永井 良三

OBJECTIVES: To determine whether immune injury during acute cardiac rejection induces phenotypic modulation of arterial smooth muscle cells and lesion formation, we studied the expression of embryonic myosin heavy-chain isoform and degrees of intimal proliferation in aortas and coronary arteries of allografted rabbit hearts. Modulation of phenotype in arterial smooth muscle cells during acute vascular injury is a widely reported phenomenon, and proliferation and migration of medial smooth muscle cells contribute to development of intimal hyperplasia of arteries in response to immune injury. METHODS: Rabbit hearts were heterotopically transplanted to the neck without immunosuppression. Hearts were harvested at 2, 5, 7, and 10 days after transplantation. Proliferation of smooth muscle cells was assessed by bromodeoxyuridine labeling. Staining for immunohistochemical indicators was done with use of monoclonal antibodies that recognize T lymphocytes and all types of smooth muscle cells (SM1), adult type of smooth muscle cells (SM2), and embryonic myosin heavy-chain isoform. Intimal thickening and luminal narrowing were assessed with a computer-assisted video image analysis system. RESULTS: Intimal thickening and luminal narrowing in aortas and coronary arteries gradually increased in a time-dependent manner. The neointima thus formed consisted of proliferating smooth muscle cells positive for both SM1 and embryonic myosin heavy-chain isoform and massive T lymphocyte accumulation. Intimal proliferation was more prominent in aortas and large epicardial coronary arteries than in the intramyocardial small coronary arteries. CONCLUSIONS: These findings suggest that allogeneic immune injury facilitates phenotypic modulation of smooth muscle cells, which may contribute to subsequent transplantation-associated atherosclerosis.

We report a case of restricted meningeal infection by atypical mycobacteria, identified by the polymerase chain reaction, in a non-immunocompromised adult successfully treated by multiple antibiotics including fluoroquinolone. New quinolones should be considered as a therapeutic option for such mycobacterial meningitis.

Background. We reported that an embryonic type of non-muscle-type myosin heavy-chain isoform (SMemb) may be a molecular marker for phenotypic alteration in initial glomerular injury and that methylprednisolone has no effect on SMemb expression in glomeruli of rats with puromycin aminonucleoside (PAN) nephrosis. The present study was designed to assess whether SMemb mRNA and protein expression in glomeruli are affected by a low-protein diet in rats with PAN-induced nephrosis and in control rats. Methods. Rats were divided into four groups: group 1, PAN-injected rats fed a standard diet containing 22% protein; group 2, PAN-injected rats fed a low-protein diet containing 6% protein, starting on the day of PAN injection; group 3, control rats fed a standard diet; group 4, control rats fed a low-protein diet for the same period. We prepared glomerular RNA and performed Northern blot analysis and immunohistochemistry in all groups. Results. Glomerular SMemb mRNA increased on days 2 and 4 (prior to and soon after the onset of proteinuria), but declined on day 8 (the peak of proteinuria). Myosin heavy-chain protein expression was evaluated immunohistochemically by use of three antibodies against SM1, SM2, and SMemb. SM1 and SM2 were absent from the glomeruli of rats with PAN nephrosis until day 20. The SMemb isoform was barely detectable in normal glomeruli, but substantial amounts of SMemb were demonstrated in the glomeruli of rats with PAN nephrosis. In the latter condition, the number of SMemb-positive glomerular epithelial cells increased on days 3 and 4, then decreased in subsequent days. Moreover, some mesangial cells became SMemb-positive transiently, returning to barely detectable levels on day 20. In addition, alpha-smooth-muscle actin, type I and III collagens were absent from the glomeruli of rats with PAN nephrosis until day 20. Urinary protein excretion was markedly suppressed by the 6% protein diet in PAN nephrosis. The low-protein diet reduced the increased mRNA expression of SMemb as well as the increased number of SMemb-positive cells in the glomeruli of rats with PAN nephrosis. However, the low-protein diet did not affect SMemb mRNA and protein levels in the glomeruli of control rats. Conclusions. In rats with PAN nephrosis, findings suggest that restriction of dietary protein leads to a reduction in glomerular SMemb expression.

BACKGROUND: Diagnosis of acute rejection and graft arteriosclerosis (chronic rejection) is critical to the success of cardiac transplantation, but accurate diagnosis is often difficult. We have reported that there are three types of vascular myosin heavy chain (MHC) isoforms: SM1, SM2, and SMemb. SM2 is specifically expressed in differentiated smooth muscle cells (SMCs). SMemb is a nonmuscle-type MHC abundantly expressed in SMCs of fetal aorta. METHODS AND RESULTS: To evaluate the usefulness of MHC expression for diagnosis and analysis of acute and chronic rejection, heterotopic cardiac transplantation was performed in rats and monkeys. Immunohistochemistry, electron microscopy, and Northern blot assay were performed to evaluate MHC expression. SMemb was expressed in spindle-shaped cells located in acutely rejected myocardium in the rats and monkeys. These cells were also observed in areas lacking cellular infiltration. These SMemb-positive cells were activated fibroblasts or myofibroblasts. SMemb mRNA was enhanced parallel to the progression of acute rejection. In the coronary arteries of chronically rejected allografts, enhanced SMemb and reduced SM2 expression was observed in both thickened intima and media. The reduced medial SM2 expression was observed before the intimal thickening occurred. These cells were phenotypically modulated SMCs. CONCLUSIONS: Altered expression of MHC isoforms is a sensitive indicator in the diagnosis of acute and chronic cardiac rejection. The pathophysiology of this alteration in MHC isoform expression should be studied further to elucidate the pathogenesis of cardiac rejection.

The angiotensin-converting enzyme (ACE) inhibitor enalapril was administered to 13 patients with chronic heart failure (New York Heart Association class II or III) for 12 weeks, and its effects on resting cardiac function (shown by echocardiogram), exercise capacity, and biochemical factors sere investigated, Left ventricular end-diastolic and end-systolic diameters improved significantly from 60.3 +/- 5.4 mm at baseline to 57.2 +/- 5.7 mm after 12 weeks of treatment and from 50.2 +/- 5.9 mm to 46.8 +/- 5.6 mm, respectively; however left atrial dimension did not change significantly after treatment, Peak oxygen uptake also improved significantly after. enalapril treatment (20.0 +/- 6.9 mL/kg/ min at baseline to 22.4 +/- 7.6 mL/kg/min after treatment), as did anaerobic threshold by was exchange (17.1 +/- 6.4 mL/kg/min vs 18.4 +/- 5.8 mL/kg/min). The serum levels of atrial natriuretic peptide and beta-endorphin decreased significantly after treatment (47.6 +/- 42.1 pg/mL before vs 31.6 +/- 29.2 pg/mL, after treatment and 11.6 +/- 3.6 pg/mL vs 5.9 +/- 2.8 pg/mL, respectively). These results indicated that enalapril is useful for improving resting cardiac function, exercise capacity, and biochemical factors in patients with chronic heart failure.

Changes in contractile and relaxation properties of heart muscle in the cardiac hypertrophy induced by pressure overload have been attributed to alterations in intracellular Ca2+ transport as well as the phenotypic and quantitative changes in contractile protein. However, contradictory data have been reported regarding Ca2+ uptake, release and storage by the sarcoplasmic reticulum (SR). The purpose of this study was to evaluate the changes in SR Ca(2+)-ATPase, ryanodine receptor, calsequestrin and alpha-actin gene expression, and the changes in Ca2+ uptake capacity in various degrees of hypertrophied hearts due to pressure overload. Cardiac hypertrophy was produced in rats by placing a constricting clip (0.80 mm) around the suprarenal abdominal aorta for 8 days. The mRNA levels and Ca2+ uptake capacity were then measured as a function of the severity of cardiac hypertrophy. Ca(2+)-ATPase and ryanodine receptor mRNA levels were increased in mildly hypertrophied hearts but were diminished in severely hypertrophied hearts, showing a bimodal response to pressure overload, Ca2+ uptake capacity showed similar changes along with a positive correlation with Ca(2+)-ATPase mRNA level (r = 0.67, P < 0.001). In contrast, the level of calsequestrin mRNA expression was unaltered and that of alpha-actin was markedly increased over a range of severity of cardiac hypertrophy. These findings suggest that the expression of sarcoplasmic reticulum genes for Ca2+ uptake and release is up- or downregulated dependent on the degree of pressure overload. The gene for the SR Ca2+ storage protein, calsequestrin, might be under different control from these genes in pressure overload. Our findings suggest that the decrease in ratio of mRNAs encoding Ca2+ uptake and release proteins to those encoding contractile proteins could significantly contribute to the slowed contractile and relaxation properties seen in pressure-overloaded hearts.

The aim of this study was to determine the phenotypic modulation in mesangial cells of glomeruli damaged by hypertension. Salt-loaded stroke-prone spontaneously hypertensive rats were untreated or treated with a calcium antagonist, manidipine (2 mg/kg/day) for eight weeks. In normotensive Wistar-Kyoto rats, alpha-smooth muscle actin was not expressed in any glomerular cells and a non-muscle myosin heavy chain isoform, SMemb, was slightly expressed in glomerular Visceral epithelial cells. In the untreated hypertensive rats, the glomeruli showed sclerosis to various degrees and expressed alpha-smooth muscle actin and SMemb. Normal expression of SMemb in the epithelial cells disappeared. Notably, alpha-smooth muscle actin-positive fibroblast-like cells appeared in the interstitium, especially around the Bowman's capsules. Manidipine ameliorated the glomerulosclerosis and reduced the expression of alpha-smooth muscle actin in mesangial cells. In conclusion, the mesangial cells changed their phenotypes and expressed alpha-smooth muscle actin and SMemb in the glomeruli during the development of hypertensive renal damage. These phenotypically changed mesangial cells are considered to be activated and to produce various kinds of cytokines and extracellular matrix, which leads to glomerulosclerosis. Manidipine attenuated the glomerular damage and the phenotypic changes. The functional relevance of phenotypic changes in these cells should be elucidated in future studies.

Nitric oxide (NO) is present in the breath exhaled from healthy humans, The location and subtype of the NO synthase responsible for the expired NO are unknown, As dexamethasone inhibits the induction of NO synthase, we evaluated the effect of administering dexamethasone (4 mg/day for 2 days) on the amount of NO exhaled by eight healthy men. The amount of NO showed a significant linear correlation with the duration of exhalation, allowing the rate of NO release to be calculated, The rate of NO release was 0.047+/-0.023 nmol/s before drug administration, There was no significant change in the release rate at the end of the 2-day administration of drug or at 5 days after cessation, Serum concentrations of interferon-gamma and interleukin-1 beta were unaffected by the administration of dexamethasone, These results suggest that the NO released from the human airway under normal conditions is not generated by the action of inducible NO synthase.

We investigated the effect of amiloride, a Na(+)-H+ exchange blocker, on ventricular hypertrophy in a murine model of dilated cardiomyopathy (DCM). Mice with DCM were given orally amiloride for 60 days. The ratio of heart weight to body weight and left ventricular cavity dimension were significantly smaller in both amiloride groups than those in furosemide and control (untreated DCM) groups (p < 0.05). The fiber diameter was significantly smaller in amiloride groups than that in furosemide group (p < 0.01). Plasma and cardiac angiotensin II (AII) levels were decreased in amiloride-treated groups compared with those in furosemide or control group (p < 0.05). Our findings suggest that amiloride prevents the development of myocardial hypertrophy and left ventricular dilatation in DCM in association with a reduction of AII.

Inflammatory cytokines play a key role in the myocardial injury produced by viral myocarditis. Although interleukin-6 (IL-6) reportedly possesses antiviral properties, its effect in viral myocarditis is unclear. To investigate the role of IL-6 in viral myocarditis induced by encephalomyocarditis virus (EMCV) in mice, we evaluated (1) the survival rate following IL-6 administration, (2) the viral titer in the heart, (3) viral replication in the heart by in situ hybridization, (4) histopathological changes using immunohistochemical staining, (5) neutralizing antibody against EMCV, (6) circulating interferon and tumor necrosis factor-alpha (TNF-alpha), (7) viral suppression in vitro by IL-6, and (8) natural killer (NK)-cell activity. Eight-week-old C3H/HeJ mice were injected intraperitoneally with EMCV (day 0) and were also injected subcutaneously twice daily for 4 consecutive days with 10 micrograms/0.1 mL of human IL-6 on day -4 (group A), day 0 (group B), or day +4 (group D) for 4 days. As a control, 0.1 mL PBS instead of IL-6 was injected on day 0 for 4 days (group C). Certain mice were killed on day 4. The myocardial virus titers, viral replication in situ, and NK-cell activity in the spleen were determined. Decreased viral titer and viral replication in the heart reduced the titer of circulating TNF-alpha, and lower NK-cell activity was observed in group B versus group C (control group). The titer of neutralizing antibodies against EMCV was significantly (P < .05) increased in group B compared with group C. The remaining mice were killed on days 10 and 30 after infection. The ratio of heart weight (HW) to body weight (BW) and myocardial injury in group B were reduced versus group C on days 10 and 30. The HW of group B on day 30 did not differ from the normal control group. The ratio of splenic weight to BW and the ratio of thymic weight to BW of group B increased on day 10, with expanded follicles observed in the spleen and enlargement of the medulla observed in the thymus. Immunohistochemical study revealed an increased percentage of macrophages in the heart and spleen of group B. In summary, IL-6 reduces myocardial damage in mice with viral myocarditis. Modification of immune responses together with reduction in viral replication appears to be the mechanism of the IL-6 effect. Although IL-6 is likely important in the process of viral antigen presentation, early activation of immune responses and attenuation of viral replication appear most significant, as reflected in the limited time window during which IL-6 is effective in myocarditis.

BACKGROUND: Much past research has concerned the relationship between coronary heart disease and the angiotensin converting enzyme (ACE) genotype, with many lines of evidence demonstrating polymorphism to be an independent risk factor for myocardial infarction. Interestingly, however, association of ACE polymorphism and severity of coronary artery stenosis according to racial background has recently been proposed. OBJECTIVE: To clarify the relationship between the ACE genotype and severity of coronary artery stenosis in Japanese patients. METHODS: In 36 consecutive patients undergoing coronary catheterization, comparative examination of coronary angiography findings with the ACE genotype was conducted. RESULTS: The severity of coronary artery stenosis indeed showed a relationship with the ACE genotype, with more severe coronary artery stenosis associated with the deletion (D) allele (P < 0.05). The serum lipids, total cholesterol and triglycerides levels, were also elevated in patients with the D allele (P < 0.05). CONCLUSION: We have provided further evidence that ACE polymorphism is associated with severity of coronary heart disease in a Japanese population. A possible relationship between serum lipids and the ACE genotype is also suggested.

To characterize the phenotypic modulation of mesangial and glomerular epithelial cells, we investigated the expression of a nonmuscle type myosin heavy chain, SMemb, and alpha-smooth muscle actin (alpha-SM actin) in rat experimental glomerular diseases, which included anti-Thy 1 nephritis, 5/6 nephrectomy, diabetes, and anti-glomerular basement membrane nephritis. SMemb was only slightly expressed in normal glomerular epithelial cells but not in mesangial cells. In the anti-Thy 1 nephritis rats, both SMemb and alpha-SM actin were most conspicuously induced in mesangial cells. However, the expression profile was shifted from alpha-SM actin to SMemb dominant pattern over the course of glomerulonephritis. The expression of SMemb was also increased in epithelial cells in this model. In the other three models, glomerular cells did not express alpha-SM actin, but did so for SMemb. In the nephrectomized and the diabetic rats SMemb was newly expressed in mesangial cells at earlier stages, but at later stages was remarkably enhanced in epithelial cells when severe glomerular hypertrophy developed. In the anti-GBM nephritis rats, SMemb expression was increased in epithelial cells. In all models examined, mesangial and epithelial expression of SMemb was confirmed by immunoelectron microscopy, and enhanced expression of SMemb mRNA in glomeruli was verified by RNase protection assay. We conclude from these results that glomerular cells change their phenotypes differently depending on various types of glomerular diseases. These phenotypic changes in glomerular cells can be revealed by the combined immunostaining for SMemb and alpha-SM actin. SMemb is especially useful to detect both mesangial and glomerular epithelial cell activation in these glomerular disease models. Understanding the functional difference and regulatory mechanisms of these cytoskeletal proteins will provide insight into the pathogenesis and progression of glomerular diseases.

We investigated whether the depressed cardiac response to adrenergic stimulation is accompanied with impaired autonomic function in mildly symptomatic patients with idiopathic dilated cardiomyopathy (DCM). Twenty-seven patients with DCM (New York Heart Association class I or II) and 7 normal control subjects underwent exercise radionuclide ventriculography and 24-h ambulatory EGG. The following frequency components of heart rate variability were calculated: the areas under the low (low frequency component [LF], 0.04 to 0.15 Hz), high (high frequency component [HF], 0.15 to 0.40 Hz), and total frequency portions of the spectrum. HF and HF% (the ratio of HF to total power) were calculated as indexes of specific vagal influences, and LF% (the ratio of LF to total power) and the ratio of LF to HF were of sympathetic tone. The left ventricular ejection fraction (LVEF) increased by more than 5% in all normal control subjects during exercise, whereas 17 (63%) of patients failed to show more than a 5% increase in LVEF. The profile of the mean hourly HF% and LF/HF showed circadian variations in normal control subjects but not in patients. The HF and HF% during sleep were significantly lower and the LF/HF during sleep was higher in patients than in normal control subjects. In patients, the LVEF during exercise minus LVEF at rest was significantly correlated with HF, LF%, and LF/HF during sleep, and with the ratios of the mean values during early morning to the mean daytime values for those spectral indexes. Our results demonstrated that mildly symptomatic patients with DCM showed an attenuated cardiac response to exercise and altered autonomic function, and their close relationship, suggesting that autonomic nervous activity contributes to cardiac desensitization in DCM.

The phenotypic change of the mesangial cell is considered to play a pivotal role in the accumulation of extracellular matrix in diabetic nephropathy. This investigation was undertaken to evaluate the expression of the various isoforms of contractile proteins in the streptozocin (STZ)-induced diabetic rat kidney and in renal biopsy specimens from patients with diabetic nephropathy. Specific antibodies to myosin heavy chain isoforms (SM1, SM2, SMemb), caldesmon, and alpha-smooth muscle actin and cDNAs for SMemb were used. Increased expression of SMemb at the mRNA and protein levels was demonstrated at 1 week after STZ administration in the rat. Both levels were increased at 4 weeks. Mesangial staining of caldesmon was observed at 4 weeks and that of alpha-smooth muscle actin at 24 weeks. Immunohistochemical mesangial staining of the contractile proteins was pronounced in patients with diabetic nephropathy in contrast to the trace mesangial staining in normal control subjects. These results indicate that the phenotypic change in mesangial cells occurs in the early stages of diabetes and that several stages in phenotypic changes may exist. Expression of the contractile protein isoforms, especially SMemb, should serve as a new marker for the subsequent glomerular hypertrophy and sclerosis.

BACKGROUND: Aortic dissection is one of the most common aortic catastrophes. Although newer diagnostic methods as exemplified by image diagnostic techniques have greatly improved the diagnosis of aortic dissection, the diagnosis is still frequently missed today because the signs and symptoms of the disease are at times obscure. A reliable biochemical diagnostic method for aortic dissection would be beneficial. METHODS AND RESULTS: A novel biochemical diagnostic method for diagnosis of aortic dissection was developed that uses an immunoassay of monoclonal antibodies to smooth muscle myosin heavy chain. A prospective study was conducted to ascertain the usefulness of the method in the diagnosis of aortic dissection. Twenty-seven patients with aortic dissection admitted within the first 24 hours after onset were enrolled. Serial assay of serum smooth muscle myosin heavy chain showed significant elevations within the first 24 hours after onset of aortic dissection, with levels exceeding 10 ng/mL, with subsequent rapid reductions. The sensitivity of the assay within the first 12 hours was 90% with a specificity of 97%. Analysis of 65 patients with acute myocardial infarction showed that the method could accurately differentiate myocardial infarction from aortic dissection. CONCLUSIONS: The immunoassay of serum smooth muscle myosin heavy chain is a rapid and reliable biochemical method in the diagnosis of aortic dissection. The potential use of the method in clinical medicine is promising.

Investigation of the molecular mechanisms that control smooth muscle cell (SMC) development and differentiation is a prerequisite in understanding the regulatory mechanisms of physiological and pathological SMC-associated vascular processes. The pluripotent murine embryonal carcinoma P19 cell, whose developmental potential resembles that of early embryonic cells, can develop into cell types derived from the neuroectoderm, mesoderm, and endoderm. In the present study, we have shown a unique strategy to enhance SMC differentiation in P19 cells. Under chemical induction of high concentrations of retinoic acid (1 micromol/L), P19 cells showed optimum differentiation into SMCs. Because the P19 cells thus induced also showed differentiation into neuronal cells, a strategy to block neuronal lineage differentiation was developed using a stable transformant antisense RNA construct against Brn-2, a neuronal lineage-specific POU-domain transcription factor; thus, by specifically inhibiting neuronal differentiation, enhanced SMC differentiation by P19 cells was attained. SMC expression was confirmed by immunohistochemical staining, RNA analysis (RNase protection assay), and protein analysis (Western blot) using SMC-specific markers (eg, SM1 and calponin) and alpha-smooth muscle actin. Our results show that the pathway of SMC differentiation may provide an in vitro system useful in the investigation of SMC regulatory mechanisms (eg, transcriptional regulation) and in the further understanding of SMC development and differentiation.