7 pg/ml, n =8) ( P <0.05). There was a significant increase in expression (arbitrary units) of TACE and TNF-alpha mRNAs in circulating leucocytes obtained from patients with Killips classes I and II AMIs [TACE/glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 2.770+/-0.303; TNF-alpha/GAPDH, 2.123+/-0.475] compared with controls (TACE/GAPDH, 1.498+/-0.209; TNF-alpha/GAPDH, 1.283+/-0.274) ( P <0.01). This increase was even greater in patients with Killips classes III and IV AMIs (TACE/GAPDH, 3.086+/-0.354; TNF-alpha/GAPDH, 2.808+/-0.422) ( P <0.01). Moreover, there was a significant positive relationship between these mRNA expression levels ( r =0.60, P <0.01). The TACE-TNF-alpha system in circulating leucocytes is stimulated and may have a negative impact on clinical outcome in AMI.

Increased expression of tumor necrosis factor‐α converting enzyme and tumor necrosis factor‐α in peripheral blood mononuclear cells in patients with advanced congestive heart failure

Tumor necrosis factor‐α converting enzyme (TACE) has recently been identified as a metalloproteinase‐disintegrin, which converts pro‐tumor necrosis factor‐α (TNF‐α) to the mature form, and is an important mediator in the pathogenesis of CHF.

Activated tumour necrosis factor-α shedding process is associated with in-hospital complication in patients with acute myocardial infarction

TACE [TNF-alpha (tumour necrosis factor-alpha)-converting enzyme] plays an essential role in the shedding of TNF-alpha, which could affect the outcome of AMI (acute myocardial infarction). To investigate the clinical significance of the TACE-TNF-alpha system in AMI, we examined TACE-mediated TNF-alpha synthesis in PBMCs (peripheral blood mononuclear cells), which are a possible source of TNF-alpha in AMI. Forty-one patients with AMI and 15 healthy subjects (HS) were enrolled in the present study. PBMCs were isolated from peripheral blood on day 1 and 14 after the onset of AMI. TACE and TNF-alpha mRNA levels and intracellular median fluorescence intensity were measured by real-time RT (reverse transcriptase)-PCR and flow cytometry respectively. TACE-mediated TNF-alpha production was evaluated in cultured PBMCs with PMA, which is known to activate TACE. Spontaneous TACE and TNF-alpha levels were higher in AMI patients than in HS (P<0.001). TACE and TNF-alpha levels in PMA-stimulated PMBCs were markedly increased in AMI patients compared with HS (P<0.001). There was a positive correlation between TACE and TNF-alpha levels in AMI. Although spontaneous and stimulated levels of TACE and TNF-alpha decreased 14 days after the onset of AMI, levels in AMI patients were higher than in HS. In AMI patients with in-hospital complications (n=15; pump failure in ten, recurrent myocardial infarction in one, malignant ventricular arrhythmia in three and cardiac death in one), spontaneous and stimulated levels of TACE and TNF-alpha were higher than in patients without complications (P<0.01). These levels were higher in AMI patients with in-hospital complications 14 days after onset. These results demonstrate that TACE-mediated TNF-alpha maturation in PBMCs may play an important role in poor outcomes from AMI, suggesting that TACE may be a potential target for the inhibition of cellular TNF-alpha production in AMI.

Effects of nitric oxide inhibition on basal forearm blood flow in patients with nonischemic chronic heart failure

SummaryIncreased peripheral vascular tone in patients with chronic heart failure (CHF) is an important factor which contributes to increased cardiac afterload and reduced exercise capacity, and consequent further deterioration in CHF. The role of endogenous nitric oxide (NO) in the regulation of basal vascular tone in CHF is controversial. This study has investigated (1) whether basal NO bioavailability is reduced in the peripheral vasculature of patients with nonischemic CHF in the absence of confounding factors influencing endothelial function, and (2) if a difference is found, what clinical characteristics are related to the decreased NO-dependent vasodilation. Basal forearm blood flow (FBF) of 12 patients with CHF and 14 healthy volunteers was measured before and after administration ofNG-monomethyl-l-arginine (l-NMMA) by venous occlusion plethysmography. Afterl-NMMA administration, basal FBF in both healthy subjects and patients with CHF decreased significantly, with the decrease in CHF patients being less than that in healthy volunteers (−0.7 ± 0.2 vs −1.5 ± 0.2ml/min per 100ml tissue,P < 0.01). When both groups were considered together, basal FBF was closely related to the decrease in FBF afterl-NMMA administration (r = −0.83.P < 0.001). When CHF patients were divided into two groups according to NYHA class, thel-NMMA-induced decrease in FBF in moderate CHF (NYHA III;n = 7) was significantly less than that in mild CHF (NYHA II;n = 5) (−0.36 ± 0.13 vs −1.16 ± 0.72ml/min per 100ml tissue,P < 0.05). In conclusion, basal bioavailability of NO in the peripheral vascular bed in nonischemic CHF is impaired, with an increase in basal vascular tone and with progression of this disorder. This suggests that impaired basal NO bioactivity may make an important contribution to the elevated peripheral vascular tone and expression of symptoms seen in CHF.

Eplerenone inhibits tumour necrosis factor α shedding process by tumour necrosis factor α converting enzyme in monocytes from patients with congestive heart failure

Eplerenone, a selective mineralocorticoid receptor antagonist, attenuated the expression of systemic proinflammatory molecules in a rat model of congestive heart failure (CHF).1 However, the mechanisms by which eplerenone reduces proinflammatory molecule expression have not been elucidated. Our previous study was the first to show that expression of tumour necrosis factor α (TNFα) converting enzyme (TACE) in peripheral blood mononuclear cells may be responsible for the release of TNFα in CHF.2 To the best of our knowledge, the observation that peripheral monocytes can activate the TACE–TNFα system in CHF is without precedent. The objective of the present study was to examine whether monocytes have a role in the activated TACE–TNFα system and to determine whether eplerenone reduces the TNFα shedding process in monocytes from patients with CHF. Peripheral monocytes were isolated from blood samples at the time of admission (32 patients with CHF and 10 healthy subjects). The origin of left ventricular (LV) dysfunction was idiopathic dilated cardiomyopathy in all cases. Echocardiography …