永井 良三

UNLABELLED: Impaired cardiac sympathetic activity can be evaluated by 123I-metaiodobenzylguanidine (MIBG) imaging. METHODS: We studied the significance of MIBG imaging for 24 patients (age 58+/-12 y) with dilated cardiomyopathy (DCM). We compared 12 patients (group A) treated with metoprolol (dose from 30-60 mg/d) with 12 patients treated with angiotensin-converting enzyme (ACE) inhibitors. Patients were studied before treatment, after 5 mo of treatment (only in group A) and after 1 y of treatment. Cardiac MIBG uptake was assessed as the heart-to-mediastinum activity ratio (H/M) and total defect score (TDS) from anterior planar and SPECT MIBG images, which were acquired in 4 h after tracer injection. New York Heart Association (NYHA) class and left ventricular ejection fraction (LVEF) calculated by echocardiography were also assessed. RESULTS: TDS decreased in both groups (in group A, from 30+/-7 through 23+/-9 to 18+/-10; P < 0.01, in group B, from 30+/-6 to 24+/-8; P < 0.01) and H/M was increased in both groups (in group A, from 1.87+/-0.31 through 2.03+/-0.28 to 2.14+/-0.29; P < 0.01, in group B, from 1.82+/-0.28 to 1.94+/-0.26; P < 0.05). But TDS and H/M were more improved in group A than in group B (P < 0.05). LVEF was significantly increased in only group A (from 38+/-6 through 43+/-8 to 49%+/-9%; P < 0.01). NYHA improved in both groups (in group A, from mean 2.5 through 2.1 to 1.8; P < 0.01, in group B, from mean 2.6 to 2.1; P < 0.05) but was more improved in group A than in group B (P < 0.05). CONCLUSION: Cardiac function, symptom and cardiac sympathetic activity evaluated by MIBG images improved after the beta-blocker therapy more than with the treatment that used ACE inhibitors.

Basic research has provided strong evidence that oxidation of LDL plays an important role in the progression of atherosclerotic vascular disease. The levels of plasma oxidized two-density lipoprotein (ox-LDL) were measured by sandwich ELISA using the monoclonal antibody (DLH3) recognized oxidatively modified lipoproteins and the anti-human apolipoprotein B monoclonal antibody in healthy subjects and patients with coronary heart disease (CHD). Plasma ox-LDL could be detected in normal subjects and patients with CHD. No sex-related difference was observed in normal subjects. The levels of ox-LDL in the forties and fifties were higher than those in the thirties and twenties. The levels of plasma ox-LDL were significantly higher in patients with CHD than in controls. There was no difference between the levels of ox-LDL of patients with single vessel disease and those with multivessel disease. These results suggest that elevated levels of oxidized LDL may be a marker for CHD.

The prognosis for patients with idiopathic dilated cardiomyopathy (DCM) is poor, although clinical features are variable. Prediction of outcome has been difficult in individual patients based on laboratory data. In some patients with DCM, myocardial damage secondary to viral or immune-mediated myocardial inflammation may persist. To objectively assess inflammation, we measured plasma concentrations of C-reactive protein (CRP) in 188 patients with idiopathic DCM over 5-8 years. All had dyspnea and fatigue at rest; all patients had a left ventricular ejection fraction less than 40% by echocardiography or by contrast or radionuclide ventriculography. We divided these patients into two groups: patients dying within 5 years following admission (n = 49) and the remainder surviving for at least 5 years (n = 139). CRP concentrations in the patients dying early were significantly higher than in the long-term survivors (1. 05 +/- 1.37 vs. 0.49 +/- 1.04 mg/dl, p < 0.05). Sixty-two percent of the patients with CRP>1.0 died within 5 years. In addition to other laboratory tests including electrocardiography and echocardiography, routine CRP measurements proved to be valuable for identifying high-risk patients who require special treatment strategies.

We report a successful resection of an inflammatory aneurysm following treatment with steroids in a 23-year-old man. Suffering from fever and severe lumbago, he was admitted to our hospital. An ultrasound and computed tomography of the abdomen revealed an infrarenal abdominal aortic aneurysm surrounded by dense perianeurysmal fibrous tissue. We diagnosed it as an inflammatory abdominal aortic aneurysm based on a symptomatic inflammatory reaction and the findings of ultrasound and computed tomography. Since the aneurysmal wall strongly adhered to the surrounding tissues and surgery was ruled out when it proved impossible to expose the vessels sufficiently to obtain vascular control, steroid therapy was started to control fever and severe lumbago. Five months later, we undertook surgery. Our conclusion is that steroid therapy was very effective against a young patient with inflammatory abdominal aortic aneurysm.

The role of lymphocytes in the pathogenesis of viral myocarditis is controversial. To better understand how lymphocyte maturation controls a virus-induced myocarditic process, a murine model of viral myocarditis was utilized. Encephalomyocarditis virus (EMCV) was inoculated intraperitoneally into three kinds of mice; virus-susceptible C57BL/6, virus-resistant 129/SV and recombination activity gene (RAG)-2 knockout 129/SV mice. The RAG2 participate in the maturity of T and B lymphocytes. Survival rate, heart weight (HW), HW to body weight (BW) ratio, viral genome, cardiac inflammation and myocardial necrosis were evaluated after EMCV (500 plaque forming unit/mouse) inoculation. On post-inoculation day 10, the survival rate of C57BL/6, 129/SV and RAG2 knockout mice were 42, 90 and 0%, respectively. Myocardial viral titer was significantly (P<0.05) higher in C57BL/6 and RAG2 knockout mice than in 129/SV mice. In situ hybridization demonstrated the EMCV genome in the myocardium of RAG2 knockout and C57BL/6 mice, but not in 129/SV mice. At day 8, HW and HW/BW ratios were elevated (P<0.05) in RAG2 knockout mice as well as C57BL/6 mice compared with 129/SV mice. Myocardial necroses were more severe in RAG2 knockout mice than in wild-type 129/SV mice. In conclusion, matured lymphocytes protect the development of viral myocarditis which includes viral replication and myocardial apoptosis.

We report our experience with a patient whose mediastinal lymphadenopathy resolved after resection of a cardiac myxoma that secreted interleukin-6 (IL-6). The patient was a 68-year-old female who complained of nocturnal chest discomfort related to congestive heart failure. An echocardiogram demonstrated a large left atrial mass. A computed tomogram showed not only the left atrial mass but multiple enlarged mediastinal lymph nodes. The serum IL-6 level was markedly elevated at 13.7 pg/ml. After resection of the cardiac myxoma, serum IL-6 returned to the normal range. A repeat computed tomogram showed no mediastinal lymphadenopathy. We believe that overproduction of IL-6 by the cardiac myxoma was the cause of the mediastinal lymphadenopathy.

The tumor necrosis factor (TNF) alpha level is elevated in patients with advanced heart failure, and the phosphorylation of contractile regulatory proteins is reduced in the human heart. We hypothesized that TNFalpha affects the phosphorylation of proteins involved in regulating contraction; phospholamban (PLB), myosin light chain 2 (MLC2) and troponin I (TnI). Spontaneously beating rat neonatal cardiac myocytes, prelabelled with [32P]orthophosphate, were treated with TNFalpha for 30 min, and stimulated with isoproterenol for 5 min. 32P-labelled myofibrillar proteins were isolated by 15% SDS-PAGE. Baseline phosphorylation levels of PLB, TnI and an unknown 23kDa phosphoprotein were decreased by TNFalpha in a dose-dependent manner. Moreover, TNFalpha attenuated the phosphorylation levels of PLB and TnI increased by a concentration of 0.01 microM isoproterenol, but not by 1 microM of isoproterenol. Although TNFalpha had no effect on the cAMP content or cAMP-dependent protein kinase activity in the presence or absence of isoproterenol, an inverse relationship was observed between the concentration of TNFalpha and the cGMP content in cardiac myocytes, and treatment with TNFalpha resulted in a concentration-dependent increase in type 2A protein phosphatase activity. The observation that TNFalpha decreases phosphorylation levels of PLB and TnI in cardiac myocytes suggests that the reduction of these protein phosphorylation levels is partially responsible for alterations of intracellular Ca2+-cycling and the force of contraction in TNF alpha-treated cardiac myocytes. Furthermore, TNFalpha reduces myocyte contraction and protein phosphorylation states possibly via cAMP-independent mechanisms, at least in part, by the activation of type 2A protein phosphatase.

While a beneficial effect of hyperthermia on viral infection has been hypothesized, there are no data on viral myocarditis in vivo. To investigate whether hyperthermia might attenuate the course or severity of viral myocarditis, we studied the pathological changes in a murine model of viral myocarditis. C3H mice were inoculated i.p. with the encephalomyocarditis virus (500 pfu). They were anesthetized and heated to a body temperature of 42.5+/-0.2 degrees C for 30 min. The latter was performed 4 hr before (n=28, HB) or 4 hr after (n=28, HA) the viral inoculation; results were compared with nonheated, infected controls (n=30, Cont). Cardiac viral titers were recorded on day 3, and the body weight (BW), heart weight (HW) and pathological changes were recorded on days 5 and 10. The incidence of spontaneous mortality on day 10 was significantly higher in the HA group (all deaths occurring by day 7 post-inoculation) as compared with the HB (35%) or Cont (18%) groups. Viral titers in the HA group (n=4) were significantly (P<0.05) higher than those in the Cont (n=7) or HB (n=7) groups (4.11+/-0.54 vs 3.01+/-0.44 and 3.23+/-0.45 LogTCID50/mg, respectively). On day 5, the HW, the BW/HW ratio, and the severity of myocardial necrosis were all significantly higher in the HA than in the Cont and HB groups. To confirm the effect of hyperthermia on the expression of heart shock protein (HSP), immunohistochemical staining was done in the virus-infected hearts. The nucleus and cytoplasm of the injured myocardium in the HA group strongly expressed HSP70, whereas the HB and Cont groups were negative for this protein. In conclusion, induction of hyperthermia after viral inoculation aggravated the viral-induced myocardial necrosis and increased the mortality rate in a murine model of viral myocarditis and induced myocardial heat shock protein 70.

The purpose of this study was to examine the role of superoxide anions in modulating the vascular tone. The effects of unmodified and lecithinized superoxide dismutase (SOD) on vascular tone were determined in aortic ring preparations of mice. In lecithinized SOD, 4 molecules of a phosphatidylcholine derivative were covalently bound to each dimer of recombinant human copper-zinc SOD to facilitate tissue accumulation. Unmodified SOD did not change vascular tone. However, lecithinized SOD induced dose-dependent vasodilation of aortic ring preparations. The pretreatment with NG-nitro-L-arginine methylester (L-NAME) 10(-4) mol/L abolished the vasodilation induced by lecithinized SOD. The results of this study indicate that superoxide anions play a prominent role in modulating the vascular tone by enhancing the breakdown of nitric oxide.

The degree of involvement of the renin-angiotensin system in endothelial dysfunction was investigated by using a one-kidney, one-clip (1K,1C) model of renal hypertension. Male Wistar rats received 0.02% enalapril, 0.02% losartan, or tap water for 1 day before and for 48 h after the induction of renal artery stenosis or sham operation. The aorta of 1K,1C rats showed increased contraction and decreased relaxation responses produced by norepinephrine and acetylcholine, respectively, vs. control responses. Exposure to 10(-5) mol/l NG-monomethyl-L-arginine acetate augmented the contractile responses to norepinephrine to a greater extent in control rats than in the 1K,1C rats. The increased contraction and decreased relaxation responses to these agonists in the 1K,1C rats were normalized by enalapril or losartan. The addition of HOE-140 to the bath did not alter these normalized responses. Results suggest that angiotensin II causes endothelial dysfunction and reduces nitric oxide levels in 1K,1C rats. Such endothelial dysfunction enhanced the norepinephrine-induced contraction during the early-stage hypertension in 1K,1C rats.

We investigated the ability of the ETA receptor antagonist T-0115 and the angiotensin-converting enzyme (ACE) inhibitor imidapril hydrochloride to prevent hypertensive complications induced in rats by chronic inhibition of nitric oxide (NO). Male Wistar rats were given distilled water (control), N-G-nitro L-arginine methyl ester (L-NAME) 500 mg/l, or L-NAME plus imidapril 10 mg/l in the drinking water. In rats treated with L-NAME 500 mg/l plus T-0115, T-0115 was given in the food at a dose of 0.2 mg/g food or 0.6 mg/g food. We then collected 24-h urine samples at 2, 4, and 6 wk, obtained blood samples at 6 wk, and histologically examined the kidney and heart, L-NAME markedly reduced the levels of NO metabolites in serum and urine while increasing the tail-cuff blood pressure, the urinary albumin level (1.90 +/- 0.65 us. 0.05 +/- 0.02 mg/d/100 g in control), and the area of the left ventricular wall (83.3 +/- 3.0 cs. 69.8+/-1.8 mm(2) in control). The plasma renin activity was significantly higher in rats treated with L-NAME than in the control rats. The concomitant administration of T-0115 0.6 mg/g food with L-NAME ameliorated the tail-cuff pressure and the albuminuria (0.56+/-0.23 mg/d/100 g), although to a lesser extent than the changes seen with imidapril 10 mg/l. T-0115 0.6 mg/g food prevented left ventricular hypertrophy as effectively as imidapril 10 mg/l (70.8 +/- 1.8 with T-0115 vs. 68.3 +/- 2.7 mm2 with imidapril). Chronic inhibition of NO synthesis produced left ventricular hypertrophy and nephrosclerosis. Our results demonstrate that inhibition of the renin-angiotensin system merely effectively prevents nephrosclerosis than does the blockade of ETA receptors in a model of hypertension induced by chronic NO blockade. However, inhibition of the ET-1 pathway appeared to be as effective as ACE inhibitors in preventing left ventricular hypertrophy in this model.

Smooth muscle cells (SMCs) in the atherosclerotic intima characteristically differ from those in the arterial media, for example, by reduced expression of SMC differentiation/maturation markers such as smooth muscle myosin heavy chain isoforms (SM1 and SM2). This study tested the hypothesis that lipid lowering promotes maturation of intimal SMCs in 33 rabbits subjected to balloon injury and cholesterol feeding (0.3%) for 4 months (Baseline group, n=15); some of which then were switched to a low-cholesterol diet for 8 months (Low group at 8 months, n=3) or 16 months (Low group at 16 months, n=10). The remaining rabbits continued to consume a high-cholesterol diet for 16 months (High group, n=5). We monitored SMC phenotype by expression of immunoreactive alpha-smooth muscle actin, SM1, and SM2. alpha-Actin is an early marker, and SM1 and SM2 are late markers for SMC differentiation/maturation. Only fully differentiated or mature SMCs express SM2. Data are reported as the percentage of the alpha-actin-positive intimal area occupied by smooth muscle myosin-positive SMCs determined by color image analysis of immunostained sections. Levels of SM1 and SM2, highly expressed by SMCs in the normal aortic media (n=5) decreased in the aortic intima of the Baseline and High groups, indicating a less mature phenotype. In contrast, SM1 and SM2 increased in the Low (16 months) group, indicating that intimal SMCs exhibit a more mature phenotype after lipid lowering. Electron microscopy also showed the presence of mature intimal SMCs with abundant myofilaments. Furthermore, lipid lowering reduced levels of platelet-derived growth factor-B in the arterial intima, a factor known to suppress smooth muscle myosin expression. These data demonstrate that lipid lowering favors accumulation of mature SMCs in the atherosclerotic intima in association with reduced levels of platelet-derived growth factor-B expression. Intimal SMCs in the Low group also displayed reduced expression of matrix metalloproteinases-3 and -9 compared with the Baseline and High groups. These findings shed new light on the effects of lipid lowering at the level of the vascular wall, which may influence the biology of the atheroma.

We have recently identified a novel gene, termed klotho that is involved in the suppression of several aging phenotypes, The gene encodes a membrane protein that shares sequence similarity with the p-glucosidases of bacteria and plants. In this study, we isolated rat klotho cDNA and examined its tissue distribution in rats. The deduced amino acid sequence of rat Klotho protein was 1014 amino acids in length and 94 and 85% homologous to those of mouse and human Klotho proteins, respectively. Northern blot analysis using the rat klotho cDNA probe identified a single transcript of 5.2 kb in size expressed predominantly in the kidney, while RT-PCR detected low levels of expression also in the brain, lung, intestine, and ovaries. During development, klotho expression in the kidney was markedly augmented after birth. Chromosomal localization of rat klotho was mapped to 12q12, Northern blot analysis showed that expression of klotho was markedly decreased by Lipopolysaccharide (LPS) in vivo suggesting that expression of klotho is affected by acute inflammatory stress. The present study leads to a better understanding of the physiologic and pathophysiologic roles Of Klotho. (C) 1998 Academic Press.

In heart failure with low cardiac output, exercise tolerance is reduced despite modulated regional blood distribution and oxygen extraction. However, low cardiac output does not necessarily lead to reduced exercise tolerance especially during mild exercise. In the present study, in order to understand the mechanisms regulating exercise tolerance in heart failure, we measured oxygen consumption ((V) over dotO(2)) and cardiac output (CO) during both mild and intense exercise. Patients with heart failure were divided into 2 groups; group L (n=8) consists of patients with low anaerobic threshold (AT) &lt;13 ml/min per kg and group H (n=7) consisting of patients with AT &gt;13 ml/min per kg. At rest, (V) over dotO(2) was Similar between groups L and H, whereas CO was lower in group L than in group H (3.5+/-0.3 vs 4.8+/-1.4ml/min, p&lt;0.01). Increase in (V) over dotO(2) during warm-up exercise was not significant between the 2 groups (7.4+/-0.5 (group L) vs 6.2+/-0.3 ml/min per kg (group H), ns), but increase in CO was lower in group L than in group H (2.5+/-0.6 vs 3.4+/-0.4 ml/min, p&lt;0.01). After warm-up to the AT point, however, the increase in not only (V) over dotO(2) but also CO was markedly reduced in group L than in group H ((V) over dotO(2): 0.5+/-0.4 vs 3.7+/-0.8 ml/min per kg, p&lt;0.01, CO: 0.2+/-0.3 vs 1.1+/-0.3 L/min, p&lt;0.01). Based on these measurements, we calculated the arteriovenous oxygen difference (c(A-V)O-2 difference) during exercise in individual patients using Fick's equation. The c(A-V)O-2 difference was markedly increased in severe heart failure during the warm-up stage, but between the end of warm-up and the AT point, it remained at the same level as that of group H. These results suggest the presence of a unique mechanism regulating the c(A-V)O-2 difference in severe heart failure patients, activation of which may, at least during mild exercise, contribute to efficient oxygen delivery to the peripheral tissues thus compensating for the jeopardized exercise tolerance in those patients.

To elucidate the role of the KVLQT1 gene in the pathogenesis of long QT syndrome (LQTS), we have established a screening system for detecting KVLQT1 mutations by the polymerase chain reaction-single strand conformation polymorphism technique (PCR-SSCP). We first determined exon/intron boundaries and flanking intronic sequences, and found that the KVLQT1 gene consists of 17 coding exons. Subsequently, we synthesized oligonucleotide primers to cover the coding region and the flanking intronic sequences, and searched for mutations in 31 Japanese LQTS families. When genomic DNA from each proband was examined by PCR-SSCP followed by direct DNA sequencing, mutations were detected in five families; two independent families carried the same mutation and three of the four were novel. Each mutation was present in affected relatives of the respective proband. This work will enable us to search more thoroughly for LQTS mutations associated with KVLQT1, and eventually will help us in finding genotype/phenotype relationships.

In a Xenopus oocyte heterologous expression system, we characterized the electrophysiology of 3 novel missense mutations of HERG identified in Japanese LQT2 families: T474I (within the S2-S3 linker), A614V, and V630L (in the outer mouth of pore-forming region). For each of the 3 mutations, injection of mutant cRNA alone did not express detectable currents. Coinjection of wild-type (WT) along with each mutant cRNA (T474I/WT, A614V/WT, and V630L/WT) suppressed HERG current in a dominant-negative manner, and the order of magnitude of current suppression was V630L/WT>A614V/WT>T474I/WT. In addition to decreases in slope conductance for all 3 mutants, the voltage dependence of steady-state inactivation was shifted to negative potentials for V630L/WT and A614V/WT. Consequently, channel availability at positive potentials was diminished, and inward rectification was enhanced for these 2 mutants. Thus, missense mutations of HERG caused dominant-negative suppression through multiple mechanisms. The shift in voltage dependence of HERG inactivation and the resulting enhanced inward rectification in A614V/WT and V630L/WT provide a novel mechanism for suppression of the HERG current carrying outward current during the repolarization phase of the action potential.

We recently reported the isolation of the klotho gene, which is predominantly expressed in the kidney and involved in human aging phenotypes. In our previous studies, we demonstrated that the Klotho protein or its metabolites may possibly function as humoral factor(s) and protect against endothelial dysfunction because acetylcholine-mediated NO production in arteries was impaired in heterozygous klotho deficient mice (kl/+). However, the pathophysiological significance of the Klotho protein has not been clarified yet. In the present study, we examined expression of the klotho gene in the kidney of the following rat models for human diseases: (1) spontaneously hypertensive rat, (2) deoxycorticosterone acetate-salt hypertensive rat, (3) 5/6 nephrectomized rat, (4) non-insulin-dependent diabetes mellitus rat (the Otsuka Long-Evans Tokushima Fatty rat), and (5) rat with acute myocardial infarction. The expression levels of klotho mRNA in the kidney in these models were significantly lower than controls except for MI rats. This is the first report showing that expression of the klotho gene in the kidney is regulated under sustained circulatory stress such as long-term hypertension, diabetes mellitus, and chronic renal failure. (C) 1998 Academic Press.

Human immunodeficiency virus (HIV) and its clinical syndrome, acquired immune deficiency syndrome (AIDS), are one of the world's most prominent health problems. To understand the mechanisms underlying HIV transcription and thereby its propagation, we have focused on the molecular interactions at the GC-rich binding sites of the HIV-1 core promoter, a region important for HIV-1 transcription. Previous biochemical studies have shown that Sp1, a zinc finger transcription factor initially isolated as a cellular factor binding that binds to the SV40 early promoter GC-rich sequence, binds to the HIV-1 GC-rich binding sites due to sequence similarities. However, the HIV-1 GC-rich binding sites are considerably different from the Sp1 consensus binding sequence, and recent genetic studies have shown the lack of regulation by Sp1 in numerous genes thought to be regulated by that factor in the past. We reasoned that other factors may bind to the HIV-1 GC-rich binding sites. Using the native HIV-1 GC-rich binding sequence as the bait, genetic screening for interacting factors was performed by the yeast one-hybrid method. A cDNA encoding a novel zinc finger protein named GBF, GC-rich sites binding factor, was isolated from a human peripheral blood leukocyte library. Primary structure analysis of GBF revealed a C2H2 Krüppel-type zinc finger at its C-terminus, and putative acidic and proline-rich domains at its N-terminus. We also show that GBF belongs to a subgroup of Krüppel-type zinc fingers distinct from Sp1. By directly addressing interactions at the HIV-1 GC-rich binding sites, our present study sheds new light on molecular interactions at the HIV-1 promoter.

BACKGROUND: Cardiotoxicity leading to congestive heart failure is a complication of the anthracyclines. Biochemical methods to diagnose and monitor cardiac function after anthracycline administration would be most useful. We examined the diagnostic role of B-type natriuretic peptide (BNP), a potent biochemical marker of left ventricular dysfunction, in patients administered anthracyclines. METHODS: Twenty-seven consecutive patients receiving anthracyclines were investigated by serial measurements of BNP levels and other cardiac neurohormones (A-type natriuretic peptide, renin, aldosterone, angiotensin II, norepinephrine, and epinephrine) and myocardial markers (creatine kinase-MB and myosin light chain). Echocardiography was done to assess systolic (ejection fraction) and diastolic (mitral inflow A/E ratio) functions. RESULTS: Of the examined cardiac biochemical markers, BNP levels alone showed marked elevations to abnormal levels after anthracycline administration. Most patients showed transient increases (peak at 3 to 7 days). Patients with persistent elevations showed a poor prognosis. A/E ratio also correlated with increases in BNP levels in selected patients, which may suggest that raised BNP levels are reflective of induced diastolic dysfunction. CONCLUSIONS: Our studies suggest the possible use of BNP levels to assess the cardiac state after anthracycline administration. BNP levels most likely reflect cardiac tolerance to the cardiotoxic agent. Serial BNP profiles also suggest persistent elevations to be associated with potentially decompensatory states in contrast to tolerable transient increases. Diagnosis of degree of cardiac tolerance by response to drug administration may be analogous to use of stress testing (exercise) to help define underlying left ventricular dysfunction.

We previously reported a clinical study in which probucol reduced the restenosis rate. The mechanism of this effect is unclear. Restenosis is characterized by neointimal hyperplasia caused by proliferation of smooth muscle cells (SMCs), which increases the expression of Platelet-derived growth factor (PDGF)-A and SMemb. SMemb, a non-muscle-type myosin heavy chain most predominantly expressed in embryonic smooth muscle, can be used as a good molecular marker for dedifferentiated SMC. The aim of this study was to analyze the effect of probucol on neointimal proliferation and the level of expression of PDGF-A and SMemb after balloon injury in rabbits. Probucol was given orally 1.3 g/d from 2 weeks prior to carotid balloon injury to the time of killing (2 or 4 weeks after balloon injury). Intimal area was determined histologically using a computerized morphometry program. For quantification of SMC proliferation, alpha-actin-positive cells and proliferating cell nuclear antigen (PCNA)-labeled cells were counted. The expression of PDGF-A and SMemb mRNA was analyzed by the RNase protection assay. SMemb expression was also examined by immunohistochemistry. Probucol remarkably decreased intimal area by 70% and the number of SMC and PCNA-labeled cells in the intima. The expression of PDGF-A mRNA was significantly increased after balloon injury in untreated rabbits, whereas it was markedly suppressed with probucol treatment. The expression of SMemb was significantly increased in injured arteries at mRNA and protein levels. However, probucol did not suppress SMemb expression. Probucol is effective in preventing SMC proliferation, which is possibly due to a decrease in the expression of PDGF.

Restenosis is caused by excessive neointima formation resulting from smooth muscle cell (SMC) proliferation and migration from the arterial media into the subendothelial space, stimulated by growth factors. A long-acting somatostatin analog, octreotide, activates protein tyrosine phosphatases and can inhibit the stimulatory effects of growth factors. In this study, we evaluated the effect of octreotide on SMC outgrowth from in vitro explants of both coronary and carotid arterial tissues in a canine endothelium-injury model. After 6 days of culture, SMC grew out of 33.3% and 58.3% of the explants from the injured canine carotid and coronary arterial tissues, respectively. In contrast, SMC outgrowth was not observed from any of the explants from normal canine carotid arterial tissue. Octreotide completely inhibited SMC outgrowth from injured canine carotid arterial tissue at a concentration of 10(-6) M. This agent also inhibited SMC outgrowth from injured canine coronary arterial tissue by 57% and 71% of the control value at concentrations of 10(-8) M and 10(-6) M, respectively. We conclude that our explant cell-culture model may prove to be valuable for assessing the effect of agents designed to reduce intimal proliferation, and that the use of the somatostatin analog octreotide in clinical settings may modify the high incidence of restenosis after coronary interventions by reducing SMC proliferation.

This study was designed to assess how the glomerular expression of the nonmuscle-type myosin heavy-chain isoform, SMemb, is regulated in rats with focal glomerulosclerosis induced by puromycin aminonucleoside. SMemb was barely detectable in control glomeruli. On day 48 of focal glomerulosclerosis, SMemb was expressed in mesangial area and glomerular epithelial cells. When glomerulosclerosis became prominent on day 80, SMemb stained immunohistochemically in a focal segmental pattern in the sclerotic glomeruli. SMemb-expressing cells did not always express a-smooth muscle actin. In Northern blot analysis, SMemb mRNA was not detected in control glomeruli, whereas it was transiently upregulated in glomeruli on day 48 in rats with focal glomerulosclerosis. The mRNA levels of SMemb were thereafter gradually downregulated by day 80; however, they remained higher than those of control glomeruli. These data suggest that glomerular embryonic nonmuscle-type myosin heavy chain is abnormally regulated in glomerulosclerosis and that glomerulosclerosis may be associated with dedifferentiation of not only the mesangial cells, but also the other resident glomerular cells.

Arteriosclerosis caused by aging is recognized to be a crucial risk factor of cardiovascular disease. We recently established klotho mouse which causes age-related disorders including arteriosclerosis. However; no information on endothelial function of klotho mouse or the physiological role of klotho protein as a circulating factor is available. In this report, we demonstrate that 50% effective dose of aortic relaxation in response to acetylcholine in heterozygous klotho mice is significantly greater (4 x 10(-5) M) than in wild-type mice (8 x 10(-6) M, n = 7, p &lt; 0.05) and that the vasodilator response of arterioles to acetylcholine is significantly attenuated in heterozygous (20% effective dose; 2 x 10(-6) M) and homozygous klotho mice (&gt;1 x 10(-5) M) as compared with wild-type mice (1 x 10(-7) M, n = 7, p &lt; 0.05). Nitric oxide metabolites (NO2- and NO3-) in urine are significantly lower in heterozygous klotho mice (142 +/- 16 nmol/day) than wild-type mice (241 +/- 28 nmol/day, n = 13, p &lt; 0.05), Parabiosis between wildtype and heterozygous klotho mice results in restoration of endothelial function in heterozygous klotho mice. We conclude that the klotho protein protects the cardiovascular system through endothelium-derived NO production by humoral pathways. (C) 1998 Academic Press.