永井 良三

We measured the serum concentration of interleukin-8 (IL-8) in 56 patients within 6 hours of chest pain onset. Results show that IL-8 appears in the systemic circulation in the active inflammatory phase of coronary artery disease, and could thus be considered a marker for leukocyte-mediated tissue damage in patients with acute coronary lesions.

We evaluated the effects of oral administration of 10, 30, and 100 mg/kg/day of E4021, a type V phosphodiesterase inhibitor, on development of monocrotaline-induced right ventricular overload and medial thickening of pulmonary arteries. Right ventricular systolic pressure, mass ratio of right ventricle to left ventricle, right ventricular wall thickness, right ventricular myocardial fiber diameter, medial thickness, and smooth muscle area in pulmonary arteries were significantly less in rats that received E4021 30 and 100 mg/kg/day than in the control with monocrotaline on day 28. Myofiber diameter, medial thickness, and smooth muscle area were significantly lower in rats treated with E4021 100 mg/kg/day than in those of E4021 30 mg/kg/day. E4021 100 mg/kg/day protected development of right ventricular overload and medial thickening of pulmonary arteries.

The effect of interleukin-6 (IL-6) secreted by a human atrial myxoma in vitro was investigated in C3H female mice with acute viral myocarditis. A culture medium containing IL-6 (100 ng/ml) and IL-8 (250 ng/ml), was prepared; viral myocarditis was induced by exposure to the encephalomyocarditis virus. Mice were assigned to four groups: 1) intraperitoneal (i.p.) injection of supernatant with IL-6 and IL-8 (0.2 ml/mice) given simultaneously with virus, 500 pfu for 4 days (Group 1); 2) i.p. injection of supernatant with IL-6 starting on Day 4 for 4 days in the same manner (Group 2); 3) i.p. injection of culture medium simultaneously with the virus (Group 3); and 4) i.p. injection of PBS in the same manner (Group 4). Uninfected control mice were administered medium only (Group 5) or supernatant with IL-6 and IL-8 (Group 6) for 4 days without virus. The survival rate on Day 14 in Group 1 was 90% significantly (p < 0.01) prolonged. The ratio of heart weight-to-day weight in the Group 1 was significantly (p < 0.01) lower. Histopathological examination revealed that cardiac necrosis and cellular infiltration in Group 1 was reduced compared with Group 3. Moreover, the radio of spleen weight/body weight in Group 1 was significantly (p < 0.01) higher than that of Group 3 and of Group 4. To confirm the effect of IL-6 or IL-8, mice were treated with recombinant IL-6 to IL-8 simultaneously with virus for 4 days. IL-6 treated mice survived significantly compared with IL-8 treated mice and untreated mice. The viral titer on day 4 of IL-6 treated mice was significantly lower than IL-8 treated or untreated mice. Thus, IL-6 derived from human myxoma improved the survival of murime viral myocarditis and reduced myocardial necrosis when the myxoma-derived IL-6 was administered simultaneously with the virus, due to eliciting cellular immunity in the spleen.

This study was designed to evaluate the effects of amlodipine, a new calcium channel blocker, on the development of right ventricular hypertrophy and thickening of the media of the pulmonary arteries in a rat model of pulmonary hypertension. Pulmonary hypertension was induced in rats by administering a single injection of monocrotaline, 80 mg/kg. The oral administration of amlodipine, 3, 10, or 30 mg/kg/day, was initiated 24 hours later (day 1). On day 28 of therapy, we determined the right ventricular systolic pressure (RVSP), the mass ratio of the right ventricle (RV) to the left ventricle, the thickness of the wall of the RV, the diameter of myocardial fibers in the RV, the percent thickness of the media of the pulmonary artery, and the percent area of smooth muscle in the pulmonary arteries. The magnitude of all parameters was significantly less in the rats administered amlodipine, 30 mg/kg/day, vs. the control group given monocrotaline alone. RVSP, the percent medial thickness, and the percent smooth muscle area, were significantly lower in rats administered a dose of amlodipine, 30 mg/kg/day vs. 10 mg/kg/day. The oral administration of amlodipine, 30 mg/kg/day, inhibited the development of RV hypertrophy and medial thickening of the pulmonary arteries in rats exposed to monocrotaline significantly more effectively vs. the untreated control exposed only to monocrotaline.

A smooth muscle layer exists in the parietal endocardium. Notwithstanding previous pioneer work, little has been known on the phenotypes of these smooth muscles. In humans as in animals, smooth muscles show two distinct phenotypes, the 'synthetic' and 'contractile' states, which can be differentiated on the basis of the expression of two types of myosin heavy chain isoforms, SM1 and SM2. In this study, using SM1- and SM2-specific monoclonal antibodies, we performed immunohistochemical examinations to determine the phenotype of the endocardial smooth muscles. We report here that the endocardial smooth muscles have the contractile phenotype.

Vascular smooth muscles contain at least three types of developmentally regulated myosin heavy-chain (MHC) isoforms; SM1, SM2, and SMemb. By investigating the expression of the three MHC isoforms, we previously demonstrated in rabbits that smooth muscles proliferating in the neointima of arterio- and atherosclerotic lesions regain an "embryonic" phenotype. In the present study, we examined if neointimal cells are morphologically similar to embryonal smooth muscles and if dedifferentiation of neointimal smooth muscles is a reversible process. Vascular injury was produced in rabbits either by endothelial cell denudation of the aorta or by poststenotic dilation of the carotid artery. We have demonstrated in this study that the proliferating neointimal cells expressed SM1 and SMemb, but not SM2, indicating smooth muscles of an "embryonic" phenotype. The dedifferentiation of neointimal smooth muscles was found to be reversible; at 4 to 8 weeks after injury, a majority of the cells reexpressed both SM1 and SM2, but not SMemb. By electron microscopy, we have revealed smooth-muscle phenotypes determined by MHC isoforms to correspond to the morphologic phenotypes as an increase in membranous organelles, and a decrease in myofilaments was associated with the reexpression of SMemb. Interestingly, we also found that in the medial wall at 4 to 8 weeks after ballooning injury, a number of SM1-negative cells proliferated rapidly, replacing normal smooth muscles. These cells were negative against SM1 and SM2 but positive for SMemb. These SM1-negative cells contained abundant membranous organelles and few myofilaments. These cells did not express SM1 or SM2 even after 8 weeks postinjury. We conclude from these results that the proliferating synthetic-type smooth muscles after vascular injury are composed of SM1-positive cells that are morphologically similar to embryonal smooth muscle and that maintain ability to redifferentiate, and SM1-negative cells that contain few myofilaments and remain dedifferentiated.

We previously reported a clinical study in which (1) Probucol prevented restenosis after coronary angioplasty and (2) Pravastatin reduced the risk of restenosis associated with plasma cholesterol concentration. To examine these mechanism, we analyzed the effect of probucol and pravastatin on the intimal proliferation, the cellular makeup of lesion and the expression of PDGF after balloon injury in rabbits. Probucol study: Probucol was given orally 1.3 g/day from 2 weeks prior to carotid balloon injury to the time of killing (2 or 4 weeks after balloon injury). Probucol remarkably decreased intimal area by 70%, the number of Smooth Muscle Cell (SMC) and Proliferating Cell Nuclear Antigen (PCNA)-labeled cells in the intima. The expression of PDGF-A mRNA was markedly suppressed with probucol treatment. However, probucol did not suppress SMemb expression. SMemb is a good molecular marker for de-differentiated SMC. Probucol is effective in preventing SMC proliferation, which is possibly due to a decrease in the expression of PDGF.<br>Pravastatin study: Pravastatin was received orally 20mg/day with 0.5% cholestrol diet 2 weeks before iliac balloon injury. After 2 or 4 weeks, the rabbits were killed. Pravastatin reduced plasma cholesterol concentration, neointimal macrophage content and intimal area. The neointimal area was related to macrophage content. Moreover, macrophage content was correlated to plasma cholesterol level. Pravastatin effectively inhibits myointimal proliferation, which may be partly explained by its hypocholesterolemic activity.

In diabetic nephropathy there is accumulation of matrix proteins, Overproduction of these matrix proteins considered to be due to the phenotypic change of mesangial cell. In order to detect the phenotypic change of the mesangial cell, renal biopsy specimens from patients with diabetic nephropathy were stained with antibodies against various types of collagens and contractile-associated protein, caldesmon, Type III collagen was not stained in the glomerulus and type VI collagen showed mesangial pattern from normal controls, In diabetes, mesangial staining of type III collagen and increases in type VI collagen were observed in the mesangium. Increased mesangial staining of caldesmon was noted in the glomerulus from diabetic nephropathy in contrast to only vessel staining from normal controls. These results indicate that phenotypic changes are noted in the mesangium in diabetes. Expression of contractile-associated protein such as caldesmon, would serve as a useful marker to predict glomerulosclerosis.

We have developed a double monoclonal sandwich enzyme immunoassay to measure smooth muscle myosin heavy chain (MHC). Analytical performance of the assay showed reliable detection of smooth muscle MHC in human sera. The mean of the smooth muscle MHC level in normal human sera was 0.9 ± 0.9 ng/ml. In sera of patients with aortic dissection, the smooth muscle MHC level sharply elevated at the onset and rapidly decreased to normal levels. Immunoassay of smooth muscle MHC in serum is a promising method for biochemical diagnosis of smooth muscle disorders. © 1995.

Here we report a rare case of T-cell prolymphocytic leukemia in whom leukemic killer cells, expressing a cytolytic factor perforin, infiltrated the heart. Perforin may have directly injured myocardial cells which showed marked expression of human leukocyte antigens (HLAs) and intercellular adhesion molecule-1 (ICAM-1) as well as costimulatory molecules B7 and B70, which are ligands for CD28 expressed on T-cells. In spite of chemotherapy against leukemic cells, this autoimmune process finally caused fatal congestive heart failure.

We have previously shown that mechanical stress induces activation of protein kinases and increases in specific gene expression and protein synthesis in cardiac myocytes, all of which are similar to those evoked by humoral factors such as growth factors and hormones. Many lines of evidence have suggested that angiotensin II (Ang II) plays a vital role in cardiac hypertrophy, and it has been reported that secretion of Ang II from cultured cardiac myocytes was induced by mechanical stretch. To examine the role of Ang II in mechanical stress-induced cardiac hypertrophy, we stretched neonatal rat cardiac myocytes in the absence or presence of the Ang II receptor antagonists saralasin (an antagonist of both type 1 and type 2 receptors), CV-11974 (a type 1 receptor-specific antagonist), and PD123319 (a type 2 receptor-specific antagonist). Stretching cardiac myocytes by 20% using deformable silicone dishes rapidly increased the activities of mitogen-activated protein (MAP) kinase kinase activators and MAP kinases. Both saralasin and CV-11974 partially inhibited the stretch-induced increases in the activities of both kinases, whereas PD123319 showed no inhibitory effects. Stretching cardiac myocytes increased amino acid incorporation, which was also inhibited by approximately 70% with the pretreatment by saralasin or CV-11974. When the culture medium conditioned by stretching cardiocytes was transferred to nonstretched cardiac myocytes, the increase in MAP kinase activity was observed, and this increase was completely suppressed by saralasin or CV-11974. These results suggest that Ang II plays an important role in mechanical stress-induced cardiac hypertrophy and that there are also other (possibly nonsecretory) factors to induce hypertrophic responses.

The contractility and distensibility of renal arterioles are important in the regulation of glomerular filtration. However, little is known regarding the characteristics of contractile proteins in these arterioles. Recently it was demonstrated that vascular smooth muscles contain two types of myosin heavy chain (MHC) isoforms, SM1 and SM2, which are unique molecular markers of smooth muscle cell phenotypes. SM1 is constitutively expressed in all types of smooth muscles, whereas SM2 exists only in mature smooth muscles. We characterized the expression of MHC isoforms as well as the ultrastructural myofilament assembly of renal arteriolar smooth muscles in human, rat and rabbit by immunohistochemical techniques. SM1 and alpha-smooth muscle actin were localized in both the preglomerular vessels (including the afferent arterioles) and efferent arterioles, whereas SM2 was present only in the preglomerular vessels. Renin-producing cells in the afferent arterioles (juxtaglomerular granular cells, JG cells) were positive for alpha-smooth muscle actin but negative for SM2. When renin synthesis was stimulated, the more proximal afferent arteriolar smooth muscles turned renin-positive and SM2 disappeared. Glomerular mesangial cells did not show immunoreactivities for SM1, SM2 or alpha-smooth muscle actin. The difference in MHC isoform expression in these arterioles was also reflected by ultrastructures; the afferent arteriolar smooth muscles contained abundant myofilaments including thick filaments, whereas the efferent arteriolar smooth muscles had a few myofilaments composed only of thin microfilaments. The JG cells displayed a myofilament assembly similar to that in the efferent arteriolar smooth muscles. We conclude from these observations that smooth muscles in pre-and postglomerular arterioles, the glomerular mesangial cells and JG cells differ in phenotypes, suggesting that they may have different contractile properties which may be critically involved in the regulation of glomerular filtration.

Endothelin-1 (ET-1) is a 21-amino acid peptide with various biological activities including vasoconstriction and cell proliferation. To clarify the physiological and pathophysiological role of ET-1, we disrupted the mouse Edn1 locus encoding ET-1 by gene targeting and demonstrated that ET-1 is essential to the normal development of pharyngeal arch-derived tissues and organs. In this study, we focused on the phenotypic manifestations of Edn1-/- homozygous mice in the cardiovascular system. Edn1-/- homozygotes display cardiovascular malformations including interrupted aortic arch (2.3%), tubular hypoplasia of the aortic arch (4.6%), aberrant right subclavian artery (12.9%), and ventricular septal defect with abnormalities of the outflow tract (48.4%). The frequency and extent of these abnormalities are increased by treatment with neutralizing monoclonal antibodies or a selective ETA receptor antagonist BQ123. At an earlier embryonic stage, formation of pharyngeal arch arteries and endocardial cushion is disturbed in Edn1-/- homozygotes. In situ hybridization confirmed ET-1 expression in the endothelium of the arch arteries and cardiac outflow tract and the endocardial cushion as well as in the epithelium of the pharyngeal arches. Thus, ET-1 is involved in the normal development of the heart and great vessels, and circulating ET-1 and/or other ET isoforms may cause a functional redundancy, at least partly, through the ETA receptor.

We have previously shown that smooth muscle contains three types of myosin heavy chains: SM1, SM2, and SMemb. The present study was designed to assess how glomerular expression of mRNA for these isoforms is regulated and whether their expression is affected by enalapril treatment in diabetic rats. Animals were divided into 4 groups: (1) untreated diabetic rats; (2) enalapril-treated diabetic rats; (3) untreated control rats, and (4) enalapril-treated control rats. Enalapril treatment was continued for 24 weeks. The glomerular mRNA levels for SM1 and SM2 showed little change in all groups throughout the experimental period. In contrast, SMemb mRNA in group 1 increased significantly with age compared to levels found in untreated controls (4.6-fold higher at 4 weeks, p &lt; 0.01; 6.8-fold higher at 12 weeks, p&lt;0.01, and 10.6-fold higher at 24 weeks, p&lt;0.001). Enalapril reduced both creatinine clearance (p&lt;0.01) and urinary protein excretion (p&lt;0.01) in diabetic rats. Moreover, enalapril significantly attenuated the increase in the glomerular SMemb mRNA level in diabetic rats (the difference between treated and untreated rats was significant at p&lt;0.01 from week 4 to 24). However, enalapril had no effect on SMemb mRNA levels in controls. These data suggest that SMemb is a molecular marker for phenotypic alteration and that the beneficial effect of enalapril on proteinuria and renal function may be, at least in part, associated with reducing SMemb mRNA expression in diabetic glomeruli.

1. We investigated the glomerular expression of three types of myosin heavy-chain isoforms, including S-myosin heavy-chain 40 (SM1), S-myosin heavy-chain 29 (SM2) and FS-myosin heavy-chain 34 (SMemb) in puromycin aminonucleoside nephrosis. 2. There was little change in SM1 and SM2 mRNA levels throughout the experiment. In contrast, glomerular SMemb mRNA increased on days 2 and 4 (before and soon after the onset of proteinuria, respectively), but declined on day 8 (the peak of proteinuria). 3. Histological myosin heavy-chain expression was examined using three antibodies against SM1, SM2 and SMemb. Immunohistochemically, SM1 and SM2 were absent in the glomeruli associated with puromycin aminonucleoside 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 puromycin aminonucleoside nephrosis. In the puromycin aminonucleoside-treated rats, the number of SMemb-positive glomerular cells increased on days 2 and 4. 4. We examined whether levels of alpha-smooth-muscle actin or proliferating cell nuclear antigen correlated with myosin heavy-chain levels in the glomeruli of rats with puromycin aminonucleoside nephrosis. None of the cellular components in the glomeruli was positive for either alpha-smooth-muscle actin or proliferating cell nuclear antigen in puromycin aminonucleoside nephrosis. 5. Administration of methylprednisolone to puromycin aminonucleoside-treated rats resulted in the rapid disappearance of proteinuria. However, methylprednisolone did not affect SMemb mRNA or immunostaining in a glomeruli of rats with puromycin aminonucleoside nephrosis.(ABSTRACT TRUNCATED AT 250 WORDS)

We have previously shown that stretching cardiac myocytes evokes activation of protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), and 90-kD ribosomal S6 kinase (p90(rsk)). To clarify the signal transduction pathways from external mechanical stress tb nuclear gene expression in stretch-induced cardiac hypertrophy, we have elucidated protein kinase cascade of phosphorylation by examining the time course of activation of MAP kinase kinase kinases (MAPKKKs), MAP kinase kinase (MAPKK), MAPKs, and p90(rsk) in neonatal rat cardiac myocytes. Mechanical stretch transiently increased the activity of MAPKKKs. An increase in MAPKKKs activity was first detected at 1 min and maximal activation was observed at 2 min after stretch, The activity of MAPKK was increased by stretch from 1-2 min, with a peak at 5 min after stretch, In addition, MAPKs and p90(rsk) were maximally activated at 8 min and at 10 similar to 30 min after stretch, respectively, Raf-1 kinase (Raf-l) and (MAPK/extracellular signal-regulated kinase) kinase kinase (MEKK), both of which have MAPKKK activity, were also activated by stretching cardiac myocytes for 2 min, The angiotensin II receptor antagonist partially suppressed activation of Raf-1 and MAPKs by stretch, The stretch-induced hypertrophic responses such as activation of Raf-1 and MAPKs and an increase in amino acid uptake was partially dependent on PKC, while a PKC inhibitor completely abolished MAPK activation by angiotensin II. These results suggest that mechanical stress activates the protein kinase cascade of phosphorylation in cardiac myocytes in the order of Raf-1 and MEKK, MAPKK, MAPKs and p90(rsk), and that angiotensin II, which may be secreted from stretched myocytes, may be partly involved in stretch-induced hypertrophic responses by activating PKC.

We immunocytochemically stained the bovine brain with monoclonal antibodies against 155 kDa MLCK and myosin heavy chain (MHC) and a polyclonal antibody against calmodulin. In the bovine brain, a strong immunoreactivity with the anti-MLCK antibody was observed in neurons in all layers of the cerebral cortex and basal ganglia, and astrocytes in the white matter. The antibody also stained Purkinje, granular and molecular cells in the cerebellum. An anti MHC antibody stained neurons and astrocytes in the cerebral cortex and Purkinje cells similarly to the anti-MLCK antibody, whereas the MHC immunoreactivity was detected in the cerebellar glomerulus and there was no immunostaining of MHC in the granular and outer stellate cells. These results suggest that the 155 kDa MLCK might be related not only to the Ca-calmodulin-myosin system but also other intracellular system.

圧負荷や炎症による障害を受けた心筋は心筋細胞形質を変換し,収縮装置やカルシウム制御蛋白の分子種のスイッチングや量を変化させる.収縮装置の変化としては,心筋ミオシンのα アイソフォームからβ アイソフォームへの変換,カルシウム制御機構の変化としては,粗面小胞体のCa2+-ATPaseの遺伝子発現の減弱(カルシウムポンプ数の減少)などがあげられる.このような変化を起こす細胞内シグナルとして,リン酸化カスケードや心筋内レニン・アンギオテンシン系の役割が注目されており,in vitro心筋細胞ストレッチングによる細胞内情報伝達系の解析,あるいは高血圧自然発症ラットの心肥大に対するアンギオテンシン変換酵素阻害薬やアンギオテンシンII受容体拮抗薬の効果などから,解明されてきた.

Homeobox-containing genes play critical roles in regulating tissue-specific gene expression essential for tissue differentiation, as well as determining the temporal and spatial patterns of development. Recently, a human cardiac homeobox-containing gene, CSX, has been isolated. CSX is abundantly expressed in the human heart from fetal stages, suggesting that CSX plays an important role in human heart formation. In the present study, we have determined the chromosomal localization of CSX by fluorescence in situ hybridization techniques and systemic screening of a yeast artificial chromosome library using polymerase chain reaction. By these methods, CSX was mapped to 5q34 of human chromosome 5 near the boundary of 5q34 and 5q35. In this region, another homeobox-containing gene MSX2, which is expressed in various tissues including the conduction system of the developing heart, has been assigned. Localization of CSX and MSX2 to the same region of the human chromosome suggests that these genes may be coordinately regulated during human heart formation.

Mechanical stress induces cardiac hypertrophy and expression of specific genes in the cardiac myocytes. External stimuli are generally transduced into the nucleus through the activation of a protein kinase cascade. We have previously shown that stretching cardiomyocytes stimulates the activity of protein kinase C (PKC), mitogen-activated protein (MAP) kinase and S6 protein kinase. In the present study, we examined two other kinases, Raf-1 kinase and MAP kinase kinase, which are supposed to lie between PKC and MAP kinase in the protein kinase cascade. Stretching cardiocytes by using the in vitro system induced hyperphosphorylation of Raf-1 kinase and activation of MAP kinase kinase. The protein kinases activated by mechanical stress are similar to those activated by growth factors. We examined the possible involvement of angiotensin II (Ang II) in the protein synthesis and gene expression induced by mechanical stress. CV11974, an Ang II-receptor antagonist, partially suppressed the increases in amino acid incorporation, c-fos gene expression and MAP kinase activity induced by stretching. These results suggest that a variety of protein kinases are activated by mechanical stress and that locally produced Ang II may in part play important roles in converting mechanical stimuli into biochemical signals.