Increased serum levels and expression of S100A8/A9 complex in infiltrated neutrophils in atherosclerotic plaque of unstable angina.

BACKGROUND: The S100A8/A9 complex is expressed in a subset of activated neutrophils and macrophages in acute inflammatory lesions associated with various diseases. OBJECTIVE: To investigate (a) whether serum S100A8/A9 levels are increased in patients with unstable angina (UA); and (b) whether S100A8/A9 expression is upregulated in coronary atherosclerotic plaques of patients with UA. DESIGN: Serum S100A8/A9 levels in 39 patients with stable angina (SA) and 53 patients with UA were measured. In addition, the presence of the S100A8/A9 complex in directional coronary atherectomy specimens was studied immunohistochemically. Cell types which stain positive for S100A8/A9 were identified by immunodouble staining with neutrophils and macrophages. RESULTS: Mean (SD) serum S100A8/A9 levels were significantly higher in patients with UA than in those with SA (3.25 (3.08) microg/ml vs 0.77 (0.31) microg/ml, p<0.05). In patients with UA, immunodouble staining clearly showed that the S100A8/A9 complex was expressed in infiltrated neutrophils and occasional macrophages. The S100A8/A9-positive area was significantly higher in UA than in SA (mean (SD) 18.3 (14.2)% vs 1.3 (2.4)%, respectively, p<0.001). CONCLUSIONS: The S100A8/A9 complex may be involved in the inflammatory process of coronary atherosclerotic plaques in patients with UA.

Plasma leptin levels and cardiac sympathetic function in patients with obstructive sleep apnoea-hypopnoea syndrome.

BACKGROUND: The control of body weight and cardiac sympathetic function in patients with obstructive sleep apnoea-hypopnoea syndrome (OSAHS) are important because both factors have significant effects on the mortality of these patients. It has recently been reported that OSAHS has a significant effect on the secretion of leptin, a hormone involved in the control of body weight and sympathetic nerve activity. In addition to the circadian rhythm of leptin secretion, the effects of one night of treatment with nasal continuous positive airway pressure (nCPAP) and the mechanism of the effects of nCPAP on nocturnal leptin secretion in patients with OSAHS has not yet been elucidated. METHODS: Blood samples were obtained at 21.00 hours, 00.00 hours, 03.00 hours, and 06.30 hours from 21 subjects with OSAHS (mean apnoea and hypopnoea index 52.4/h), with and without nCPAP treatment. Iodine-123 (I(123))-meta-iodobenzylguanidine (MIBG) imaging was used to evaluate myocardial sympathetic function before nCPAP treatment. RESULTS: Plasma leptin reached a peak level at 00:00 hours (p<0.01) in patients with OSAHS, both with and without nCPAP treatment. The first night of nCPAP treatment significantly decreased the plasma leptin levels at 03.00 hours (without nCPAP: mean (SE) 21.6 (4.7) ng/ml; with nCPAP: 19.3 (4.1) ng/ml, p<0.02) and at 06.30 hours (without nCPAP: 17.6 (3.8) ng/ml; with nCPAP: 15.2 (3.2) ng/ml, p<0.01). The magnitude of the decrease in leptin levels after nCPAP treatment was significantly correlated with cardiac sympathetic function measured before nCPAP treatment (p<0.03). CONCLUSIONS: Patients with OSAHS undergo nocturnal increases in leptin levels in spite of interruption of sleep due to apnoea and hypopnoea, a trend seen in normal subjects. Plasma leptin levels in patients with OSAHS decreased significantly after the first night of nCPAP treatment. Enhanced cardiac sympathetic function in these patients may contribute to the leptin levels before nCPAP treatment and vice versa.

Epidemiology of idiopathic cardiomyopathy in Japan: results from a nationwide survey.

OBJECTIVE: To estimate the total number of patients with idiopathic cardiomyopathy in Japan and the prevalence of the disorder. DESIGN: A nationwide epidemiological survey. SETTING: Hospitals selected randomly from among all hospitals in Japan. PATIENTS: Patients presenting with any of the three types of idiopathic cardiomyopathy: dilated cardiomyopathy, hypertrophic cardiomyopathy, and restrictive cardiomyopathy. MAIN OUTCOME MEASURES: The total number of patients in Japan was estimated using the sampling and response rates in each stratum with respect to hospital size. The second survey was conducted for patients reported in the first survey in order to obtain detailed information, including age, sex, and specific clinical data. RESULTS: Estimated patient totals and 95% confidence intervals (CI) were 17 700 (95% CI 16 500 to 18 800) for dilated cardiomyopathy, 21 900 (95% CI 20 600 to 23 200) for hypertrophic cardiomyopathy, and 300 (95% CI 250 to 350) for restrictive cardiomyopathy. Crude prevalence per 100 000 population was estimated as 14.0 for dilated cardiomyopathy, 17.3 for hypertrophic cardiomyopathy, and 0.2 for restrictive cardiomyopathy; crude incidence per 100 000 person-years was estimated as 3.58, 4.14, and 0.06, respectively. CONCLUSIONS: The total number and prevalence of patients with idiopathic cardiomyopathy in Japan are estimated for the first time in a nationwide survey. The prevalence of dilated cardiomyopathy in Japan appears to be about half that of Western populations, while that of hypertrophic cardiomyopathy is about the same.

Activation of lectin-like oxidized low-density lipoprotein receptor-1 induces apoptosis in cultured neonatal rat cardiac myocytes.

BACKGROUND: Lectin-like oxidized LDL receptor-1 (LOX-1) was originally identified as a receptor expressed predominantly in endothelial cells. LOX-1 can also be expressed in other cell types, and the activation of the LOX-1 pathway has been implicated in apoptosis. There have been no reports, however, about LOX-1 expression in cardiac myocytes or regulation of myocardial cell apoptosis by LOX-1. METHODS AND RESULTS: In primary cardiac myocytes from neonatal rats, immunohistochemical analyses using a specific monoclonal antibody against LOX-1 demonstrated that LOX-1 expression was markedly induced by stimulation with norepinephrine and endothelin-1. LOX-1 expression was upregulated in cardiac myocytes as well as in vessel walls of failing rat hearts in vivo. In the presence of a low concentration of oxidized LDL that did not induce apoptosis by itself, artificial overexpression of LOX-1 in cardiac myocytes in culture resulted in apoptosis. LOX-1 overexpression induced activation of p38 mitogen-activated protein kinase (MAPK) and oxidative stress in cardiac myocytes, as demonstrated by an increase in positive immunostaining for 8-hydroxy-2'-deoxyguanosine. Inhibition of p38 MAPK by cotransfection of a dominant-negative form of MKK6 as well as by administration of a specific inhibitor, SB203580 or FR167653, almost completely blocked the induction of apoptosis by LOX-1 activation. Antioxidant catalase also blocked LOX-1-induced apoptosis as well as activation of p38 MAPK. CONCLUSIONS: These findings demonstrate that LOX-1 expression in cardiac myocytes is induced by neurohormonal factors activated in heart failure and that LOX-1-dependent apoptosis in these cells requires p38 MAPK, a component of oxidant stress-sensitive signaling pathways.

Altered cultured mesangial cell phenotypes from RF/J mice: a spontaneous immune complex mediated glomerulonephritis with progressive glomerulosclerosis.

BACKGROUND/AIM: RF/J mice are a model of spontaneous immune complex mediated glomerulonephritis showing massive extracellular matrix accumulation and progressive glomerulosclerosis. The aim of this study was to investigate whether there is an altered cultured mesangial cell (MC) phenotype in RF/J mice associated with these glomerular changes. METHODS: The nature of cultured MCs from RF/J mice in the proliferative response to platelet-derived growth factor (PDGF) BB was compared with that of normal mice (BALB/c) by 3H-thymidine incorporation. The binding of PDGF-BB was examined with Scatchard analysis, and the messenger RNAs (mRNAs) of PDGF beta-receptor, collagen I, collagen IV, and fibronectin were detected using Northern blot analysis in the MCs of each mouse. RESULTS: The 3H-thymidine incorporation of MCs from RF/J mice showed significantly lower responses to PDGF-BB stimulations with concentrations ranging from 0.5 to 10.0 ng/ml in comparison with those of BALB/c mice which exhibited a proportional dose- dependent increase of the incorporation (p < 0.05 for 0.5 ng/ml PDGF-BB, p < 0.01 for 1.0-10.0 ng/ml). According to the Scatchard analysis, MCs from BALB/c mice showed aKD of 105 pM of PDGF-BB binding to its receptors, and the density of receptors was 5.82 fmol/10(5) cells. However, no binding PDGF-BB site on the surface of MCs from RF/J mice was noted. Northern blot analysis of MCs from RF/J mice indicated negative expression of detectable PDGF-beta receptor mRNA. As for matrix protein messages, MCs from RF/J mice did not express mRNA of type I collagen, but did express a higher amount of type IV collagen and fibronectin in comparison with MCs from normal BALB/c mice. CONCLUSIONS: An altered phenotype in MCs of RF/J mice was demonstrated, possibly contributing to the characteristic pathological glomerular changes. However, the precise association remains to be clarified.

Myocardial metabolism of 123I-BMIPP during low-flow ischaemia in an experimental model: comparison with myocardial blood flow and 18F-FDG.

Risk stratification of coronary artery disease may provide a basis for selection of treatment to prevent myocardial events and to assist functional recovery. Iodine-123 (rho-iodophenyl)-3-R,S-methylpentadecanoic acid (123I-BMIPP) is a radioiodinated fatty acid analogue for single-photon emission tomographic (SPET) imaging, and several reports have demonstrated that the abnormal uptake of 123I-BMIPP is associated with wall motion abnormality and severe coronary artery stenosis. Clarification of the contribution of fatty acids to myocardial metabolism would be highly valuable in recognising this critical condition. In this study, we investigated the myocardial uptake of 123I-BMIPP under low-flow ischaemia, and compared it with the uptake of fluorine-18 fluorodeoxyglucose (18F-FDG). Using open chest dogs, the flow of the left anterior descending coronary artery was controlled using a pneumatic occluder in order to maintain a 30%-40% reduction of Doppler flow. 123I-BMIPP and 18F-FDG were injected into the left atrium after 90 min of ischaemia (protocols 1 and 3). Canine hearts were excised after 120 min of ischaemia for the measurement of radioactivity. In protocol 2, 123I-BMIPP alone was injected and hearts were excised 8 min after the injection. A time-course biopsy study was also performed at the same time (protocol 3). Wall thickening was evaluated using a wall tracker module. The uptake of 18F-FDG increased significantly in the ischaemic region (232%+/-135% vs non-ischaemic, P<0.05 in protocol 1) even on mild reduction of myocardial blood flow (MBF). The increased uptake of 18F-FDG did not correlate well with the severity of MBF. On the other hand, 123I-BMIPP uptake decreased gradually (78.9%+/-23.6%, P<0.05 in protocol 1, and 85.9%+/-24.3% in protocol 2) in the ischaemic region, specifically in the endocardium (64.0%+/-28.9%, P<0.05 in protocol 1, and 75.1%+/-28.8%, P<0.05 in protocol 2), and correlated strongly with MBF (r=0.93 in protocol 1 and r=0.97 in protocol 2) as a logarithmic function. This indicated that the abnormal uptake of 123I-BMIPP was associated not only with wall motion abnormality but also with the severity of MBF. In the biopsy study (protocol 3), the radioactivity of either 123I-BMIPP or 18F-FDG correlated well with the MBF at the time of tracer injection and was similar to post-mortem analysis. It is concluded that 18F-FDG is a valid tool for identifying ischaemic myocardium even in its earliest stages. On the other hand, 123I-BMIPP might be used to detect moderately to severely ischaemic myocardium such as hibernation, suggesting the potential value of 123I-BMIPP in the risk stratification of patients with severe coronary artery disease who require revascularisation without delay.

Neutralization of interleukin-1beta in the acute phase of myocardial infarction promotes the progression of left ventricular remodeling.

OBJECTIVES: We sought to examine the role of the pro-inflammatory cytokine, interleukin-1-beta (IL-1beta), in the process of left ventricular (LV) remodeling in the early phase after myocardial infarction (MI). BACKGROUND: Studies have shown that pro-inflammatory cytokines are closely related to the progression of LV remodeling after MI. METHODS: Mice underwent coronary artery ligation, and the time course of LV remodeling was followed up to 20 weeks. The gene expression level of IL-1beta was examined. In a second set of experiments, the mice underwent coronary artery ligation followed by treatment with anti-IL-1beta antibody (100 microg, intravenously), versus control immunoglobulin G (100 microg, intravenously) immediately after the operation. RESULTS: Rapid hypertrophy of noninfarcted myocardium was observed by four weeks, and interstitial fibrosis progressed steadily up to 20 weeks. Anti-IL-1beta treatment increased the occurrence of ventricular rupture and suppressed collagen accumulation in the infarct-related area. At four and eight weeks after the operation, total heart weight and LV end-diastolic dimension were significantly greater in the anti-IL-1beta-treated mice than in the other groups. In the infarct-related area, collagen accumulation was suppressed, whereas in the noninfarcted area, pro-collagen gene expression levels, particularly type III, were decreased in the anti-IL-1beta-treated mice. CONCLUSIONS: Anti-IL-1beta treatment suppressed pro-collagen gene expression and delayed wound healing mechanisms-properties that are likely to lead to progression of LV remodeling. In the acute phase of MI, IL-1beta appears to play a protective role.

A rat model of dilated cardiomyopathy to investigate partial left ventriculectomy.

BACKGROUND: This study is the first to assess a small animal model of dilated cardiomyopathy (DCM) for evaluation of partial left ventriculectomy. METHOD: Eighteen Dahl salt-sensitive (DS) rats were divided into three groups. Six rats were fed an 8% high-salt diet from the age of 7 weeks (Group 1), and similarly six rats from 8 weeks (Group 2) and six from 9 weeks (Group 3). Blood pressure (BP) was measured by the tail-cuff method and left ventricular (LV) dimensions by echocardiography. RESULTS: In Groups 1 and 2, systolic BP rose and reached 200 mmHg by the 10th to 11th week, when all rats died within a week without signs of heart failure. However, in Group 3, systolic BP gradually rose to 196+/-15 mmHg (mean +/- SD) at the age of 14 weeks, when LV end-diastolic diameter (EDD) was 6.2+/-0.4 mm (control 5.1+/-0.7 mm) and LV fractional shortening (FS) was 77+/-3% (control 68+/-3%). At the age of 25 to 30 weeks, all rats in Group 3 showed signs of congestive heart failure, systolic BP remained high, EDD markedly increased (8.7+/-0.6 mm), and LVFS decreased (38.9+/-8.1%). From this stage, rats survived for 13.7+/-5.9 days. We employed the Group 3 model for our pilot PLV study. Eight rats had PLV with a beating heart by plicating the LV area between the papillary muscle bases. Two rats died perioperatively but the rest survived (60% survival 3 weeks after PLV). Postoperatively, the rats' LVEDD decreased and FS improved significantly. CONCLUSIONS: Using DS rats, we developed a DCM model for investigating PLV. The model may contribute to scientific investigation of PLV.

Coagulation process proceeds on cultured human mesangial cells via expression of factor V.

BACKGROUND: In a previous clinicopathological study, we observed mesangial factor V expression accompanied by the intact form of cross-linked fibrin deposition in the active type of IgA nephropathy. The conversion of prothrombin to thrombin by factor Xa is potently accelerated more than 104-fold by the presence of factor V, which is a membrane-bound cofactor. Another membrane-bound cofactor, tissue factor, is known to play an initiating role in the coagulation cascade and to be synthesized in mesangial cells (MCs) by the stimulation of tumor necrosis factor-alpha (TNF-alpha). However, the synthesis of factor V, which plays on the terminating stage of prothrombin activation, has not been reported previously in MCs by in vitro study. Our current study tested the coagulation process via expression of factor V by the stimulation of proinflammatory cytokine, TNF-alpha, in cultured human MCs. METHODS: To evaluate factor V protein expression, immunoperoxidase staining with densitometric evaluation and Western blot analysis were conducted after stimulation of TNF-alpha. To test factor V activity, stimulated MCs were incubated in combination with factor Xa, prothrombin, fibrinogen and factor XIII, and fibrin production on MCs was assessed after immunoperoxidase staining on the cell surface. In a blocking test using an antibody against factor V, suppression of fibrin production was evaluated to clarify the role of factor V activity. For the evaluation of factor V mRNA expression in cultured human MCs, in situ hybridization and Northern blot analysis were performed. RESULTS: Factor V protein expression in MCs after TNF-alpha stimulation increased both time- and dose-dependently. As a marker of factor V activity with exogenous factor Xa, fibrin production on TNF-alpha-stimulated MCs was increased in a time-dependent manner and was inhibited by the addition of anti-factor V antibody. Factor V mRNA was identified in MCs by in situ hybridization and showed an increase after stimulation with TNF-alpha on Northern blot analysis. CONCLUSIONS: Our data suggest that the coagulation process proceeds on MCs as the result of increased expression of endogenous factor V activity on its cell surface in cooperation with exogenous factor Xa.

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.

Disopyramide and its metabolite enhance insulin release from clonal pancreatic beta-cells by blocking K(ATP) channels.

In an insulin-secreting pancreatic beta-cell line (MIN6), insulin release was caused by disopyramide, an antiarrhythmic drug with Na-channel blocking action, and its main metabolite mono-isopropyl disopyramide (MIP). Insulin secretion, measured as immunoreactive insulin (IRI), was accelerated to 265.7% of the control by disopyramide and to 184.4% by MIP, with half-effective concentrations (EC50) of 30.9 +/- 1.5 microM and 92.4 +/- 2.2 microM. We tested the possibility that these drugs induce insulin release by inhibiting ATP-sensitive K+ (K(ATP)) channels of MIN6 cells. In the cell-attached or ATP-free inside-out mode with patch membranes on MIN6 cells, K-selective channels were recorded with unitary conductance of 70.5 +/- 3.5 pS (150 mM external K+ ions at room temperature). The channels were concluded to be MIN6-K(ATP) channels because they were closed by extracellular high glucose (11.0 mM) or glibenclamide (200 nM) and were reversibly activated by diazoxide (50 microM). In the inside-out patch mode, they were inhibited by micromolar ATP. In both cell-attached and insideout mode, disopyramide and MIP inhibited single MIN6-K(ATP) channels. In the inside-out mode, they produced a dose-dependent inhibition of channel activity: the half-blocking concentrations (IC50) were 4.8 +/- 0.2 microM for disopyramide and 40.4 +/- 3.1 microM for MIP. It was therefore concluded that both agents exert insulinotrphic effect through the inhibition of membrane K(ATP) channels in MIN6 cells.

Enhanced external counterpulsation improved myocardial perfusion and coronary flow reserve in patients with chronic stable angina; evaluation by(13)N-ammonia positron emission tomography.

AIMS: The mechanism by which enhanced external counterpulsation therapy exerts its beneficial effects on chronic and symptomatic stable angina is largely unknown. To clarify the mechanism of action of enhanced external counterpulsation, we used(13)N-ammonia positron emission tomography to evaluate myocardial perfusion. METHODS AND RESULTS: This was not a randomized controlled study. Eleven patients (eight male, age: 61.6+/-9.7) with angina pectoris underwent enhanced external counterpulsation therapy for 35 1 h sessions. They underwent a treadmill exercise test and(13)N-ammonia positron emission tomography, both at rest and with dipyridamole, before and after enhanced external counterpulsation therapy. Neurohumoral factors and nitric oxide were also evaluated. Myocardial perfusion increased at rest after therapy (0.69+/-0.27 to 0.85+/-0.47 ml x min(-1) x g(-1), P<0.05). In ischaemic regions, particularly the anterior region, myocardial perfusion at rest and with dipyridamole and coronary flow reserve improved significantly after therapy (at rest: 0.71+/-0.26 to 0.86+/-0.31;P<0.05, with dipyridamole: 1.26+/-0.65 to 1.84+/-0.94;P<0.02, coronary flow reserve: 1.75+/-0.24 to 2.08+/-0.28;P<0.04). Exercise time was prolonged and the time to 1-mm ST depression improved markedly (P<0.01). After therapy, nitric oxide levels increased (P<0.02) and neurohumoral factors decreased. CONCLUSIONS: Enhanced external counterpulsation therapy improved myocardial perfusion at rest and with dipyridamole and was associated with an increased exercise tolerance with(13)N-ammonia positron emission tomography and increased nitric oxide levels. These results suggest that one of the enhanced external counterpulsation mechanisms is development and recruitment of collateral vessels.

Serial evaluation of fatty acid metabolism in rats with myocardial infarction by pinhole SPECT.

BACKGROUND: Iodine 123-labeled 15-(p-iodophenyl)-3-(R,S)-methylpentadecanoic acid (BMIPP) is mainly trapped in the myocardium as triglyceride, depending on the adenosine triphosphate level. Ten percent to 20% of it is metabolized through alpha-oxidation after beta-oxidation; however, the precise mechanism of the regulatory pathways of BMIPP is yet to be clarified. METHODS AND RESULTS: A brief left coronary artery occlusion (10-30 minutes) was performed in 28 male Wistar-Kyoto rats. Dual single photon emission computed tomography images of BMIPP and thallium 201 were obtained 3 days and 24 days after the operation. The activities of 3-hydroxyacyl-coenzyme A dehydrogenase (HAD), citrate synthase (CS), and alpha-glycerol-phosphate dehydrogenase (GPD) were then measured in both ischemic and nonischemic regions. BMIPP and Tl-201 chloride severity scores were also evaluated conventionally. CS and HAD levels were significantly lower in the ischemic region than in the nonischemic region in the chronic group (CS, 102.9 +/- 28.1 vs 138.7 +/- 33.7 micromol/g/min, respectively, P =.0051; HAD, 54.7 +/- 20.1 vs 78.6 +/- 18.7 micromol/g/min, respectively, P =.0031). There was no difference in GPD between the ischemic and nonischemic regions. The BMIPP severity score had closer inverse relations with HAD (acute, r = -0.82; chronic, r = -0.80) and CS (acute, r = -0.87; chronic, r = -0.81), but not with GPD, than did Tl-201 chloride severity score. CONCLUSIONS: BMIPP imaging correlates well with the activities of HAD and CS, suggesting that a decrease in BMIPP uptake reflects deterioration of both fatty acid metabolism and citrate cycle and shows information other than regional myocardial perfusion.

Differential effects of angiotensin II versus endothelin-1 inhibitions in hypertrophic left ventricular myocardium during transition to heart failure.

BACKGROUND: In view of their mutual crosstalk, the roles of angiotensin II (Ang II) and endothelin-1 (ET-1) in the myocardium are assumed to be synergistic and supplemental. METHODS AND RESULTS: In the phase of compensated left ventricular (LV) hypertrophy of Dahl salt-sensitive rats, Ang II peptide and the ACE mRNA in the LV were increased by 1.6- and 3.8-fold, respectively. In contrast, ET-1 peptide and the preproET-1 mRNA remained unchanged. In subsequent congestive heart failure (CHF), Ang II and ACE mRNA did not show further increases. But ET-1 and the mRNA were increased de novo by 5.3- and 4.1-fold, respectively. In ascending aorta-banded rats, the local activations of Ang II and ET-1 also showed a differential time course between LV hypertrophy and CHF. Long-term treatments of Dahl salt-sensitive rats with temocapril (an ACE inhibitor) and with bosentan (a mixed ET receptor blocker) equally improved long-term survival. Temocapril reduced the LV/body weight ratio and ameliorated LV fractional shortening. Conversely, although bosentan equally improved fractional shortening, it did not reduce the increase in LV mass. Combined treatment with these 2 drugs further ameliorated the animal's survival without additional decreases in systolic pressure. CONCLUSIONS: The pathophysiological roles in the myocardium during the transition to CHF differ qualitatively between Ang II and ET-1. Thus, long-term therapy with a combination of ACE inhibition and ET antagonism may provide a new approach for heart failure in humans.

Up-regulated TGF-beta mRNA expression in splenic T cells of high IgA-prone mice: a murine model of IgA nephropathy with glomerulosclerosis.

BACKGROUND/AIMS: Recently, we established a high serum IgA-prone inbred (HIGA) mouse strain as a murine model of spontaneous IgA nephropathy by selective mating of high serum IgA ddY mice, and found that they showed enhanced production of glomerular extracellular matrix components with increased expression of TGF-beta mRNA and protein in the kidneys. In this study, we examined the roles of lymphocytes in the development of high serum IgA in this strain. METHODS: We performed flow cytometric analyses of T and B cells in splenic mononuclear cells (SMNCs) from these mice using BALB/c mice as normal controls. We also compared serum TGF-beta1 concentrations and TGF-beta mRNA expression levels in the B-cell-depleted (T-cell-rich) fraction of SMNCs in these mice. RESULTS: HIGA mice showed significantly fewer CD3-positive cells compared with BALB/c mice when young, but not when aged. The CD4/CD8 ratio of HIGA mice was lower than that of BALB/c mice, but this difference was not significant. Although the number of B220-positive cells did not vary significantly, the ratio of surface IgA-positive B cells was significantly increased in both young and adult HIGA mice. The B-cell-depleted SMNCs from HIGA mice exhibited higher levels of expression of TGF-beta and TGF-beta1 mRNA than controls from a young age, which were maintained throughout life, but there were no differences in PDGF, MCP-1 or bFGF expression between these two strains. In contrast to local mRNA expression, serum TGF-beta1 concentration was decreased in HIGA mice compared with BALB/c controls. CONCLUSION: These findings suggest that the mating procedure performed to establish HIGA mice selected for a unique phenotype of local up-regulation of TGF-beta production in the kidneys, as well as T cells that may contribute to both the early and consistently high serum IgA expression and enhanced production of renal extracellular matrix components in HIGA mice. Additionally, TGF-beta1 may act locally, not systemically, in a paracrine or autocrine manner.

ESDN, a novel neuropilin-like membrane protein cloned from vascular cells with the longest secretory signal sequence among eukaryotes, is up-regulated after vascular injury.

A novel cDNA has been isolated from primary culture of human coronary arterial cells by a signal sequence trap method, and designated ESDN (endothelial and smooth muscle cell-derived neuropilin-like molecule). ESDN is a type-I transmembrane protein with the longest cleavable secretory signal sequence among eukaryotes. ESDN contains a CUB domain and a coagulation factor V/VIII homology domain, which reminds us of the structure of neuropilins. ESDN also harbors an LCCL domain, which is shared by Limulus factor C and Coch. Mouse and rat counterparts were also identified revealing >84% amino acid identity with human ESDN. The human ESDN gene was mapped between D3S1552 and D3S1271. Northern blot analysis showed that ESDN mRNA was expressed in various tissues; particularly highly expressed in cultured vascular smooth muscle cells. The ESDN expression was up-regulated in platelet-derived growth factor-BB-stimulated vascular smooth muscle cells in vitro and neointima of the balloon-injured carotid artery in vivo. Overexpression of ESDN in 293T cells suppressed their bromodeoxyuridine uptake. In addition, ESDN protein was strongly expressed in nerve bundles in rodents. Thus, ESDN is considered to play a role in regulation of vascular cell growth and may have a wide variety of functions in other tissues including the nervous system, like neuropilins.

Calcineurin-GATA4 pathway is involved in beta-adrenergic agonist-responsive endothelin-1 transcription in cardiac myocytes.

Increases in the expression of endothelin-1 (ET-1) in cardiac myocytes play a critical role in the development of heart failure in vivo. Whereas norepinephrine (NE) is a potent inducer of ET-1 expression in cardiac myocytes, the signaling pathways that link NE to inducible cardiac ET-1 expression are unknown. Adrenergic stimulation results in an increase in intracellular calcium levels, which in turn activates calcineurin. Here, we have shown that stimulation with NE markedly increased the expression of the ET-1 gene in primary cardiac myocytes from neonatal rats. This increase was severely attenuated by a beta-adrenergic antagonist, metoprolol, but not by an alpha-adrenergic antagonist, prazosin. Consistent with these data, the beta-adrenergic agonist isoproterenol (ISO) activated the rat ET-1 promoter activity to an extent that was similar to NE. The ISO-stimulated increase in promoter activity was significantly inhibited by a Ca(2+)-antagonist, nifedipine, and an immunosuppressant, cyclosporin A, which blocks calcineurin. Mutation analysis indicated that the GATA4 binding site is required for ISO-responsive ET-1 transcription. Stimulation with ISO enhanced the interaction between NFATc and GATA4 in cardiac myocytes. Consistent with this interaction, overexpression of GATA4 and NFATc synergistically activated the ET-1 promoter. These findings demonstrate that NE-stimulated ET-1 expression in cardiac myocytes is mediated predominantly via a beta-adrenergic pathway, and that calcium-activated calcineurin-GATA4 plays a role in this process.

Cytokine gene therapy for myocarditis by in vivo electroporation.

Cytokines are important pathophysiologic and pathogenic factors in cardiovascular disorders, including viral myocarditis. We attempted to treat viral myocarditis with cytokine gene therapy by transferring an inhibitory cytokine, IL-1 receptor antagonist (IL-1ra) or viral IL-10 (vIL-10), by in vivo electroporation, a new method for gene transfer into muscle. Four-week-old male DBA/2 mice were inoculated intraperitoneally with 10 PFU of encephalomyocarditis virus. Immediately after virus inoculation, an expression plasmid carrying IL-1ra or vIL-10 was injected into tibialis anterior muscles followed by electroporation. Serum levels of IL1ra and vIL-10 reached 10.5 and 2.3 ng/ml, respectively, on day 5, when gene expression reached its peak. Histopathological examination showed that myocardial cellular infiltration was improved in mice treated with IL-1ra or vIL-10 compared with the control group. On day 14 after the onset of myocarditis, transfer of IL1ra or vIL-10 expression plasmid had significantly improved the survival rates of the animals. The expression of TNF-alpha was decreased to 0.60-fold (p < 0.005) and inducible nitric oxide synthase (iNOS) 0.43-fold (p < 0.005) by IL-1ra treatment, and the expression of IFN-gamma in the heart was decreased to 0.35-fold (p < 0.05), and iNOS 0.21-fold (p < 0.005), by vIL-10 relative to the controls. These results show that gene therapy with IL-1ra or vIL-10 expression plasmid was effective in the treatment of viral myocarditis, and in vivo electroporation may be a useful method by which to deliver cytokine therapy in cardiovascular diseases.

Effects of posture on cardiac autonomic nervous activity in patients with congestive heart failure.

OBJECTIVES: We aimed to clarify which recumbent position is preferred by patients with congestive heart failure (CHF) and to evaluate whether cardiac autonomic nervous activity is different among three recumbent positions (supine, left lateral decubitus, right lateral decubitus) in patients with CHF. BACKGROUND: It remains unclear whether cardiac autonomic nervous activity is different among three recumbent positions in patients with CHF. METHODS: We studied 17 male CHF patients (66+/-7 years) and 17 age- and gender-matched healthy subjects (66+/-7 years). Each subject underwent 24-h ambulatory electrocardiographic monitoring. A channel was used to record the CM5 lead, and another to record the signal of the patient's posture with use of a newly developed small-sized detector (3.2 cm x 3.2 cm). By using spectral analysis of heart rate variability, frequency-domain measures were calculated and compared among the three recumbent positions. Normalized high-frequency (HF: 0.15 to 0.40 Hz) power was used as an index of vagal activity and the low frequency (0.04 to 0.15 Hz)/HF power ratio was used as an index of sympathovagal balance. RESULTS: In patients with CHF, the time for the right lateral decubitus position was two-fold longer than that for the supine and left lateral decubitus positions. The increased cardiac sympathetic activity and decreased vagal tone in CHF patients were normalized in the right lateral decubitus position. CONCLUSIONS: The right lateral decubitus position in patients with CHF may be a self-protecting mechanism of attenuating the imbalance of cardiac autonomic nervous activity.