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PurposeThe density of mast cells increases in the myocardium of patients suffering from heart failure. However, their function remains unclear. In this study, preformed angiotensin II (ANG II), a potent growth factor, was found to be contained in, and released by, human mast cells. Methods:The human mast cell line (HMC-1) was incubated with 0 to 10−6M calcitonin gene-related peptide (CGRP) or culture medium. The expression of renin-angiotensin system mRNA was examined using RT-PCR analysis. ELISA and immunohistochemistry with monoclonal antibody against human ANG II were performed to detect the presence of ANG II in HMC-1. The effect of CGRP on the expression of angiotensinogen mRNA was examined by quantitative RT-PCR analysis. Results:Preformed ANG II was detected in a human mast cell line (HMC-1) which is a neoplastic cell line of mast cells by ELISA and immunohistochemistry. Presence of mRNA of angiotensinogen and renin was confirmed by polymerase chain reaction in HMC-1, while mRNA of angiotensin converting enzyme (ACE) was undetectable. Since myocardial mast cells are interfaced with nerve fibers and functionally associated with CGRP, the effect of CGRP on ANG II release from HMC-1 was examined. CGRP induced the release of ANG II and increased angiotensinogen mRNA in HMC-1. ConclusionsThe presence of preformed ANG II and gene expression of the renin-angiotensin system were detected in human mast cells. The release and synthesis of ANG II in mast cells was regulated by CGRP.

Concentrations of the circulating hepatocyte growth factor (HGF) increase in the very early phase of acute myocardial infarction, and are a marker of arterial thrombosis. A recently developed, highly sensitive HGF assay can detect the early stages of arterial thrombosis in patients with unstable angina pectoris, acute aortic dissection and pulmonary thromboembolism. Heparin rapidly induces the release of HGF into the circulation, and HGF is a major factor involved in heparin-induced angiogenesis. Furthermore, the activation of mast cells by thrombus formation releases HGF into the circulation. This new pathway, thrombus formation-mast cell activation- degranulation-heparin-HGF-angiogenesis, may be both diagnostically useful and a therapeutic target.

Background:Tubulointerstitial damage is recognized as a determinant of the prognosis of kidney disease. Various types of viral infection have been reported to induce tubulointerstitial lesions; however, that caused by hepatitis C virus (HCV) remains unclear, although glomerular lesions caused by this viral infection have been well documented. Methods:To identify any association, we retrospectively investigated 320 patients who underwent renal biopsy and did not have extrarenal diseases causing tubulointerstitial nephritis. Results:Of these patients, 13 patients had HCV infection and 307 patients did not. In a case-control study, HCV infection showed a significant association with the prevalence of tubulointerstitial injury. To offset the secondary tubulointerstitial change caused by advanced glomerulopathy, we performed a glomerular stage-matched comparison of patients with membranous nephropathy (MN). Nine patients with MN among the 13 HCV-infected patients and 18 HCV-negative patients with electron microscopic glomerular stage-matched MN were randomly selected from the overall pool of patients. Comparing areas of interstitial fibrosis and inflammatory cell infiltration, both were greater in HCV-infected than HCV-negative patients. In biopsy tissues from HCV-infected patients, positive signal for HCV was observed in the perinuclear area of tubular epithelial cells and infiltrating cells on immunohistochemistry and in situ hybridization. By a strand-specific reverse-transcription polymerase chain reaction for HCV, both genomic- and replicative-strand RNA were detected in renal tissues. Conclusion:These results suggest that HCV infection is a potent pathogenic factor of tubulointerstitial injury.

Mast cells are believed to be involved in the pathophysiology of heart failure, but their precise role in the process is unknown. This study examined the role of mast cells in the progression of heart failure, using mast cell-deficient (WBB6F1-W/Wv) mice and their congenic controls (wild-type [WT] mice). Systolic pressure overload was produced by banding of the abdominal aorta, and cardiac function was monitored over 15 wk. At 4 wk after aortic constriction, cardiac hypertrophy with preserved left ventricular performance (compensated hypertrophy) was observed in both W/Wv and WT mice. Thereafter, left ventricular performance gradually decreased in WT mice, and pulmonary congestion became apparent at 15 wk (decompensated hypertrophy). In contrast, decompensation of cardiac function did not occur in W/Wv mice; left ventricular performance was preserved throughout, and pulmonary congestion was not observed. Perivascular fibrosis and upregulation of mast cell chymase were all less apparent in W/Wv mice. Treatment with tranilast, a mast cell–stabilizing agent, also prevented the evolution from compensated hypertrophy to heart failure. These observations suggest that mast cells play a critical role in the progression of heart failure. Stabilization of mast cells may represent a new approach in the management of heart failure.

Background:The reduction in nephrons in IgA nephropathy is critical to the prognosis of this disease. However, the immunopathological mechanism of the modifications seen in glomerular lesions is not clear. We thus investigated the influence of nephron reduction by heminephrectomy on renal lesions in a high immunoglobulin A inbred strain of ddY mouse (HIGA mouse), which shows progressive mesangial sclerosis with elevated renal expression of transforming growth factor (TGF)-β. Methods:Five-week-old HIGA mice were heminephrectomized (Nx), and were evaluated in comparison with a sham-operated group (S) at 40 weeks old. Histological findings, immunoglobulin depositions (IgG, IgA, and IgM), and expressions of cytokine and extracellular matrix proteins (TGF-β, fibronectin, collagen type I and IV) were analysed. PCNA and TUNEL stainings were performed with electron microscopic detection of apoptosis. Tissue renin-angiotensin systems (RAS) were also investigated by real-time quantitative RT-PCR. Results:In the Nx group, the glomerular tuft area and ratio of mesangial matrix area per tuft were significantly increased, and the glomerular cell count per tuft area was significantly decreased. Glomerular immunoglobulin deposits of IgG, IgA, and IgM in Nx were all significantly expanded in the paramesangium. The glomerular expressions of TGF-β and the extracellular matrix proteins were significantly increased in Nx mice. In contrast to the significant decrease of PCNA-positive cells, TUNEL-positive cells were significantly increased in Nx. Angiotensin-converting enzyme (ACE) was significantly increased in the renal cortex of Nx. Conclusion:Simple heminephrectomy, other than 5/6 renal ablation, of HIGA mice may be a potential model for research into the progressive glomerulosclerosis of human IgA nephropathy. The pathological role of apoptosis is apparently involved in these disease processes, possibly through upregulated RAS.

A novel cDNA has been isolated from primary culture of human coronary arterial cells by a signal sequence trap method, and designated ESDN (endothelial andsmooth muscle cell-derivedneuropilin-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 ESDNgene 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 vitroand 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.

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-α 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-γ 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.

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 α-oxidation after β-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 a-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 μmol/g/min, respectively, P = .0051; HAD, 54.7 ± 20.1 vs 78.6 ± 18.7 μmol/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.

Congestive heart failure may be produced by a variety of disorders, including dilated cardiomyopathy, hypertension, and ischemic heart disease. We have developed experimental models of these diseases, and found gene expressions of proinflammatory cytokines increased in the hearts of these animals. Various drugs for heart failure modulate the production of cytokines in experimental models of heart failure. Pimobendan, an inhibitor of phosphodiesterase III prolonged survival, attenuated inflammatory lesions, and decreased the production of cytokines and nitric oxide. Recent studies have shown that these inhibitory effects are due to inhibition of activation of NF-κB. In contrast, digitalis increased the production of cytokines and exacerbated myocarditis. Interleukin-10 prolonged survival, attenuated myocardial injury, and appears promising as a treatment of heart failure due to viral myocarditis. Endothelin-1 plays an important pathophysiological role in heart failure, and treatment with an endothelin antagonist had a cardioprotective effect in experimental models of heart failure.

Previous studies have indicated that cardiac events in young patients with hypertrophic cardiomyopathy (HCM) are related to ischaemia rather than to arrhythmia. We measured coronary flow reserve in paediatric HCM and compared the values with those in adult HCM. We studied 12 patients with HCM including six paediatric (<20 years old; mean 13 years) and six adult patients (>20 years old: mean 62 years), and six healthy young adults (mean 29 years) as controls. Every patient underwent magnetic resonance imaging (MRI) for anatomical assessment. Myocardial blood flow at rest and after dipyridamole infusion was measured with dynamic nitrogen-13 ammonia positron emission tomography (PET). Partial volume effect was corrected for using the anatomical data obtained with MRI. In adult patients with HCM, coronary flow reserve in the hypertrophied septal region was not significantly different from that in the non-hypertrophied lateral wall (1.38±0.29 vs 1.77±0.39, respectively). In the paediatric patients, coronary flow reserve in the hypertrophied septal region was significantly lower than in the non-hypertrophied lateral wall (0.84±0.33 vs 2.74±0.90, respectively, P<0.01). In addition, coronary flow reserve in adult patients was lower than in control subjects both in the septal wall (1.38±0.29 vs 2.94±0.35, respectively, P<0.0001) and in the lateral wall (1.77±0.39 vs 2.85±0.69, respectively, P<0.05). In contrast, coronary flow reserve in paediatric patients was not significantly different from that in control subjects in the lateral wall (2.74±0.90 vs 2.85±0.69, respectively), while absolute reduction of myocardial blood flow was noted after pharmacological vasodilatation in the hypertrophied septal region. In conclusion, significant regional differences of coronary flow reserve were present in the paediatric patients with HCM. These results suggest that paediatric patients with HCM intrinsically have the potential to experience significant regional ischaemia even in the absence of coronary stenosis.

Abstract The importance of hepatitis C virus infection has been recently noted in patients with hypertrophic cardiomyopathy or dilated cardiomyopathy. In a collaborative research project of the Committees for the Study of Idiopathic Cardiomyopathy, hepatitis C virus antibody was found in 74 of 697 patients (10.65) with hypertrophic cardiomyopathy and in 42 of 663 patients (6.3%) with dilated cardiomyopathy; these prevalences were significantly higher than that found in volunteer blood donors in Japan. Hepatitis C virus antibody was detected in 650 of 11,967 patients (5.4%) seeking care in 5 academic hospitals. Various cardiac abnormalities were found, and arrhythmias were the most frequent. These observations suggest that hepatitis C virus infection is an important cause of a variety of otherwise unexplained heart diseases. As a collaborative research with National Cardiovascular Center and Juntendo University, we tried to detect hepatitis C virus genomes using paraffin section of autopsied hearts. Among 106 hearts examined, β-actin gene was amplified in 61 hearts (52.6%). Among these, hepatitis C virus RNA was detected in 13 hearts (21.3%), and negative strands in 4 hearts (6.6%). Hepatitis C virus RNA was found in 6 hearts (26.0%) with hypertrophic cardiomyopathy, 3 hearts (11.5%) with dilated cardiomyopathy, 4 hearts (33.3%) with myocarditis. These hepatitis C virus RNA positive samples were obtained between 1979 and 1990, indicating that hepatitis C virus RNA can be amplified from paraffin-embedded hearts preserved for many years. More recently, we examined the effect of interferon on myocardial injury associated with active hepatitis C. As TL-201-SPECT was a more sensitive method to detect myocardial injury induced by hepatitis C virus than electrocardiography or echocardiography, we used to T1-SPECT scores to evaluate the effect of interferon on myocardial injury. SPECT scores improved in 8 patients (53%) out of 15 patients in whom interferon therapy was completed. Circulating hepatitis C virus disappeared after interferon therapy in all 11 patients with improvement or no change, but hepatitis C virus genomes persisted in the blood in 2 aggravated patients. Although this study is preliminary, interferon therapy is a promising treatment for myocardial diseases caused by hepatitis C virus infection. Zusammenfassung Der Zusammenhang zwischen einer Hepatitis-C-Virus-Infektion und dem Auftreten einer hypertrophischen und einer dilatativen Kardiomyopathie wurde erst vor kurzem erkannt. In einem gemeinsamen Forschungsprojekt der "Committees for the Study of Idiopathic Cardiomyopathy" wurden Hepatitis-C-Virus-Antikörper bei 74 (10,6%) von 697 Patienten mit hypertrophischer Kardiomyopathie und bei 42 (6,3%) von 663 Patienten mit dilatativer Kardiomyopathie gefunden, wobei diese Prävalenz signifikant höher ist als bei freiwilligen Blutspendern in Japan. Hepatitis-C-Virus-Antikörper wurden bei 650 (5,4%) von 11 967 Patienten, die zur Behandlung in fünf Universitätskliniken waren, gefunden. Verschiedene kardiale Veränderungen wurden beobachtet, wobei Arrhythmien am häufigsten auftraten. Diese Beobachtungen legen nahe, daß eine Hepatitis-C-Virus-Infektion eine wichtige Ursache für eine Reihe bisher ungeklärter Herzerkrankungen ist. In einem gemeinsamen Forschungsprojekt mit dem "National Cardiovascular Center" und der Juntendo-Universität versuchten wir Hepatitis-C-Virus-Genome in Paraffinscnitten von autoptischen Herzen nachzuweisen. Von 106 untersuchten Herzen wurde das β-Aktin Gen bei 61 Herzen (52,6%) nachgewiesen. Bei diesen Proben wurden die Hepatitis-C-Virus-RNA in 13 Herzen (21,3%) und Negativstränge bei vier Herzen (6,6%) gefunden. Eine Hepatitis-C-Virus-RNA wurde bei sechs Herzen (26,0%) mit hypertrophischer Kardiomyopathie, drei Herzen (11,5%) mit dilatativer Kardiomyopathie und bei vier Herzen (33,3%) mit Myokarditis identifiziert. Diese Hepatitis-C-Virus-RNA-positiven Proben wurden zwischen 1979 und 1990 eingebettet, und daher kann gefolgert werden, dass Hepatitis-C-Virus-RNA noch aus paraffineingebettetem Gewebe, das mehrere Jahre gelagert wurde, amplifiziert werden kann. Vor kurzem untersuchten wir den Einfluss von Interferon auf eine Myokardschädigung, die mit einer akuten Hepatitis C einhergeht. Da TL-201-SPECT sensitiver war als die Elektrokardiographie oder Echokardiographie, um eine Hepatitis-C-Virus-induzierte Myokardschädigung nachzuweisen, verwandten wir T1-SPECT-Scores, um den Einfluss von Interferon auf eine Myokardschädigung zu untersuchen. SPECT-Scores verbesserten sich bei acht (53%) von 15 Patienten nach einer Interferonbehandlung. Zirkulierende Hepatitis-C-Viren verschwanden nach einer Interferontherapie bei allen elf Patienten, die entweder eine Besserung oder keine Zustandsveränderung aufwiesen; bei zwei Patienten mit schwerer Erkrankung waren Hepatitis-C-Virus-Genome im Blut noch nachweisbar. Obwohl diese Studie noch vorläufig ist, erscheint eine Interferontherapie erfolgversprechend bei Myokarderkrankungne, die durch eine Hepatitis-C-Virus-Infektion verursacht werden.

Summary. We have previously demonstrated that turkey poults fed furazolidone (Fz) in high concentrations (700 ppm) develop dilated cardiomyopathy (DCM) which approximates the human condition [1–3]. We wanted to study the effects of a calcium channel blocker in an animal model with a documented decrease in β-receptor density, increased levels of circulating catecholamines, and abnormal calcium metabolism. The effects of a third generation calcium channel blocker has not been studied in our model. We hypothesized that the model would be predictive of the human condition and provide additional insights into the potential use of Ca2+channel blockers in the setting of DCM. In the present study, we examined the effect of pranidipine, a new dihydropyridine calcium antagonist, in the setting of DCM on the gross and microscopic morphology of the heart and the overall contractile performance of the myocardium. A state of symptomatic to mild cardiomyopathy was induced in Broad-Breasted White turkey poults by administration of Fz for three weeks. Blood pressure, heart rate, fractional shortening, and body weight were monitored and compared in DCM animals treated with pranidipine and those given a placebo. After four weeks of treatment or no treatment with pranidipine, animals were euthanized and heart weight, cardiac dimensions, and microscopic morphology were compared. Progressive left ventricular (LV) dilatation and wall thinning was prevented with pranidipine treatment. In addition, microscopic examination demonstrated myocyte hypertrophy regression in DCM animals treated with pranidipine. In DCM animals, treatment with pranidipine resulted in significantly smaller left ventricular dimensions. We conclude that the calcium channel blocker pranidipine was not detrimental to global cardiac function in animals with dilated cardiomyopathy.