永井 良三

To investigate the role of insulin receptor substrate (IRS)-2 in vivo, we generated IRS-2-deficient mice by gene targeting. Although homozygous IRS-2-deficient mice (IRS-2-/- mice) had a body weight similar to wild-type mice, they progressively developed type 2 diabetes at 10 weeks. IRS-2-/- mice showed insulin resistance and a defect in the insulin-stimulated signaling pathway in liver but not in skeletal muscle. Despite insulin resistance, the amount of beta-cells was reduced to 83% of that in wild-type mice, which was in marked contrast to the 85% increase in the amount of beta-cells in IRS-1-deficient mice (IRS-1-/- mice) to compensate for insulin resistance. Thus, IRS-2 plays a crucial role in the regulation of beta-cell mass. On the other hand, insulin secretion by the same number of cells in response to glucose measured ex vivo was significantly increased in IRS-2-/- mice compared with wild-type mice but was decreased in IRS-1-/- mice. These results suggest that IRS-1 and IRS-2 may play different roles in the regulation of beta-cell mass and the function of individual beta-cells.

Genetically modified mice serve as a powerful tool to determine the role of specific molecules in a wide variety of biological phenomena including vascular remodeling. Several models of arterial injury have been proposed to analyze transgenic/knock-out mice, but many questions have been raised about: their reproducibility and physiological significance. Here, we report a new mouse model of vascular injury that resembles balloon-angioplasty. A straight spring wire was inserted into the femoral artery via arterioctomy in a small muscular branch. The wire was left in place for one minute to denude and dilate the artery. After the wire was removed, the muscular branch was tied off and the blood flow of the femoral artery was restored. The lumen was enlarged with rapid onset of medial cell apoptosis. While the circumference of the external elastic lamina remained enlarged, the lumen was gradually narrowed by neointimal hyperplasia composed of smooth muscle cells. At 4 weeks, a concentric and homogeneous neointimal lesion was formed reproducibly in the region where the wire had been inserted. Similar exuberant hyperplasia could be induced in all strains examined (C57BL/6J, C3H/HeJ, BALB/c, and 129/SVi). This model may be widely used to study the molecular mechanism of postangioplasty restenosis at the genetic level. (C) 2000 Academic Press.

Interleukin-1 beta (IL-1 beta) is a multipotent cytokine participating in a variety of cardiovascular diseases. In this study, we examined the effects of IL-1 beta on the expression of vascular endothelial cell growth factor (VEGF) and pursued the molecular mechanisms underlying this effect. Treatment of cultured neonatal rat cardiac myocytes with IL-1 beta increased the levels of VEGF mRNA in a time- and a concentration-dependent manner. These effects were completely abolished by SB203580 and SB202190 (p38 MAPK inhibitors) but not by PD98059 (MEK1 inhibitor), calphostin C (protein kinase C inhibitor), or genistein (tyrosine kinase inhibitor). While IL-1 beta phosphorylated c-Jun N-terminus protein kinase (JNK) rapidly and transiently, the effect of IL-1 beta on p38 mitogen-activated protein kinase (MAPK) was gradual and persistent. Transient transfection assays showed that IL-1 beta increases the transcription from the VEGF promoter. A series of 5;-deletion and site-specific mutation analyses indicated that IL-1 beta as well as overexpression of p38 MAPK and JNK activate VEGF promoter activity through two G+C-rich sequences located at -73 and -62. Electrophoretic mobility shift and supershift assays showed Sp1 and Sp3 proteins specifically bind to the G+C-rich sequences. The half-life of VEGF mRNA was significantly increased in cells treated with IL-1 beta. Together, these results indicate that IL-1 beta induces VEGF gene expression at both transcriptional and post-transcriptional levels, and IL-1 beta evokes p38 MAPK and JNK signalings, which in turn stimulate the transcription of the VEGF gene through Sp1-binding sites. These findings suggest the role of IL-1 beta as a cytokine inducing VEGF in cardiac myocytes, and imply that activation of stress-activated MAP kinases regulate Sp1 sites-dependent transcription.

Overexpression of the oncogene for ErbB-2 is an unfavorable prognostic marker in human breast cancer. Its oncogenic potential appears to depend on the state of tyrosine phosphorylation. However, the mechanisms by which ErbB-2 is constitutively tyrosine-phosphorylated in human breast cancer are poorly understood. We now show that human breast carcinoma samples with ErbB-2 overexpression have higher proliferative and metastatic activity in the presence of autocrine secretion of prolactin (PRL). By using a neutralizing antibody or dominant negative (DN) strategies or specific inhibitors, we also show that activation of Janus kinase Jak2 by autocrine secretion of PRL is one of the significant components of constitutive tyrosine phosphorylation of ErbB-2, its association with Grb2 and activation of mitogen-activated protein (MAP) kinase in human breast cancer cell lines that overexpress ErbB-2. Furthermore, the neutralizing anti-PRL antibody or erbB-2 antisense oligonucleotide or DN Jak2 or Jak2 inhibitor or DNRas or MAP kinase kinase inhibitor inhibits the proliferation of both untreated and PRL-treated cells. Our results indicate that autocrine secretion of PRL stimulates tyrosine phosphorylation of ErbB-2 by Jak2, provides docking sites for Grb2 and stimulates Ras-MAP kinase cascade, thereby causing unrestricted cellular proliferation. The identification of this novel cross-talk between ErbB-2 and the autocrine growth stimulatory loop for PRL may provide new targets for therapeutic and preventive intervention of human breast cancer.

BACKGROUND: It remains unclear how hemodynamic overload induces cardiac hypertrophy. Recently, activation of calcium-dependent phosphatase, calcineurin, has been elucidated to induce cardiac hypertrophy. In the present study, we examined the role of calcineurin in load-induced cardiac hypertrophy by using Dahl salt-sensitive (DS) rats, which develop both pressure and volume overload when fed a high salt diet. METHODS AND RESULTS: In the DS rat heart, the activity of calcineurin was increased and cardiac hypertrophy was induced by high salt diet. Treatment of DS rats with the calcineurin inhibitor FK506 (0.1 or 0.01 mg/kg twice daily) from the age of 6 weeks to 12 weeks inhibited the activation of calcineurin in the heart in a dose-dependent manner and attenuated the development of load-induced cardiac hypertrophy and fibrosis without change of hemodynamic parameters. Additionally, treatment with 0.1 mg/kg twice daily but not with 0.01 mg/kg twice daily of FK506 from the age of 12 weeks to 16 weeks induced regression of cardiac hypertrophy in DS rats. Load-induced reprogramming of gene expression was also suppressed by the FK506 treatment. CONCLUSIONS: These results suggest that calcineurin is involved in the development of cardiac hypertrophy in rats with salt-sensitive hypertension and that inhibition of calcineurin could induce regression of cardiac hypertrophy.

BACKGROUND: A rapid 30-minute assay of circulating smooth-muscle myosin heavy-chain protein has been developed as a biochemical diagnostic tool for aortic dissection. OBJECTIVE: To determine the sensitivity and specificity of this assay. DESIGN: Cross-sectional study. SETTING: 8 major cardiovascular centers in Japan. PATIENTS: 95 patients with acute aortic dissection, 48 patients with acute myocardial infarction, and 131 healthy volunteers. MEASUREMENTS: Levels of circulating smooth-muscle myosin heavy-chain protein. RESULTS: Patients with acute aortic dissection who presented within 3 hours after onset had elevated levels of circulating smooth-muscle myosin heavy-chain protein. In these patients, the assay had a sensitivity of 90.9%, a specificity of 98% compared with healthy volunteers, and a specificity of 83% compared with patients who had acute myocardial infarction; the clinical decision limit was 2.5 microgram/L. All patients with proximal lesions had elevated levels of smooth-muscle myosin heavy-chain protein, and only patients with distal lesions had decreased levels (<2.5 microgram/L). CONCLUSIONS: Levels of smooth-muscle myosin heavy-chain protein can be used to diagnose aortic dissection soon after symptom onset. The assay had the greatest diagnostic value in patients with proximal lesions.

Recent studies have established oxidative modification of low density lipoprotein (LDL) as an important atherogenic factor. We examined the clinical relevance of circulating oxidized LDL (OxLDL) levels in atherosclerotic disease by an enzyme immunoassay with use of specific antibodies against OxLDL (FOH1a/DLH3) and apolipoprotein B. Plasma OxLDL levels were significantly higher in patients with coronary heart disease (n=65) than in control subjects (n=181; 201. 3+/-11.2 versus 112.4+/-3.3 U/dL, respectively; P<0.01). OxLDL levels were not associated with age, sex, total cholesterol, or apolipoprotein B levels in normal control subjects. Our results suggest that circulating OxLDL may be a possible biochemical risk marker for coronary heart disease.

In vivo studies suggest that sterol regulatory element-binding protein (SREBP)-1 plays a key role in the upregulation of lipogenic genes in the livers of animals that have consumed excess amounts of carbohydrates. In light of this, we sought to use an established mouse hepatocyte cell line, H2-35, to further define the mechanism by which glucose regulates nuclear SREBP-1 levels, First, we show that these cells transcribe high levels of SREBP-1c that are increased 4-fold upon differentiation from a prehepatocyte to a hepatocyte phenotype, making them an ideal cell culture model for the study of SREBP-1c induction. Second, we demonstrate that the presence of precursor and mature forms of SREBP-1 protein are positively regulated by medium glucose concentrations ranging from 5.5 to 25 mM, and are also regulated by insulin, with the amount of insulin in the fetal bovine serum being sufficient for maximal stimulation of SREBP-1 expression. Third, we show that the increase in SREBP-1 protein is due to an increase in SREBP-1 mRNA, Reporter gene analysis of the SREBP-1c promoter demonstrated a glucose-dependent induction of transcription. In contrast, expression of a fixed amount of the precursor form of SREBP-1c protein showed that glucose does not influence its cleavage. Fourth, we demonstrate that the glucose induction of SREBP could not be reproduced by fructose, xylose, or galactose nor by glucose analogs a-deoxy glucose and 3-O-methyl glucopyranose. These data provide strong evidence for the induction of SREBP-1c mRNA by glucose leading to increased mature protein in the nucleus, thus providing a potential mechanism for the up-regulation of lipogenic genes by glucose in vivo.

The klotho gene, originally identified by insertional mutagenesis in mice, suppresses multiple aging phenotypes (e.g., arteriosclerosis, pulmonary emphysema, osteoporosis, infertility, and short life span). We have previously shown that mice heterozygous for a defect in the klotho gene upon parabiosis with wild-type mice show improved endothelial function, suggesting that the klotho gene product protects against endothelial dysfunction. In the present study, using the Otsuka Long-Evans Tokushima Fatty (OLETF) rat which demonstrates multiple atherogenic risk factors (e.g., hypertension, obesity, severe hyperglycemia, and hypertriglyceridemia) and is thus considered an experimental animal model of atherosclerotic disease, we show that adenovirus-mediated klotho gene delivery can (1) ameliorate vascular endothelial dysfunction, (2) increase nitric oxide production, (3) reduce elevated blood pressure, and (4) prevent medial hypertrophy and perivascular fibrosis. Based on these findings, klotho gene delivery improves endothelial dysfunction through a pathway involving nitric oxide, and is involved in modulating vascular function (e.g., hypertension and vascular remodeling). Our findings establish the basis for the therapeutic potential of klotho gene delivery in atherosclerotic disease.

We examined the mechanism(s) for HERG channel dysfunction in an S818L mutation in the HERG C-terminus using the heterologous expression system in Xenopus oocytes. Injection of S818L cRNA alone did not produce expressed currents. Coinjection of an equal amount of S818L cRNA with wild-type (WT) cRNA into oocytes did not exhibit apparent dominant-negative suppression. However, coinjection of excess amounts of S818L cRNAs with WT cRNA into oocytes decreased HERG current amplitudes and shifted the voltage dependence of activation to negative potentials, accelerated its activation and deactivation. The data suggest that S818L alone cannot form functional channels, whereas S818L subunits can, at least in part, coassemble with WT subunits to form heterotetrameric functional channels, and imply that the HERG C-terminus may contain a domain involving the activation-deactivation process of the channel. These findings may provide new insights into the structure-function relationships of the HERG C-terminus.

Angiotensin II (Ang II) evokes a variety of hypertrophic responses such as activation of protein kinases, reprogramming of gene expressions and an increase in protein synthesis in cardiac myocytes. In this study, we examined the role of Rho family small GTP binding proteins (G proteins) in Ang II-induced cardiac hypertrophy. Ang II strongly activated extracellular signal-regulated protein kinases (ERKs) in cardiac myocytes of neonatal rats. Although Ang II-induced activation of ERKs was completely suppressed by an Ang II type 1 receptor antagonist, CV-11974, this activation was not inhibited by the pretreatment with C3 exoenzyme, which abrogates Rho functions. Overexpression of Rho GDP dissociation inhibitor (Rho-GDI), dominant negative mutants of Rac1 (D.N.Rac1), or D.N.Cdc42 had no effects on Ang II-induced activation of transfected ERK2. The promoter activity of skeletal alpha-actin and c-fos genes was increased by Ang II, and the increase was partly inhibited by overexpression of Rho-GDI and the pretreatment with C3 exoenzyme. Ang II increased phenylalanine incorporation into cardiac myocytes by approximately 1.4 fold as compared with control, and this increase was also significantly suppressed by the pretreatment with C3 exoenzyme. These results suggest that the Rho family small G proteins play important roles in Ang II-induced hypertrophic responses in cardiac myocytes.

We have recently identified a novel gene, klotho (kl), which may suppress several aging phenotypes. A defect of kl gene expression in the mouse results in a syndrome resembling human aging, such as arteriosclerosis, skin atrophy, osteoporosis, and pulmonary emphysema. To determine whether mouse homozygotes for the kl mutation (kl/kl) show abnormal glucose metabolism, an oral glucose tolerance test (OGTT) was performed at 6 to 8 weeks of age. Blood glucose levels during the OGTT were significantly lower in kl/kl mice versus wild-type mice. The insulin content of the pancreas was significantly lower in kl/kl mice compared with wild-type mice. Decreased insulin production was also supported by Northern blot analysis showing lower levels of insulin mRNA in kl/kl mice. To examine how lower blood glucose levels may exist in kl/kl mice despite decreased insulin production, insulin tolerance tests (ITTs) were performed. The glucose decline following insulin injection was more severe in kl/kl mice versus wild-type mice, suggesting that insulin sensitivity was higher in kl/kl mice versus wild-type mice. In kl/kl mice, an augmented expression of GLUT4 in skeletal muscle was demonstrated by both Northern blot analysis and Western blot analysis. Thus, we conclude that insulin production is decreased and insulin sensitivity is increased in the klotho mouse, a novel animal model for human aging. Copyright (C) 2000 by W.B. Saunders Company.

To further investigate the immunological mechanisms involved in Takayasu's arteritis, we analyzed the T-cell receptor (TCR) V gamma and V delta gene usage by infiltrating gamma delta T-cells and the expression of costimulatory molecules B7-1, B7-2, CD40, CD27 ligand (CD27L), CD30L, OX40L in the arterial tissue of a patient with Takayasu's arteritis. We found that the repertoires of TCR V gamma as well as V delta gene transcripts of the infiltrating cells were restricted as compared with those of peripheral blood lymphocytes from a patient with Takayasu's arteritis. This strongly suggests that gamma delta T-cells as well as alpha beta T-cells, as we previously reported, were specifically involved in the pathogenesis of Takayasu's arteritis. We also found that B7-1, B7-2, CD40, CD27L, CD30L, and OX40L were expressed in the arterial tissue, suggesting the roles for these costimulatory molecules in T-cell-mediated vascular injury in Takayasu's arteritis. Our findings strongly support the involvement of T-cell-mediated immunological mechanisms in the pathogenesis of Takayasu's arteritis. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.

This report describes a case of cardiac tamponade as the initial manifestation of unclassified connective tissue disease (UCTD). A 68-year-old Japanese woman was admitted to hospital because of dyspnea and edema. She had undergone a radical left mastectomy for the treatment of breast cancer 18 years before. On admission, bilateral leg edema, hepatomegaly, and a paradoxical pulse were noted on physical examination. The erythrocyte sedimentation rate was elevated and the C-reactive protein was 2.8 mg/dl. Antinuclear antibodies and anti-SS-A/Ro antibodies were present. The scl-70 and anticentromere antibodies were elevated. Chest radiography showed cardiomegaly. Echocardiography revealed a large pericardial effusion, but the pericardial fluid did not contain malignant cells or bacteria. She did not meet the diagnostic criteria for any known connective tissue diseases, so was diagnosed with cardiac tamponade due to UCTD. Prednisolone (30 mg/day) was administered, which resulted in a gradual resolution of the pericardial effusion. Although connective tissue diseases are known to cause pericardial effusion, cardiac tamponade as the initial manifestation of the disease in the absence of other symptoms is quite rare.

Endothelin (ET)-1 is a 21-amino acid peptide that induces a variety of biological activities, including vasoconstriction and cell proliferation, and its likely involvement in cardiovascular and other diseases has recently led to broad clinical trials of ET receptor antagonists. ET-1 is widely distributed in the central nervous system (CNS), where it is thought to regulate hormone and neurotransmitter release. Here we show that CNS responses to emotional and physical stressors are differentially affected in heterozygous ET-1-knockout mice, which exhibited diminished aggressive and autonomic responses toward intruders (emotional stressors) but responded to restraint-induced (physical) stress more intensely than wild-type mice. This suggests differing roles of ET-1 in the central pathways mediating responses to different types of stress. Hypothalamic levels of ET-1 and the catecholamine metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG) were both increased in wild-type mice subjected to intruder stress, whereas MHPG levels were not significantly affected in ET-1-knockout mice. Furthermore, immunohistochemical analysis showed that ET-1 and tyrosine hydroxylase, an enzyme in the catecholamine synthesis pathway, were colocalized within certain neurons of the hypothalamus and amygdala. Our findings suggest that ET-1 modulates central coordination of stress responses in close association with catecholamine metabolism.

Background: Using Doppler color flow imaging, abnormal flow patterns were report-ed to occur with pulmonary artery (PA) dilation, We have frequently observed red signals in the main PA, suggesting reversed flow (RF) in patients without overt pulmonary hypertension. The clinical implication of these signals has not been extensively studied. Patients and methods: We studied 191 of 412 patients referred for echocardiography (99 men and 92 women; mean +/- SD age, 62 +/- 13 years), in whom the main PA diameter had been adequately measured. If a red signal was observed by color flow imaging, a pulsed Doppler echocardiogram of the red signal was recorded simultaneously. The presence of the red signal was correlated with the PA diameter and the PA systolic pressure determined using the modified Bernoulli equation. In 54 patients who also underwent cardiac catheterization studies, the red signal was correlated with PA and pulmonary capillary wedge (PCW) pressures, and with pulmonary vascular resistance. Results: Red signals adjacent to the medial PA border were detected in parallel with systolic blue signals in 127 patients (66%), Pulsed Doppler recordings revealed that they were caused by RF occurring immediately after the forward systolic signal and persisted in diastole. The PA diameter (28 +/- 4.8 mm) and the estimated PA systolic pressure (34 +/- 16 mm Hg) of patients with the RF signal were significantly greater (p &lt; 0.001 and p &lt; 0.05, respectively) than those of patients without the signal (22 +/- 2.5 mm and 28 +/- 6.0 mm Hg, respectively). Among patients who had hemodynamic studies, PA and PCW pressures were significantly higher (p &lt; 0.05) in the 41 patients with the RF signal (22 +/- 12 mm Hg vs 15 +/- 2.6 mm Hg and 11 +/- 5.5 mm Hg vs 8 +/- 3.1 mm Ng respectively). Conclusion: RF signals in the main PA occur mostly as a result of PA dilation, which may be caused by primary pulmonary hypertension or chronic elevation of left atrial pressure in left-sided cardiac abnormalities.

The effects of bile acids on intracellular Ca(2+) concentration [Ca(2+)](i) and nitric oxide production were investigated in vascular endothelial cells. Whole-cell patch clamp techniques and fluorescence measurements of [Ca(2+)](i) were applied in vascular endothelial cells obtained from human umbilical and calf aortic endothelial cells. Nitric oxide released was determined by measuring the concentration of NO(2)(-). Deoxycholic acid, chenodeoxycholic acid and the taurine conjugates increased [Ca(2+)](i) concentration-dependently, while cholic acid showed no significant effect. These effects resulted from the first mobilization of Ca(2+) from an inositol 1,4,5-triphosphate (IP(3))-sensitive store, which was released by ATP, then followed by Ca(2+) influx. Both bile acids and ATP induced the activation of Ca(2+)-dependent K(+) current. Oscillations of [Ca(2+)](i) were occasionally monitored with the Ca(2+)-dependent K(+) current in voltage-clamped cells and Ca(2+) measurements of single cells. The intracellular perfusion of heparin completely abolished the ATP effect, but failed to inhibit the bile acid effect. Deoxycholic acid and chenodeoxycholic acid enhanced NO(2)(-) production concentration-dependently, while cholic acid did not enhance it. The bile acids-induced nitric oxide production was suppressed by N(G)-nitro-L-arginine methyl ester, exclusion of extracellular Ca(2+) or N-(6-aminohexyl)-5-chloro-l-naphthalenesulphonamide hydrochloride (W-7) and calmidazolium, calmodulin inhibitors. These results provide novel evidence showing that bile acids increase [Ca(2+)](i) and subsequently nitric oxide production in vascular endothelial cells. The nitric oxide production induced by bile acids may be involved in the pathogenesis of circulatory abnormalities in liver diseases including cirrhosis.

STUDY OBJECTIVE: Previous studies have suggested that serum markers of smooth muscle destruction have utility in predicting ectopic pregnancy. Our goal was to determine whether a novel marker of muscle destruction, smooth muscle heavy-chain myosin (SMHC), is elevated in the serum of patients with ectopic pregnancy. METHODS: We conducted a prospective cohort study, with consecutive enrollment, of all women in the first trimester of pregnancy who presented to our urban emergency department with complaints of lower abdominal pain with or without vaginal bleeding. Patients were excluded if there was a history of recent surgery or major trauma. Means were compared using 2-tailed Student's t test with P values less than.05 set for significance. Data analysis included calculation of receiver operating characteristic (ROC), 95% confidence intervals (CIs), and a regression model. RESULTS: A total of 175 patients were enrolled; ectopic pregnancy was diagnosed in 29, and 146 had other diagnoses. Patients with ectopic pregnancy had a mean serum SMHC concentration of 2.53 ng/dL (95% CI 1.84 to 3.22), whereas those in the non-ectopic pregnancy group had a mean concentration of 1.41 ng/dL (95% CI 1.23 to 1.60; P <.0001). ROC analysis demonstrated an area under the curve of 0.72 (95% CI 0.65 to 0.79). Regression analysis to examine confounders in each group analyzed the effects of race, maternal age, estimated gestational age, and serum levels of human chorionic gonadotropin beta-subunit. Our analysis identified only a positive correlation between estimated gestational age and SMHC in the non-ectopic pregnancy group. CONCLUSION: There is a statistically significant elevation of serum SMHC levels in tubal pregnancy, although our data suggest that the assay has limited clinical utility as a lone marker for ectopic pregnancy. Further investigation is needed to determine whether the assay has a role as an adjunct in the evaluation of suspected ectopic pregnancy.

Heme oxygenase (HO) is a heme-catabolizing enzyme that converts heme into biliverdin, iron, and carbon monoxide. HO-1, an inducible form of HO, is thought to act as an endogenous antioxidant defense mechanism. To determine whether chronic administration of angiotensin II affects HO-1 expression in the heart, expression and localization of HO-1 were investigated in the heart of rats receiving angiotensin II infusion (0.7 mg. kg(-1). day(-1)) via osmotic minipump for up to 7 days. Angiotensin II induced formation of granulation tissue, characterized by myofibroblast proliferation, fibrous deposition, and inflammatory cell migration. Angiotensin II also upregulated cardiac HO-1 expression. Immunohistochemistry revealed that HO-1 was intensively expressed in the granulation tissue. The selective AT(1)-receptor antagonist, losartan, completely, but hydralazine only partially, suppressed angiotensin II-induced granulation tissue formation and HO-1 upregulation. Chronic norepinephrine infusion (2.8 mg. kg(-1). day(-1)) did not induce granulation tissue formation or HO-1 upregulation. Our data suggest that angiotensin II upregulates cardiac HO-1 expression in the newly formed inflammatory lesion, which may represent an adaptive response to angiotensin II-induced cardiac damage.

We investigated whether application of hydrostatic pressure facilitates ex vivo adenovirus gene transfer into rabbit aortas. Assay of beta-galactosidase activity was performed in the aortas subjected to adenovirus encoding LacZ gene (Ad-LacZ) transfer. Application of hydrostatic pressure at 8 atmospheres during Ad-LacZ gene transfer (10(10) pfu/mL, 10 min) to aortic segments resulted in an approximately 4.5-fold increase in transgene efficiency. X-Gal staining showed predominant beta-galactosidase activity in the endothelial and the adventitial cells in the aorta subjected to pressure-supported Ad-LacZ gene transfer. Then we examined the effect of heme oxygenase-l (HO-1), which catabolizes heme to carbon monoxide and biliverdin, by transferring adenovirus encoding HO-1 (Ad-HO-1) into rabbit aortas using this pressurization system, The Ad-MO-I-infected aortic segment showed a significantly decreased contractile response to phenylephrine compared to the Ad-LacZ-infected aortic segment. Pressure-supported adenovirus gene transfer may increase the feasibility of exploiting intraoperative adenovirus-mediated gene transfer. (C) 2000 Academic Press.

Doxorubicin (Dox), an anthracyclin antineoplastic agent, causes dilated cardiomyopathy. CARP has been identified as a nuclear protein whose mRNA levels are exquisitely sensitive to Dox. In this study we investigated the molecular mechanisms underlying the repression of CARP expression by Dox in cultured neonatal rat cardiac myocytes. Dox (1 mu mol/l)-mediated decrease in CARP mRNA levels was strongly correlated with BNP but not with ANP mRNA levels. Hydrogen peroxide scavenger catalase (1 mg/ml) but not hydroxyl radical scavengers dimethylthiourea (10 mmol/l) or mannitol (10 mmol/l) blunted the Dox-mediated decrease in CARP and BNP expression. Superoxide dismutase inhibitor diethyldithiocarbamic acid (10 mmol/l), which inhibits the generation of hydrogen peroxide from superoxide metabolism, attenuated the repression. PD98059 (MEK1 inhibitor, 50 mu mol/l), SB203580 (p38 MAP kinase inhibitor, 10 mu mol/l), calphostin C (protein kinase C (PKC) inhibitor, 1 mu mol/l), non-selective protein tyrosine kinase inhibitors genistein (50 mu mol/l) or herbimycin A (1 mu mol/l) failed to abrogate the downregulation of CARP and BNP expression by Dox. In contrast. H7 (30 mu mol/l), a potent inhibitor of serine/threonine kinase, significantly blocked Dox-mediated downregulation of CARP and BNP expression. Transient transfection of a series of 5'-deletion and site-specific mutation constructs revealed that M-CAT element located at -37 of the human CARP promoter mediates Dox-induced repression of CARP promoter activity. These results suggest that a genetic response to Dox is mediated through the generation of hydrogen peroxide, which is selectively linked to the activation of H7-sensitive serine/threonine kinase distinct from PKC and well characterized mitogen-activated protein (MAP) kinases (ERK and p38MAP kinase). Furthermore, our data implicated M-CAT element as a Dox-response element within the CARP promoter in cardiac myocytes. (C) 2000 Academic Press.

The effects of endothelin (ET)-1 on cultured human bronchial smooth muscle cells (HBSMC) were investigated and compared with those of histamine, using the patch clamp techniques and measurements of intracellular Ca(2+) ([Ca(2+)](i)). Both ET-1 and histamine caused an initial transient elevation of [Ca(2+)](i) by Ca(2+) mobilization, followed by a sustained rise due to Ca(2+) entry. Nicardipine inhibited the sustained phase, but La(3+) abolished it. With low ethyleneglycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) and K(+) internal solutions, both ET-1 and histamine induced a sustained depolarization from approximately -40 to -20 mV. Under voltage clamp conditions, both drugs transiently activated an outward K(+) current at a holding potential of 0 mV. Additionally, with a Cs(+) internal solution, they elicited another transient inward current, frequently followed by current oscillations. These transient currents were blocked by high EGTA or heparin. With high EGTA and Cs(+) internal solutions, both drugs activated a long-lasting inward current. The reversal potential of these agonist-induced currents was approximately 0 mV and was not altered by the replacement of internal or external concentration of Cl(-), suggesting that the inward current was a nonselective cation current (I(cat)). The half-maximal effective concentration to activate I(cat) was 12 nM for ET-1 and 11 microM for histamine. La(3+) and Cd(2+) abolished these agonist-induced I(cat). The effects of ET-1 on [Ca(2+)](i) and I(cat) could be blocked by combined pretreatment with BQ-123 and BQ-788. Sarafotoxin S6c also increased [Ca(2+)](i) and activated I(cat). By polymerase chain reaction of reverse transcribed RNA, we detected both ET-A and ET-B receptor messenger RNA. These results provide the first evidence that ET-1 is a potent activator of I(cat) in HBSMC via ET-A and ET-B receptors, and the activation of I(cat) plays an important role in ET-1-induced Ca(2+) entry in human airways.

Abetalipoproteinemia (ABL) is an inherited disease characterized by the virtual absence of apolipoprotein B (apoB)-containing lipoproteins from plasma. Only limited numbers of families have been screened for mutations in the microsomal triglyceride transfer protein (MTP) gene. To clarify the genetic basis of clinical diversity of ABL, mutations of the MTP gene have been screened in 4 unrelated patients with ABL, Three novel mutations have been identified: a frameshift mutation caused by a single adenine deletion at position 1389 of the cDNA, and a missense mutation, Asn780Tyr, each in homozygous forms; and a splice site mutation, 2218-2A--&gt;G, in a compound heterozygous form. The frameshift and splice site mutations are predicted to encode truncated forms of MTP, When transiently expressed in Cos-l cells, the Asn780Tyr mutant MTP bound protein disulfide isomerase (PDI) but displayed negligible MTP activity. It is of interest that the patient having the Asn780Tyr mutation, a 27-year-old male, has none of the manifestations characteristic of classic ABL even though his plasma apoB and vitamin E were virtually undetectable.jlr These results indicated that defects of the MTP gene are the proximal cause of ABL.

Recent studies have suggested that prolonged hypoxia results in increased production of reactive oxygen species in cardiomyocytes, which leads to apoptosis of these cells. We previously showed that lecithinized recombinant human copper, zinc-superoxide dismutase (rhSOD) showed increased bioavailability through greater membrane affinity and a longer half-life than unmodified SOD. The purpose of this study was to investigate whether lecithinized SOD plays a protective role against hypoxic injury in cardiomyocytes. Cultured rat cardiomyocytes incubated with lecithinized SOD (100 U/ml), unmodified SOD (100 U/ml), or vehicle alone were subjected to hypoxia for up to 72 h. Lecithinized SOD, but not unmodified SOD, was successfully delivered intracellularly, which was verified by Western blot and confocal laser-scanning microscopy. Treatment of cells with lecithinized SOD significantly suppressed hypoxia-induced cell damage. Since lecithinized SOD also suppressed hypoxia-induced DNA fragmentation, the improved cell survival provided by lecithinized SOD is thought to be mediated by its antiapoptotic effect. In summary, lecithinization resulted in a facilitated rhSOD delivery into cultured cardiomyocytes, which reduced mortality of cardiomyocytes exposed to prolonged hypoxia. (C) 2000 Elsevier Science Inc.

Long QT syndrome (LQTS) is a prime example of interplay between molecular biology, cellular physiology, and organ physiology. Both the congenital and acquired forms of LQTS are due to intrinsic and/or acquired abnormalities of the ionic currents responsible for cardiac repolarization. We analyzed the QTc interval, QRS axes and interventricular septal thickness (IVST) in 41 patients who had a prolonged QT interval on routine electrocardiography (ECG) (5 females and 36 males, mean age 65+/-13 years). The QRS axis of patients in the LQTS group (27+/-49 degrees) was significantly lower (p < 0.05) than in the control group (46+/-26 degrees). However, the IVST in the LQTS group (10+/-2 mm) was significantly thicker than in the control group (9+/-1 mm) (p < 0.05), while the WTd was not significantly different. The QTc interval in patients with ventricular septal hypertrophy (IVST > or = 12 min, 478.8+/-7.9 msec) was significantly longer (p < 0.05) than in the normal group (IVST < 12 mm, 472.1+/-17.5 msec). In conclusion, the results of this preliminary study suggest that prolongation of the QT interval on ECG should prompt screening for electrocardiographic evidence of ventricular hypertrophy in patients with this disease.

After compensating for two kinds of motion artifacts caused by cardiac beating and respiration, cardiac magnetic resonance (MR) imaging is now feasible for the diagnosis of various cardiac diseases. Taking cost-effectiveness into consideration, this paper reviews the experiences of preferable indications of cardiac MR imaging by demonstrating the characteristic preciseness and uniqueness that play an important role in obtaining time-volume curves consisting of the theoretically most accurate measurements of left and right ventricular volumes, in overall evaluation of the left ventricular apex and the right ventricle, in delineating the wide range of the corollary arterial tree, in measuring the most precise blood flow volume through the cross-sectional images of the vessels, and in assessing the spatial derivative of the blood flow velocity at the vessel wall, i.e., wall shear rate.

Recent studies have suggested that prolonged hypoxia results in increased production of reactive oxygen species in cardiomyocytes, which leads to apoptosis of these cells. We previously showed that lecithinized recombinant human copper, zinc-superoxide dismutase (rhSOD) showed increased bioavailability through greater membrane affinity and a longer half-life than unmodified SOD. The purpose of this study was to investigate whether lecithinized SOD plays a protective role against hypoxic injury in cardiomyocytes. Cultured rat cardiomyocytes incubated with lecithinized SOD (100 U/ml), unmodified SOD (100 U/ml), or vehicle alone were subjected to hypoxia for up to 72 h. Lecithinized SOD, but not unmodified SOD, was successfully delivered intracellularly, which was verified by Western blot and confocal laser-scanning microscopy. Treatment of cells with lecithinized SOD significantly suppressed hypoxia-induced cell damage. Since lecithinized SOD also suppressed hypoxia-induced DNA fragmentation, the improved cell survival provided by lecithinized SOD is thought to be mediated by its antiapoptotic effect. In summary, lecithinization resulted in a facilitated rhSOD delivery into cultured cardiomyocytes, which reduced mortality of cardiomyocytes exposed to prolonged hypoxia. (C) 2000 Elsevier Science Inc.

We examined the relationship between plasma homocysteine levels and restenosis after PTCA (Percutaneous transluminal coronary angioplasty) to investigate whether plasma homocysteine levels can be a predictor of restenosis after PTCA. One hundred and twelve male patients who have undergone a successful elective PTCA were consecutively enrolled and plasma homocysteine levels were measured at the time of follow-up angiography. Plasma homocysteine levels in patients with restenosis were significantly higher than those in patients without restenosis (15.0 +/- 3.9 vs. 13 +/- 2.9 micromol/L; P = 0.011). The difference was augmented when diabetic patients were selectively studied. The comparison between restenosis group and non-restenosis group indicated the threshold effect of hyperhomocysteinemia. These results suggest that plasma homocysteine is a potential risk factor of restenosis after PTCA, and therapeutic strategy targeted against hyperhomocysteinemia may be beneficial for preventing restenosis.

Adrenomedullin, which was discovered as a vasodilating peptide, has been reported to be produced in various organs, in which adrenomedullin regulates not only vascular tone but also cell proliferation and differentiation in an autocrine/paracrine manner. We evaluated the effect of adrenomedullin on endothelial cell apoptosis. Human umbilical vein endothelial cells underwent apoptosis when cultured in serum-free medium. Treatment with adrenomedullin reduced the number of cells with pyknotic nuclei (Hoechst 33258 staining) and inhibited cell death (dimethylthiazo-diphenyltetrazolium bromide assay) in a dose-dependent manner. The administration of adrenomedullin did not alter the expression levels of Bcl-2 family proteins. Experiments with analogs of cAMP or a cAMP-elevating agonist demonstrated that elevation of the intracellular cAMP concentration does not mediate the antiapoptotic effect of adrenomedullin. The coadministration of N-nitro-L-arginine methyl ester (2 mmol/L), an inhibitor of nitric oxide synthase, abrogated the effect of adrenomedullin. Lower doses of sodium nitroprusside (1 to 10 mu mol/L), a nitric oxide donor, mimicked the antiapoptotic effect of adrenomedullin. The antiapoptotic effect of sodium nitroprusside was not attenuated by the inhibition of soluble guanylyl cyclase with 1 mu mol/L oxadiazolo-quinoxalin-1-one nor could apoptosis be inhibited by the incubation of human umbilical vein endothelial cells with 1 mmol/L 8-bromo-cGMP, a cell-permeant cGMP analog. These results indicate that adrenomedullin and nitric oxide inhibit endothelial cell apoptosis via a cGMP-independent mechanism.

Glycosyl phosphatidylinositols (GPIs) are used to anchor many proteins to the cell surface membrane and are utilized in all eukaryotic cells. GPI anchoring units are attached to proteins via a transamidase reaction mediated by a GPI transamidase complex. We isolated one of the components of this complex, mGPAA1 (murine GPI anchor attachment), by the signal sequence trap method. mGPAA1 cDNA is about 2 kb in length and encodes a putative 621 amino acid protein. The mGPAA1 gene has 12 small exons and 11 small introns. mGPAA1 mRNA is ubiquitously expressed in mammalian cells, and in situ hybridization analysis revealed that it is abundant in the choroid plexus, skeletal muscle, osteoblasts of rib, and occipital bone in mouse embryos. Its expression levels and transamidation efficiency decreased with differentiation of embryonic stem cells. The 3T3 cell lines expressing antisense mGPAA1 failed to express GPI-anchored proteins on the cell surface membrane.

The antiphospholipid-antibody syndrome is associated with an increased incidence of arterial and venous thrombosis. Although renal infarction has been observed in these patients, stenotic lesions of the renal artery associated with the antiphospholipid-antibody syndrome have not been reported. A 47-year-old male with a history of hypertension for 7 years developed blurred vision secondary to thrombotic occlusion of the central retinal artery. Laboratory and radiologic examinations revealed renal dysfunction, a positive anticardiolipin antibody, and narrowing of the right renal artery. Successful percutaneous transluminal renal artery angioplasty resulted in normalization of the blood pressure and recovery of renal function.

CARP, a cardiac doxorubicin (adriamycin)-responsive protein, has been identified as a nuclear protein whose expression is downregulated in response to doxorubicin. In the present study, we tested the hypothesis that CARP serves as a reliable genetic marker of cardiac hypertrophy in vivo and in vitro. CARP expression was markedly increased in 3 distinct models of cardiac hypertrophy in rats: constriction of abdominal aorta, spontaneously hypertensive rats, and Dahl salt-sensitive rats. In addition, we found that CARP mRNA levels correlate very strongly with the brain natriuretic peptide mRNA levels in Dahl rats. Transient transfection assays into primary cultures of neonatal rat cardiac myocytes indicate that transcription from the CARP and brain natriuretic peptide promoters is stimulated by overexpression of p38 and Rac1, components of the stress-activated mitogen-activated protein kinase pathways. Mutation analysis and electrophoretic mobility shift assays indicated that the M-CAT element can serve as a binding site for nuclear factors, and this element is important for the induction of CARP promoter activity by p38 and Rac1. Thus, our data suggest that M-CAT element is responsible for the regulation of the CARP gene in response to the activation of stress-responsive mitogen-activated protein kinase pathways. Moreover, given that activation of these pathways is associated with cardiac hypertrophy, we propose that CARP represents a novel genetic marker of cardiac hypertrophy.