永井 良三

It is known that mechanical stress directly changes the conformation of the functional proteins, or directly activates enzymes such as phospholipase in the plasma membrane. The integrin-cytoskeleton complex may be an alternative candidate structure for a mechanoreceptor and a transducer. The cytoskeleton has been also shown to play an important role in secretion. Mechanical stress may stimulate the secretion of some cytokines or angiotensin II, which may generate multiple intracellular signals as a secondary event. External stimuli are generally transduced into the nucleus through the activation of protein kinase cascade. Stretching of cardiac myocytes stimulates the activity of PKC, Raf-1 kinase, MAP kinase kinase. MAP kinase and S6 kinase. In cardiac myocytes, mechanical stress directly induces gene expression as well as protein synthesis. Immediate early genes are first induced, and then fetal-type genes are reinduced. Both in hypertrophied hearts and in the experimental model of cardiac hypertrophy induced by pressure overload. Ca(2+)-ATPase content of cardiac myocytes is depressed. Reduced function of sarcoplasmic reticulum causes insufficient decrease of intracellular calcium in diastole and induces slowing of ventricular relaxation. In the interstitium of pressure overloaded hearts, the accumulation of collagen fiber is increased. The abnormal deposit leads to increased chamber stiffness and diastolic dysfunction. Furthermore, TGF-beta and tissue renin-angiotensin system are up-regulated in pressure overloaded hearts, both of which accelerate the interstitial fibrosis.

エンドセリン(ET)-1遺伝子欠損マウスより血管内皮細胞を培養した.ET-1を欠損した血管内皮細胞は正常の形態を呈し,アセチルLDLの取り込みも認めた.血清存在下でのET-1欠損血管内皮細胞の増殖は正常の血管内皮細胞と変化なかった.ETアイソフォームの代償的な発現はなかった.bigET-1からET-1への変換もその程度に差はなかった.これらの結果より,ET-1は血管内皮の形態,増殖,ET-1のプロセッシング機構の発現にとっては必須ではなく,ETアイソフォームは血管内皮細胞ではredunduntな発現はないことが示唆された

Smooth muscle myosin heavy chains (MHC) exist in multiple isoforms. Rabbit smooth muscle contain at least three types of MHC isoforms; SM1 (204 kDa), SM2 (200 kDa) and SMemb (200 kDa). SM1 and SM2 are specific to smooth muscle, but SMemb is a nonmuscle-type MHC abundantly expressed in the embryonic aorta and in activated mesenchymal cells. We previously reported that these three MHC isoforms are differentially expressed in rabbit during normal vascular development and in experimental arteriosclerosis and demonstrated that MHC isoforms are excellent markers for smooth muscle phenotype. In order to clarify the clinical significance of MHC isoforms, this article will focus on the expression of smooth muscle MHC isoforms in normally developing and atherosclerotic human arteries, especially in coronary arteries. We recently isolated and characterized three cDNA clones encoding human SM1, SM2, and SMemb. The expression of SM2 mRNA in the human fetal aorta was significantly lower as compared to SM1 mRNA but the ratio of SM2- to SM1-mRNA was increased after birth. SMemb mRNA in the aorta was decreased after birth. Immunohistologically, SM1 was constitutively positive from the fetal stage to adulthood in the apparently normal media of the aorta and coronary arteries, whereas SM2 was not detected in fetal arteries of early gestational stage. SM2 was recognized in well-differentiated smooth muscle after perinatal stage. In the human aorta or coronary arteries, unlike in rabbit, SMemb was detected even in the adult. Mild diffuse intimal thickening in the major coronary arteries of the young was found to be composed of smooth muscle cells, reacting equally to three antibodies for MHC isoforms. In thickened but non-atheromatous intima, the expression of well-differentiated smooth muscle-specific MHC (SM2) was reduced, especially in the deeper layer. With progression of atherosclerosis, intimal smooth muscle diminished the expression of not only SM2 but also SM1, whereas alpha-smooth muscle actin was well preserved. We conclude from these results that smooth muscle MHC isoforms are important molecular markers for studying human vascular smooth muscle cell differentiation as well as the cellular mechanisms of atherosclerosis.

Contraction of the pre- and postglomerular arteries plays an important role in the regulation of glomerular blood flow. Mesangial cells may also be involved in the mechanism of this regulation, but it has not been clarified yet whether or not mesangial cells and vascular smooth muscles show an identical phenotype, especially in terms of their contractile proteins. In this study, in order to elucidate any difference in the cellular phenotypes between mesangial cells and renal vascular smooth muscles, we investigated the localization of myosin heavy chain isoforms using a monoclonal antibody against SM1 and SM2. Both SM1 and SM2 are specific to smooth muscles. SM1 is specifically expressed in smooth muscles from early development and SM2 appears after birth. In normal renal tissues, SM1 and SM2 were expressed only in the smooth muscle cells of the arterioles and small arteries. However, glomerular cells, including mesangial cells, were not stained with either anti-SM1 antibody or anti-SM2 antibody. Localization of SM1 and SM-2 was similar to that of α-smooth muscle actin (SM α-actin). Staining for SM-1 was not observed in the mesangial areas of renal tissues with glomerular disease. These results clearly indicate that mesangial cells have a different phenotype from that of vascular smooth muscle cells in terms of their contractile proteins. © 1995, Japanese Society of Nephrology. All rights reserved.

The Na(+)-H+ antiporter is a unique transmembrane protein with multiple roles in cellular functions through intracellular alkalization. It participates in the regulation of intracellular pH, cell volume and intracellular signalling in response to various mitogenic stimuli. To clarify its role as a subcellular signal in cardiovascular remodeling like vascular hyperplasia or cardiac hypertrophy, we determined mRNA levels of the Na(+)-H+ antiporter isoform, NHE-1, in vascular smooth muscles and pressure-overloaded hearts in rabbits. The NHE-1 mRNA levels in rabbit aortas and hearts were developmentally regulated with high levels at embryonic and neonatal stages than in adults. In primary-cultured smooth muscle cells (SMC), the mRNA levels were increased during exponential growth, but decreased to initial levels at confluency. Growth of a mutant SMC line, C5, which is deficient in Na(+)-H+ antiporter activity, was markedly reduced in bicarbonate-free medium. However, when the activity was restored by transfecting cells with a full-length NHE-1 cDNA in an expression vector, the growth rate of C5 was accelerated again. After balloon injury to the vascular wall, the NHE-1 mRNA levels of the injured arteries were also increased, suggesting that Na(+)-H+ antiporter contributes to the network of the growth promoting systems in smooth muscle cells in vivo. Pressure-overload on the ventricle increased the NHE-1 mRNA levels in hearts approximately two-fold of sham-operated rabbits after 3 days and remained for at least two weeks (P < 0.05). We further demonstrated that 3-methylsulfonyl-4-piperidino-benzoyl guanidine mesylate (Hoe 694), a potent antagonist of Na(+)-H+ antiporter, partially inhibited stretch-induced activation of mitogen-activated kinase (MAP kinase) in the cultured cardiomyocytes. From these results, we conclude that activation of the Na(+)-H+ antiporter and its gene expression is involved in molecular mechanisms of both cardiac hypertrophy and vascular smooth muscle cell proliferation, indicating a potential target in developing new therapeutics for cardiovascular diseases.

Essential hypertension is one of the most common diseases that exacerbate the risk of cardiovascular or cerebrovascular attacks. Although the etiology of essential hypertension remains unclear, recent investigations have revealed that an enhancement of Na(+)-proton (Na(+)-H+) exchange activity is a frequently observed ion transport abnormality in hypertensive patients and animal models. To test the hypothesis that increased Na(+)-H+ exchange causes hypertension, we produced transgenic mice overexpressing Na(+)-H+ exchanger and analyzed their Na+ metabolism and blood pressure. Urinary excretion of water and Na+ was significantly decreased in transgenic mice, and systolic blood pressure was elevated after salt loading. The impaired urinary excretion of Na+ suggested that the Na(+)-H+ exchanger overexpressed in the renal tubules increased reabsorption of Na+, which caused a blood pressure elevation by Na+ retention after excessive salt intake. Our results demonstrate that overexpression of Na(+)-H+ exchanger can be a genetic factor that interacts with excessive salt intake and causes salt-sensitive blood pressure elevation.

Effective activation of T cells in immune responses depends on two signals, one from the CD3/TCR complex and another from accessory cell surface proteins. Very late Ag-4 (VLA-4) can exert a costimulatory function upon binding with vascular cellular adhesion molecule-1 (VCAM-1). We investigated the effects of mAbs against VCAM-1 and VLA-4 on cardiac allograft survival and humoral response to soluble Ags. BALB/c hearts were transplanted into C3H/He recipients. mAbs (100 micrograms/day) were administered i.p. for the first 6 days. Graft survival in mice treated with M/K-2 (anti-VCAM-1; median survival: 20 days, n = 6) and those treated with PS/2 (anti-VLA-4; 30 days, n = 6) was greater than that in control mice (8 days, n = 7). Eight of 18 mice treated with both M/K-2 and PS/2 accepted the grafts over 65 days, and five of them accepted the grafts over 100 days. Allografts treated with the two mAbs showed scattered infiltration of leukocytes without evidence of active rejection at 65 days. Mice with long-surviving cardiac grafts were challenged with skin grafts from donor and third-party (C57BL/6) strains. Survival of the donor-type skin was significantly greater than that of the third-party skin. One mouse specifically accepted the donor-type skin indefinitely (> 150 days). FACS analysis of splenocytes at 55 days after transplantation showed complete recovery of VLA-4 expression from a down-regulation observed at day 7. In addition, mice immunized with heat-aggregated human gamma-globulin did not produce specific Ab, even after boost immunization, if PS/2 was administered at the time of the first immunization. This unresponsiveness to xenogeneic protein lasted for more than 50 days. These results indicate that in vivo administration of anti-VCAM-1 and anti-VLA-4 mAbs induces specific immunosuppression to cardiac allografts and soluble Ags.

Objectives: To elucidate the regulation of cardiac gene expression by mechanical stress and to analyse molecular mechanisms associated with the involvement of angiotensin II (Ang II) in the development of cardiac hypertrophy and dysfunction. Methods: Neonatal rat cardiocytes were cultured in deformable silicone dishes, and mechanical stress was imposed on the cardiocytes by stretching them. In in vivo studies, spontaneously hypertensive rats (SHR) were treated with a non-peptide, specific Ang II type 1 receptor antagonist, TCV 116. Results: Expression of c-fos was rapidly induced, and fetal type genes such as skeletal alpha actin and beta myosin heavy chain genes were re-expressed by stretching. The mechanical stress decreased the expression of Ca2+-ATPase in the sarcoplasmic reticulum. With regard to signals for the development of cardiac hypertrophy, mechanical stress evoked c-fos expression via the activation of protein kinase C. The phosphorylation cascade (sequential activation of protein kinase C, Raf-1 kinase, mitogen-activated protein kinase kinase, mitogen-activated protein kinase and S6 kinase), which may be involved in protein synthesis and gene expression, was activated by mechanical stress in cardiocytes. Stretch-induced cardiac cellular hypertrophy was partially inhibited by TCV 116. TCV 116 treatment of SHR reduced left ventricular weight, left ventricular wall thickness, myocyte transverse diameter, V-3 myosin heavy chain levels and the interstitial collagen volume fraction. Conclusions: These results indicate that Ang II may, in part, mediate the stretch-induced hypertrophic growth of cardiomyocytes via the type 1 Ang II receptor.