永井 良三

We have developed an original vector library that allowed us to exploit the phenomenon of RNA interference but also allowed us to avoid the confounding effects of the interferon response. In the present work, we used our library of small interfering RNA expression vectors to examine the genes involved in apoptosis that was induced by double-stranded RNA. To our surprise, screening of our library revealed two novel double-stranded RNA-induced apoptotic pathways, a JNK/SAPK-mediated mitochondrial pathway and an ERK2-related pathway, both of which appeared to be independent of the serine-threonine protein kinase-dependent caspase pathway. We also found that MST2 and protein kinase Ca both activated the pro-apoptotic signal mediated by ERK2. The results of our screening analysis suggested the utility of large scale screenings with libraries of small interfering RNA expression vectors. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

OBJECTIVES: The present study was designed to assess aortic valve morphology and function in mice of advanced age. We also evaluated the potential contribution of bone-marrow-derived cells to the pathogenesis of aortic stenosis. BACKGROUND: Age-associated valvular degeneration is characterized by lipid accumulation, collagen deposition, and calcification containing smooth muscle-like cells and osteoblast-like cells. Cellular and molecular factors that mediate these changes remain unknown. METHODS: We extensively examined the aortic valves of senile wild-type and apolipoprotein E (ApoE)-/- mice with echocardiography. The aortic valves were analyzed by immunohistochemistry and electron microscopy. The bone marrow of wild-type and ApoE-/- mice was reconstituted with that of green fluorescent protein (GFP) or beta-galactosidase (LacZ) mice, which expressed GFP or LacZ ubiquitously. RESULTS: Transaortic flow velocity was correlated with age in wild-type and ApoE-/- mice. The aortic valves of old ApoE-/- mice showed sclerosis that resembled the pathology of human aortic stenosis. A significant number of GFP-positive cells (10.7 +/- 4.1%) in the sclerotic valves of ApoE-/- mice expressed alpha-smooth muscle actin, whereas most of the GFP-positive cells were identified as endothelial cells or macrophages in wild-type mice. There were bone-marrow-derived cells that were positive for osteoblast-related proteins near the sites of ectopic calcification. The sclerotic valves displayed frequent apoptotic cell death and chemokine expression. CONCLUSIONS: Senile ApoE-deficient mice display aortic valve sclerosis that is similar to that observed in humans. The sclerotic valves displayed frequent apoptotic cell death and chemokine expression. Smooth muscle-like cells observed in degenerative valves might derive, at least in part, from bone marrow.

AIMS/HYPOTHESIS: Secreted by adipocytes, adiponectin is a hormone that acts as an antidiabetic and anti-atherogenic adipokine. We recently cloned the genes encoding two adiponectin receptors (ADIPOR1 and ADIPOR2). The aim of this study was to examine whether ADIPOR1 and/or ADIPOR2 play a major role in genetic susceptibility to insulin resistance or type 2 diabetes in the Japanese population. METHODS: By direct sequencing and a search of public databases, we identified single nucleotide polymorphisms (SNPs) in ADIPOR1 and ADIPOR2, and investigated whether these SNPs are associated with insulin resistance and type 2 diabetes in the Japanese population. RESULTS: The linkage disequilibrium (LD) in the chromosomal region of ADIPOR1 was almost completely preserved, whereas the LD in ADIPOR2 was less well preserved. None of the SNPs in ADIPOR1 or ADIPOR2 were significantly associated with insulin resistance or type 2 diabetes. No differences in ADIPOR1 or ADIPOR2 haplotype frequencies were observed between type 2 diabetic and non-diabetic subjects. CONCLUSIONS/INTERPRETATION: Genetic variations in ADIPOR1 or ADIPOR2 are unlikely to lead to a common genetic predisposition to insulin resistance or type 2 diabetes in the Japanese population.

This study investigated acute and chronic effects of eicosapentaenoic acid (EPA) on voltage-gated Na+ current (I(Na)) expressed in cultured human bronchial smooth muscle cells (hBSMCs). The whole-cell voltage clamp technique and quantitative real-time RT-PCR analysis were applied. The alterations in the fatty acid composition of phospholipids after treatment with EPA were also examined. Extracellular application of EPA produced a rapid and concentration-dependent suppression of tetrodotoxin-sensitive I(Na) with the half-maximal inhibitory concentration of 2 microM. After washing out EPA with albumin, I(Na) returned to the control level. Similar inhibitory effects were observed regarding other fatty acids (docosahexaenoic, arachidonic, stearic, and oleic acids), but EPA was the most potent inhibitor. The effect of EPA on I(Na) was not blocked by nordihydroguaiaretic acid and indometacin, and was accompanied by a significant shift of the steady-state inactivation curve to more negative potentials. In cells chronically treated with EPA, the EPA content of the cell lipid fraction (mol%) increased time-dependently, while arachidonic acid (AA) decreased, resulting in an increase of EPA to AA ratio. Then, the level of mRNA (SCN9A) encoding I(Na) decreased significantly. These results provide novel evidence that EPA not only rapidly inhibits I(Na), but also reduces the mRNA levels of the Na+ channel after cellular incorporation of EPA in cultured hBSMCs.

A 66-year old man, who had been diagnosed with dilated cardiomyopathy and felt a progressive shortness of breath and fatigability, was admitted to hospital. Computed. tomography showed a thickening of the aortic wall from the aortic arch to the aortic bifurcation, as well as mild pleural and pericardial effusion. Intravenous pyelography showed severe ureteral stenosis, along with hydronephrosis, of the left side. There was a marked increase in C-reactive protein and the erythrocyte sedimentation rate, but the serology for connective tissue disease and perinuclear antineutrophil cytoplasmic antibodies was negative. Retroperitoneal fibrosis (RPF) with intrathoracic extension was diagnosed. After confirming the absence of malignant disease, an oral predonisolone treatment of 30 mg/day was started, and this ameliorated the ureteral obstruction, aortic wall thickening and pericardial effusion. The patient had been taking 300 mg of loxoprofen sodium for headaches every day for 16 years. The relationship between loxoprofen, cardiomyropathy and RPF remains unclear. There is a possibility of RPF in the patients with a thickening of thoracic aortic wall, as in this case.

Background Sleep apnea syndrome (SAS) and exercise hyperpnea are common in patients with chronic heart failure (CHF), and although it is not known whether they are both regulated by the same mechanisms, the hypothesis of the present study was that they are related to augmented central chemosensitivity. Methods and Results The oxygen desaturation index (ODI) was evaluated in 29 patients and those with ODI > 5 times/h underwent polysomnography. Patients with an apnea-hypopnea index (AHI) > 15/h without evidence of obstructive apnea were defined as central SAS (CSAS). Cardiopulmonary exercise testing was performed to determine peak oxygen uptake and the VE-VCO2 Slope. A hypercapnic gas mixture (7% CO2/93% O-2) was used to activate the central chemoreflex. Nine patients had central SAS (CHF-CSAS) and 20 did not have apnea (CHF-nonSAS). Patients with CHF-CSAS had a lower peak oxygen uptake than the CHF-nonSAS group (13.0 +/- 2.4 vs 16.9 +/- 4.3 ml (.) kg(-1) (.) min-, p < 0.05). There was a significant correlation between central chemosensitivity and the AHI. (r=0:63, p < 0.05), between central chemosensitivity and the VE-VCO2 slope (r=0.50, p < 0.01), whereas the VE-VCO2 slope showed an insignificant tendency to correlate with AHI (r=0.44, p=0.07). Conclusion CHF-CSAS is associated with impaired exercise tolerance and elevated central chemosensitivity is the responsible mechanism for CSAS and exercise hyperpnea.

Hepatocellular carcinoma is a very common neoplastic disease in countries where hepatitis viruses B and/or C are prevalent. Small hepatocellular carcinoma lesions detected by ultrasonography at an early stage are often hyperechoic because they are composed of well-differentiated cancer cells that are rich in triglyceride droplets. The triglyceride content of hepatocytes depends in part on the rate of lipogenesis. Key lipogenic enzymes, such as fatty acid synthase, are co-ordinately regulated at the transcriptional level. We therefore examined the mRNA expression of lipogenic enzymes in human hepatocellular carcinoma samples from 10 patients who had undergone surgical resection. All of the samples exhibited marked elevation of expression of mRNA for lipogenic enzymes, such as fatty acid synthase, acetyl-CoA carboxylase and ATP citrate lyase, compared with surrounding non-cancerous liver tissue. In contrast, the changes in mRNA expression of SREBP-1, a transcription factor that regulates a battery of lipogenic enzymes, did not show a consistent trend. In some cases where SREBP-1 was elevated, the main contributing isoform was SREBP-1c rather than SREBP-1a. Thus, lipogenic enzymes are markedly induced in hepatocellular carcinomas, and in some cases SREBP-1c is involved in this activation.

Purpose The aim of this study was to assess the prevalence of carotid atherosclerosis in people of both genders who underwent general health screening.<br>Results Of 8144 enrolled subjects (5473 men,2671 women),29.4% of the men and 16.5% of women, and 14.9% of the men and 9.2% of the women had carotid plaque and intima-media thickening, respectively, and the prevalence of both increased with age in both genders. The male-to-female rations for the prevalence of carotid plaque and intima-media thickening peaked at 41-45 years (8.62) and 46-50 years (5.72), respectively. The gender gap for these values declined thereafter, and the male-to-female ratios for the prevalence of carotid plaque and intima-media thickening was 1.29 and 0.93 at age ≥76 years. Conclusion A gender gap in terms of the prevalence of carotid plaque and intima-media thickening is present in mid-life, and thereafter the deference declines in the Japanese population.

Purpose The aim of the present study was to analyze the presence of metabolic syndrome in the Japanese population.<br>Methods Data from 49375 subjects (16886 women,32489 men) with a mean age of 52.2±10.2 years who underwent general health screening between 1994 and 2003 at our institute were analyzed. Metabolic syndrome was diagnosed using modified criteria of the National Cholesterol Education Program Adult Treatment Panel III including five components; increased body mass index, hypertension, increased serum triglycerides, decreased serum HDL cholesterol, and increased fasting glucose.<br>Results The mean overall rate of metabolic syndrome was 10.0% (women,4.1%; men,13.1%), seeming to reach a plateau after 50 years of age in men, but with a continual age-associated increase in women. HOMA-IR rose as the number of metabolic syndrome risk factors increased in both genders. After adjusting for age, serum total cholesterol levels, and smoking status, the frequency of metabolic syndrome was greatest in men (odds ratio,3.17,95% CI 2.89-3.48, p<0.0001).<br>Conclusion In this large Japanese study population undergoing general health screening, metabolic syndrome was found to be over three times more frequent in men than women.

Objective - There are few data available on possible independent association between uric acid and carotid atherosclerosis. Here we first sought to investigate association between uric acid levels and metabolic syndrome in Japanese; second, we assessed whether there is an independent association of uric acid with prevalence of carotid atherosclerosis in individuals subdivided according to gender and metabolic syndrome status. Methods and Results - Cross-sectional data from 8144 individuals who underwent general health screening were analyzed. After adjusting for age, total cholesterol, and smoking status, the odds ratios ( 95% CI) of sex-specific quartiles of serum uric acid for metabolic syndrome were 1.0, 1.06 (0.60 to 1.87), 2.18 (1.30 to 3.64), and 4.17 (2.56 to 6.79) in women, and 1.0, 0.92 (0.74 to 1.14), 1.52 (1.25 to 1.65), and 1.97 (1.61 to 2.40) in men. After adjusting for age, serum levels, total cholesterol, and smoking status, prevalence of carotid plaque was higher in subjects in the second, third, and fourth quartiles of uric acid level with odds ratios ( 95% CI) of 1.24 (1.01 to 1.52), 1.37 (1.11 to 1.68), and 1.31 (1.05 to 1.63), respectively, in men without metabolic syndrome but not in men with metabolic syndrome or in women with or without metabolic syndrome. Conclusion - The prevalence of metabolic syndrome showed a graded increase according to serum uric acid values in both genders. In men who did not have metabolic syndrome, uric acid was found to be an independent risk factor for incidence of carotid plaque.

BACKGROUND: Hyperhomocysteinemia induces vascular endothelial dysfunction, contributing to a predisposition to the onset and/or progression of atherosclerosis. The major mechanism suggested for the adverse effect of homocysteine on vascular function seems to involve oxidative stress. Thus, we hypothesized that the administration of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor fluvastatin, which is experimentally demonstrated to have antioxidative properties as one of its pleiotropic effects, is a useful strategy for eliminating the detrimental events induced by hyperhomocysteinemia. METHODS AND RESULTS: In diet-induced hyperhomocysteinemic rats, we estimated oxidative stress and assessed endothelium-dependent vasodilatation. Hyperhomocysteinemia induced significant increases in urinary 8-isoprostaglandin F2alpha-III excretion and vascular superoxide generation, and impaired endothelium-dependent vasodilatation. Additional oral administration of the antioxidant fluvastatin or vitamin E, which normalized increased oxidative stress induced by hyperhomocysteinemia, ameliorated endothelial dysfunction. CONCLUSIONS: Hyperhomocysteinemia, even mild to moderate, induces endothelial dysfunction through its oxidative effect. The antioxidant fluvastatin was able to cancel out the oxidative stress induced by hyperhomocysteinemia and ameliorate endothelial dysfunction. Clinical use of fluvastatin might be a potent strategy for eliminating the detrimental events induced by hyperhomocysteinemia as well as hyperlipidemia. In addition to lowering homocysteine by means of folate supplementation, administration of the antioxidants is expected to be a potentially effective anti-homocysteine therapy.

Transcription is regulated by a network of transcription factors and related cofactors that act in concert with the general transcription machinery. Elucidating their underlying interactions is important for understanding the mechanisms regulating transcription. Recently, we have shown that Krüppel-like factor KLF5, a member of the Sp/KLF family of zinc finger factors and a key regulator of cardiovascular remodeling, is regulated positively by the acetylase p300 and negatively by the oncogenic regulator SET through coupled interaction and regulation of acetylation. Here, we have shown that the deacetylase HDAC1 can negatively regulate KLF5 through direct interaction. KLF5 interacts with HDAC1 in the cell and in vitro. Gel shift DNA binding assay showed that their interaction inhibits the DNA binding activity of KLF5, suggesting a property of HDAC1 to directly affect the DNA binding affinity of a transcription factor. Reporter assay also revealed that HDAC1 suppresses KLF5-dependent promoter activation. Additionally, overexpression of HDAC1 suppressed KLF5-dependent activation of its endogenous downstream gene, platelet-derived growth factor-A chain gene, when activated by phorbol ester. Further, HDAC1 binds to the first zinc finger of KLF5, which is the same region where p300 interacts with KLF5 and, intriguingly, HDAC1 inhibits binding of p300 to KLF5. Direct competitive interaction between acetylase and deacetylase has been hitherto unknown. Collectively, the transcription factor KLF5 is negatively regulated by the deacetylase HDAC1 through direct effects on its activities (DNA binding activity, promoter activation) and further through inhibition of interaction with p300. These findings suggest a novel role and mechanism for regulation of transcription by deacetylase.

Long-term administration of angiotensin II causes myocardial loss and cardiac fibrosis. We previously found iron deposition in the heart of the angiotensin II-infused rat, which may promote angiotensin II-induced cardiac damage. In the present study, we have investigated whether an iron chelator ( deferoxamine) and a free radical scavenger (T-0970) affect the angiotensin II-induced upregulation of transforming growth factor-beta 1 (TGF-beta 1). Angiotensin II infusion for 7 days caused a robust increase in TGF-beta 1 mRNA expression in vascular smooth muscle cells, myofibroblast-like cells, and migrated monocytes/macrophages. T-0970 and deferoxamine suppressed the upregulation of TGF-beta 1 mRNA and reduced the extent of cardiac fibrosis in the heart of rats treated with angiotensin II. These agents blocked the angiotensin II-induced upregulation of heme oxygenase-1, a potent oxidative and cellular stress-responsive gene, but they did not significantly affect systolic blood pressure or plasma levels of aldosterone. In addition, T-0970 and deferoxamine suppressed the angiotensin II-induced upregulation of monocyte chemoattractant protein-1 in the heart. These results collectively suggest that iron and the iron-mediated generation of reactive oxygen species may contribute to angiotensin II-induced upregulation of profibrotic and proinflammatory genes, such as TGF-beta 1 and monocyte chemoattractant protein-1.

We previously found that ANG II infusion into rats causes iron deposition in the kidney and heart, which may have a role in the regulation of profibrotic gene expression and tissue fibrosis. In the present study, we have investigated whether ANG II can also induce iron accumulation in the liver. Prussian blue staining detected frequent iron deposition in the interstitium of the liver of rats treated with pressor dose ANG II for 7 days, whereas iron deposition was absent in the livers of control rats. Immunohistochemical and histological analyses showed that some iron-positive nonparenchymal cells were positive for ferritin and heme oxygenase-1 (HO-1) protein and TGF-beta 1 mRNA and were judged to be monocytes/macrophages. It was shown that ANG II infusion caused about a fourfold increase in ferritin and HO-1 protein expression by Western blot analysis and about a twofold increase in TGF-beta 1 mRNA expression by Northern blot analysis, which were both suppressed by treating ANG II-infused rats with losartan and deferoxamine. In addition, mild interstitial fibrosis was observed in the liver of rats that had been treated with pressor dose ANG II for 7 days or with nonpressor dose ANG II for 30 days, the latter of which also caused loss of hepatocytes and intrahepatic hemorrhage in the liver. Taken together, our data suggest that ANG II infusion induces aberrant iron homeostasis in the liver, which may have a role in the ANG II-induced upregulation of profibrotic gene expression in the liver.

To study the mechanisms of vascular dysfunction in diabetes mellitus, we examined the responses of the aorta to adrenomedullin (AM) and ANG II in obese Zucker (OZ), lean Zucker (LZ), and OZ rats administered fluvastatin (OZ + Flu). AM-induced endothelium-dependent vasorelaxation was impaired in OZ rats compared with LZ rats, and fluvastatin restored AM-induced, endothelium-dependent vasorelaxation (%Deltatension at 10(-7) mol/l AM; LZ, -85.1 +/- 3.1%; OZ, -50.7 +/- 2.5%; OZ + Flu, -75.6 +/- 2.7%). Expression of endothelial nitric oxide synthase (eNOS) and Akt phosphorylation in response to AM (10(-7) mol/l) were also diminished in OZ rats. Fluvastatin restored the eNOS expression and Akt phosphorylation [eNOS expression (relative intensity): LZ, 2.3 +/- 0.4; OZ, 1.0 +/- 0.2; OZ + Flu, 1.8 +/- 0.3; Akt phosphorylation (relative intensity): LZ, 2.3 +/- 0.2; OZ, 1.0 +/- 0.3; OZ + Flu, 1.9 +/- 0.2]. ANG II-induced vasoconstriction was enhanced in the aortic rings of OZ rats compared with LZ rats, and this enhanced vasoconstriction was partially normalized by fluvastatin and was abolished when the aorta of OZ rats was preincubated with the Rho kinase inhibitor Y-27632. GTPgammaS-induced contraction of permeabilized aortic smooth muscle cells, which is an indicator of the Rho-dependent Ca(2+) sensitization of contraction, was enhanced in OZ rats compared with LZ rats, and this enhanced contraction was suppressed in OZ + Flu rats. These results suggested that endothelium-dependent vasorelaxation was impaired, Ca(2+) sensitization of contraction was augmented in blood vessels of OZ rats and that fluvastatin restored vascular function by activating the Akt-dependent pathway and inhibiting the Rho-dependent pathway.

BACKGROUND: It is well known that diabetes mellitus is a major risk factor for vascular diseases such as atherosclerosis and restenosis after angioplasty. It has become clear that advanced glycation end products (AGE) and their receptor (RAGE) are implicated in vascular diseases, especially in diabetes mellitus. Nevertheless, the mechanisms by which diabetes mellitus is often associated with vascular diseases remain unclear. METHODS AND RESULTS: To study the role of endogenous cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 in the development of vascular diseases and in the expression of RAGE, we used semapimod, a pharmacological inhibitor of cytokine production, and examined its effect on neointimal formation in the femoral artery of obese Zucker (OZ) rats. We also used an adenovirus construct expressing a dominant negative mutant of the receptor for TNF-alpha (AdTNFRDeltaC) to block the action of endogenous TNF-alpha. Semapimod significantly suppressed neointimal formation and RAGE expression in OZ rats compared with untreated OZ rats. This inhibitory effect of semapimod on neointimal formation was overcome by infection of an adenovirus expressing RAGE into the femoral artery of OZ rats. Furthermore, AdTNFRDeltaC infection significantly suppressed neointimal formation and RAGE expression in the femoral artery of OZ rats. CONCLUSIONS: These results suggest that endogenous cytokines, especially TNF-alpha, were implicated in neointimal formation in OZ rats and that RAGE was a mediator of the effect of these cytokines on neointimal formation.

Carvedilol is a beta-adrenoceptor blocker and a potent antioxidant that improves cardiac function in patients with heart failure. The restoration of sarcoplasmic reticulum Ca2+-ATPase (SERCA2) gene expression may be an underlying mechanism of its beneficial effects on cardiac function. In primary cultured neonatal rat cardiac myocytes, treatment with either carvedilol or its beta-receptor inactive metabolite, BM910228, attenuated the hydrogen peroxide-mediated decrease in SERCA2 mRNA and protein levels, while metoprolol, a pure beta-blocker, had no effect. Moreover, carvedilol itself significantly enhanced SERCA2 gene transcription, suggesting that carvedilol specifically restores SERCA2 gene transcription. Site-directed mutagenesis revealed that two Sp1 sites in the SERCA2 gene promoter region mediated the response to carvedilol under oxidative stress. Further, electrophoretic mobility shift assays revealed that Sp1 and Sp3 transcription factors correlated with carvedilol-mediated changes in the promoter assays. These studies may provide a mechanistic explanation for the beneficial effects of carvedilol in heart failure.

Background - Neointimal hyperplasia is the major cause of in-stent restenosis (ISR). The sirolimus-eluting stent (SES) has emerged as a promising therapy to prevent ISR; however, the exact mechanism by which locally delivered sirolimus, an immunosuppressive agent, prevents ISR remains unknown. Recent evidence suggests that circulating progenitor cells may contribute to neointimal formation. Methods and Results - Mononuclear cells (MNCs) were isolated from peripheral blood of healthy human volunteers. Smooth muscle (SM)-like cells outgrew from the culture of MNCs (1 x 10(6)) in the presence of platelet-derived growth factor-BB and basic fibroblast growth factor, whereas endothelial cell - like cells were obtained in the presence of vascular endothelial growth factor. Sirolimus potently inhibited SM-like cell outgrowth. The number of SM-like cells was significantly reduced at a concentration as low as 0.1 ng/mL (15.9 +/- 5.8% of control, P &lt; 0.001). Sirolimus also exerted an inhibitory effect on endothelial cell - like cells that originated from MNCs. Wire-mediated vascular injury was induced in femoral arteries of bone marrow chimeric mice. Either vehicle or sirolimus was administered locally to the perivascular area of the injured arteries. Sirolimus significantly reduced neointima hyperplasia at 4 weeks (intima/media ratio 2.0 &PLUSMN; 0.3 versus 1.0 &PLUSMN; 0.2, P &lt; 0.05) with a decreased number of bone marrow - derived SM-like cells and hematopoietic cells in the lesion. Reendothelialization was retarded in the arteries treated with sirolimus. Conclusions - The potent inhibitory effects of sirolimus on circulating smooth muscle progenitor cells may mediate the clinical efficacy of SES, at least in part. Sirolimus potentially may affect reendothelialization after stent implantation.

Tako-tsubo-like cardiomyopathy is a newly-recognized enigmatic disease characterized by transient left ventricular dysfunction of a broad area of the apex with a hyperkinetic area around the cardiac base. There is ST-segment elevation with no coronary stenosis. The exact mechanism for this entity remains unknown. Here, we report a case of tako-tsubo-like cardiomyopathy that showed a marked left ventricular hypertrophy (LVH) when the wall motion returned to normal. LVH was normalized at 10 months. The cause of LVH remains unknown.

骨髄由来細胞の血管病変形成への関与を明らかにするため様々な方法を用いて検証した.異なる血管傷害モデルを用いた検討,一個の造血幹細胞を用いた骨髄置換マウスでの解析,Parabiosisモデルを用いた検討,末梢血からの血管前駆細胞の分離実験などを行い以下の結論を得た.血液中の前駆細胞は傷害血管のリモデリングに関与する.特にVaso Vasorumからの新生血管によってプラーク内部へ到達した前駆細胞は,動脈硬化巣構成細胞に分化し粥腫の進展と性状決定に重要な役割を演じる.アンジオテンシン受容体拮抗薬は血中前駆細胞数を減少させ動脈硬化を抑制する可能性がある

社会技術研究ミッションプログラムI医療安全研究グループにおいては，大学医学部附属病院における理論構築環境（研究）と，技術実装環境（大学医学部附属病院）を活用して，他の研究グループと連携を図りつつ，医療安全向上を目的とした包括的，かつ多層的なアプローチで研究を行った．その中でも，社会技術としては，医師-患者間，医師間，医療機関間において，臨床判断に資する情報共有，コミュニケーションを促進するツールとしての診療ナビゲーションシステムを開発し，東京大学医学部附属病院循環器内科において実装した．

Introduction The success of the Human Genome Project and the availability of the complete sequence of the human genome have revealed the existence of numerous genes whose functions are unknown. Although many functional genes were successfully identified using libraries of randomized ribozymes (Kruger et al., 2000 Li et al., 2000 Welch et al., 2000 Beger et al., 2001 Kawasaki et al., 2002 Kawasaki and Taira, 2002 Onuki et al., 2002 Nelson et al., 2003 Rhoades and Wong-Staal, 2003 Suyama et al., 2003a, b Chatterton et al., 2004 Kuwabara et al., 2004 Onuki et al., 2004), the randomized library naturally contained many ribozymes that could not hybridize with any human gene transcripts, resulting in some false positives. While exploitation of RNA interference (RNAi) is hampered by the induction of the interferon response upon the introduction of double-stranded RNA (dsRNA) into mammalian cells (Elbashir et al., 2001), RNAi can be a powerful tool in gene analysis and, for example, the functions of a number of genes in the nematode Caenorhabditis elegans have been identified as being associated with particular mutant phenotypes (Fraser et al., 2000 Gonczy et al., 2000). Recent progress in mammalian cells, as discussed in this chapter, suggests that it should soon be possible to extend genome-wide approaches to mammalian cells, using libraries of small interfering RNA (siRNA) oligonucleotides or siRNA-expression libraries. Efforts to exploit the phenomenon of RNA interference (RNAi) in mammalian cells have been hampered by the interferon response in cells into which foreign RNA is introduced.

Background: Therapeutic angiogenesis by using cells is being performed in Japan. However, it is unknown in how many centers and how these therapies are performed. The efficacy and side effects are also unknown. Thus, we conducted the survey by mailing questionnaire within Japan. Methods and results: Two surveys were performed in 2003. The first survey unveiled that cell therapy was performed in 32 facilities until October 2003. The second survey unveiled the followings. (1) The total number of performed cases was 221. 153 patients (69.2%) had arterio-sclerosis obliterance (ASO), 56 patients (25.3%) thromboangitis obliterans (TAO, Burger's disease), and 12 patients (5.4%) other conditions. (2) The sources of cells were bone marrow-mononuclear cells (61.5%), peripheral-mononuclear cells (9.5%), and peripheral CD34+ cells (22.1%). A few patients (6.7%) were treated with a cytokine only (granulocyte-colony stimulating factor: G-CSF). (3) Inclusion criteria were the same for most facilities, such as patients with PAOD, especially with critical limb ischemia with rest pain, non-healing ischemic ulcers and non-candidates for non-surgical or surgical revascularization. All facilities excluded patients with histories of malignant disorder during the past 5 years, proliferative diabetic retinopathy, pregnancy, proliferative blood disease, uncontrolled ischemic heart disease, rheumatic arthritis, or psychiatric disease. (4) Subjective improvement was observed in 138 of 199 patients (69%). Objective improvements for ABI, TcO2 or angiographic findings were observed in 98 of 182 patients (53.8%). (6) Three of 221 patients (1.4%) died after cell therapy. One died from cerebro-vascular attack (thrombo-embolizm), and two died from acute myocardial infarction (AMI). Conclusion: Our clinical survey has shown that cell therapy is being performed in many medical centers in Japan. It seems to be safe and effective for patients with PAOD with no surgical options © 2005 Springer-Verlag Tokyo.

Atherosclerosis is responsible for more than half of all deaths in western countries. Numerous studies have reported that exuberant accumulation of smooth muscle cells play a principal role in the pathogenesis of vascular diseases. It has been assumed that smooth muscle cells derived from the adjacent medial layer migrate, proliferate and synthesize extracellular matrix. Although much effort has been devoted, targeting migration and proliferation of medial smooth muscle cells, no effective therapy to prevent occlusive vascular remodeling has been established. Recently, we reported that bone marrow cells substantially contribute to the pathogenesis of vascular diseases, in models of post-angioplasty restenosis, graft vasculopathy and hyperlipidemia-induced atherosclerosis. It was suggested that bone marrow cells may have the potential to give rise to vascular progenitor cells that home in the damaged vessels and differentiate into smooth muscle cells or endothelial cells, thereby contributing to vascular repair, remodeling, and lesion formation. This article overviews recent findings on circulating vascular precursors and describes potential therapeutic strategies for vascular diseases, targeting mobilization, homing, differentiation and proliferation of circulating progenitor cells. © 2005 Springer-Verlag Tokyo.

Objective Skeletal muscle glucose utilization (SMGU) can be measured by F-18-FDG PET to characterize insulin resistance. The aim of this study was to determine whether femoral muscle SMGU can be measured without arterial blood sampling by sequential PET imaging of the thoracic and femoral regions. Methods Ten patients with possible insulin resistance underwent dynamic F-18-FDG PET of the femoral region during hyperinsulinaemic euglycaemic clamping (group A), and femoral muscle SMGU was calculated using PET data of various time periods and measured arterial input. SMGU was also calculated using venous plasma activity, instead of arterial activity, as input during the late phase. Another five patients underwent sequential PET of the thoracic and femoral regions after single tracer injection (group B). The input function was estimated from aorta activity on thoracic images during the early phase and from venous activity during the late phase, and SMGU with this estimated input was compared with that with measured arterial input. Results In group A, exclusion of early dynamic PET data from analysis had essentially no effect on the calculated SMGU, and partial substitution of venous activity for arterial activity only marginally changed the estimates. The difference between SMGUs with measured and estimated inputs was minimal in group B. Conclusion Femoral muscle SMGU can be calculated without femoral imaging early after tracer injection, and the input function can be assessed using data of thoracic imaging and venous blood samples. These results support the validity of measuring femoral muscle SMGU without arterial sampling, simultaneously with measurement of myocardial glucose utilization. (C) 2005 Lippincott Williams Wilkins.

The cluster of metabolic and hemodynamic risk factors known as metabolic syndrome is known to be a risk factor for ischemic cardiovascular diseases and stroke. By analyzing the cross-sectional data from 8,144 individuals (age 19-88 years) who underwent general health screening, we have investigated the prevalence of metabolic syndrome, as diagnosed by modified-National Cholesterol Education Program (NCEP) criteria corresponding to the following five categories: triglycerides &gt;= 150 mg/dl; high density lipoprotein (HDL)-cholesterol &lt; 40 mg/dl in men or &lt; 50 mg/dl in women; fasting plasma glucose &gt;= 110 mg/dl; systolic/diastolic blood pressure &gt;= 130/85 mmHg; and body mass index &gt; 25 kg/m(2). We found that the prevalence of metabolic syndrome was 19% in men and 7% in women. After adjustment for age, metabolic syndrome was found to be significantly more prevalent in men than in women, with an odds ratio of 3.08 (95% confidence interval [CI] 2.62-3.61, p &lt; 0.0001). Among the five metabolic/hemodynamic risk factor components, hypertension was observed most frequently in individuals with metabolic syndrome, at 85% in men and 87% in women. In addition, multivariate logistic regression analysis adjusted for age, sex, serum total cholesterol levels, and smoking status showed that hypertension possessed the greatest odds ratio (1.43, 95% CI 1.27-1.60) for carotid plaque among the metabolic/hemodynamic risk factors. These data emphasize the importance of controlling blood pressure for reducing the risk of both metabolic syndrome and carotid arteriosclerosis in apparently healthy individuals.

Cimetidine, a histamine type-2 receptor antagonist, has been reported to improve survival of patients with cancers. However, the exact mechanisms by which cimetidine suppresses development of cancers remain to be elucidated. Solid tumors require neovascularization for their growth. Here, we investigated the effects of cimetidine on tumor growth and angiogenesis. Syngeneic colon cancer cells, CMT93 cells, were inoculated into the subcutaneous space of C57BL/6 mice. Mice were treated with either saline or cimetidine. Tumor size was measured everyday and angiogenesis was evaluated histologically. Cimetidine markedly suppressed tumor growth with reduced neovascularization in the tumor. Cimetidine had no effect on proliferation of CMT93 cells in vitro. Vascular endothelial growth factor production by cancer cells was not affected by cimetidine, while vascular-like tube formation by endothelial cells in vitro was significantly impaired in the presence of cimetidine. Our findings suggest that cimetidine suppresses tumor growth, at least in part, by inhibiting tumor-associated angiogenesis. (c) 2004 Elsevier SAS. All rights reserved.

Endothelin-1 is known to be implicated in the pathogenesis of hepatobiliary diseases such as cirrhosis, especially in portal hypertension. This study aimed to investigate the effects of ursodeoxycholic acid on endothelin-1 production in human endothelial cells. The effects of ursodeoxycholic acid and its conjugates (tauroursodeoxycholic and glycoursodeoxycholic acids) on endothelin-1 production as well as nitric oxide (NO) in human umbilical vein endothelial cells (HUVECs) were examined. The production of endothelin-1 and nitric oxide in culture medium was measured using enzyme-linked immunosorbent assay (ELISA) and the Griess method, respectively. Endothelin-1 and endothelial nitric oxide synthase (eNOS) mRNA expression were investigated by real-time quantitative reverse transcriptase/polymerase chain reaction (RT-PCR). Ursodeoxycholic acid (30-1000 microM) inhibited endothelin-1 production in a concentration-dependent manner, and ursodeoxycholic acid at concentrations higher than 300 microM increased nitric oxide production in culture medium. The conjugates of ursodeoxycholic acid also increased nitric oxide production and decreased endothelin-1 production, which was less effective than ursodeoxycholic acid. N-nitro-L-arginine-mythel-ester (L-NAME), a nitric oxide synthase (NOS) inhibitor, suppressed the ursodeoxycholic acid-induced nitric oxide production, but it did not antagonize the inhibitory effects of ursodeoxycholic acid on endothelin-1 production. Ursodeoxycholic acid also induced a concentration-dependent decrease in endothelin-1 mRNA expression without significant changes in eNOS mRNA expression. These results provide novel evidence that ursodeoxycholic acid inhibits endothelin-1 production in human endothelial cells, but nitric oxide is not responsible for the inhibitory effect of ursodeoxycholic acid on endothelin-1. Thus, ursodeoxycholic acid therapy may prevent the development of several pathogenesis such as portal hypertension observed in patients with cirrhosis due to the improvement of endothelial function.

Since there is in sufficient evidence on patients with coronary artery disease in Japan, the Japanese Coronary Artery Disease (JCAD) Study, in which 217 institutions participate, was designed to collect basic data based on evidence-based medicine (EBM). In this study, cardiac catheterization is performed on all cases to select study subjects confirmed as having CAD diagnosed based on the criteria that he or she has stenosis in at least one branch of a coronary artery to the extent of 75% or higher according to the AHA classification. Data including background information, risk factors, clinical management, and medication are to be collected over the web. The follow-up arm of the study consists of following each subject for three years to obtain data on the long-term prognosis of patients with CAD while the other arm is for enrolling new subjects every six months who will be followed for six months only for the purpose of determining the latest trend in patients. The two arms of the study have been ongoing since April 2000. As of September 30, 2003, 15,506 subjects have been enrolled in the follow-up arm and the follow-up data have been entered in the database. The authors plan to report data showing any correlation between incidence rate, focusing mainly on cerebrocardiovascular events, and other factors such as the management of risk factors, and type and dosage of medications obtained in the largest cohort ever studied in Japan of patients with a coronary artery lesion confirmed by cardiac catheterization.

Disruption of histamine H2 receptor and gastrin receptor had different effects growth of gastric mucosa: hypertrophy and atrophy, respectively. To clarify the roles of gastrin and histamine H2 receptors in gastric mucosa, mice deficient in both (double-null mice) were generated and analyzed. Double-null mice exhibited atrophy of gastric mucosae, marked hypergastrinemia and higher gastric pH than gastrin receptor-null mice, which were unresponsive even to carbachol. Comparison of gastric mucosae from 10-week-old wild-type, histamine H2 receptor-null, gastrin receptor-null and double-null mice revealed unique roles of these receptors in gastric mucosal homeostasis. While small parietal cells and increases in the number and mucin contents of mucous neck cells were secondary to impaired acid production, the histamine H2 receptor was responsible for chief cell maturation in terms of pepsinogen expression and type III mucin. In double-null and gastrin receptor-null mice, despite gastric mucosal atrophy, surface mucous cells were significantly increased, in contrast to gastrin-null mice. Thus, it is conceivable that gastrin-gene product(s) other than gastrin-17, in the stimulated state, may exert proliferative actions on surface mucous cells independently of the histamine H2 receptor. These findings provide evidence that different G-protein coupled-receptors affect differentiation into different cell lineages derived from common stem cells in gastric mucosa.

Paradoxical outward movement of left ventricular (LV) inferior wall in systole is occasionally recognized in normal subjects and clinically important in terms of the differential diagnosis between physiological pseudo-asynergy and pathological asynergy. In this study, the potential mechanisms by which pseudo-asynergy of LV inferior wall (PLI) is observed in normal subjects were investigated. PLI was defined as the outward movement of LV inferior wall observed during more than 50% of systole. The was evaluated in 7843 consecutive subjects in routine echocardiography. The effects of body position and artificial gravity on the manifestation of PLI were also examined. PLI was observed in 0.11% (9 / 7842) of subjects on left lateral position. Measurement of the angle formed by LV long-axis and the long-axis of the body on frontal plane revealed that hearts in subjects with PLI were in relatively horizontal position. PLI was observed on sitting position in 43% (40/92) of subjects without PLI on left lateral position. The subjects with sitting position-induced PLI exhibited significantly higher obesity index. PLI was also PLI in routine echocardiography is relatively low, PLI can be induced in normal subjects by any condition that causes close contact of LV inferior wall to diaphragm. Thus, PLI should be taken into consideration in the differential diagnosis of abnormal LV inferior wall motion, especially when performing exercise echocardiography.

AIM: Vascular intimal hyperplasia is an important clinical concern in vascular diseases, such as anastomotic stricture as a possible complication of cardiovascular surgery. We recently suggested that a rat aortotomy model could be substituted for a vascular anastomotic stricture around a suture line. TNP-470 is known as an angiogenesis inhibitor and has demonstrated abilities to inhibit DNA synthesis of smooth muscle cells (SMCs) and SMCs proliferation. The aim of this study was to investigate the effect of TNP-470 on SMC proliferation using rat aortotomy models. METHODS: Longitudinal aortotomy was performed in the abdominal aorta of rats. Rats received a subcutaneous injection of materials (TNP-470, 20 mg/kg) or vehicle 3 times a week (n=10 in each group). The aorta was harvested 2 weeks after aortotomy. Serial sections from tissues were stained with hematoxylin and eosin, and the ratio of intimal to medial cross-sectional areas (I/M ratio) was determined. Values are expressed as the mean +/- the standard deviation. Results. Thickening of the intimal layer 2 weeks following aortotomy was observed in the control group however, intimal thickening was inhibited in the TNP-treated group. The I/M ratio was significantly (p = 0.0376) lower in the TNP-treated group than in the control group (8.3 +/- 4.8 vs 15.6 +/- 9.6%). Conclusion. TNP-470 significantly suppressed intimal thickening in experimental rat aortotomy models. TNP-470 might inhibit the development of anastomotic stricture after cardiovascular surgery.

Background - Carbon monoxide (CO) is postulated to protect tissues against several types of injuries. We investigated the role of CO in amelioration of cardiac ischemia - reperfusion injury in vivo and the mechanisms involved in it. Methods and Results - Rats inhaled CO ( 250 ppm, 500 ppm, or 1000 ppm) for 24 hours in a chamber after myocardial ischemia - reperfusion induced by occluding the left anterior descending coronary artery for 30 minutes. Pre-exposure to 1000 ppm of CO significantly reduced the ratio of infarct areas to risk areas and suppressed the migration of macrophages and monocytes into infarct areas, and the expression of tumor necrosis factor (TNF)-alpha in the heart; however, 250 ppm, 500 ppm of CO, or low barometric pressure hypoxia (0.5 atm) did not affect them. Exposure to 1000 ppm CO resulted in the activation of p38 mitogen-activated protein kinase (p38MAPK), protein kinase Balpha(Akt), endothelial nitric oxide synthase ( eNOS), and cyclic guanosine monophosphate ( cGMP) in the myocardium. Inhibition of p38MAPK, PI3kinase, NO, and soluble guanylate cyclase with SB203580, wortmannin, N(G)-nitro-L-arginine methyl ester (L-NAME), and methylene blue, respectively, attenuated the cytoprotection by CO. Conclusion - CO has beneficial effects on cardiac ischemia - reperfusion injury; this effect is mediated by p38MAPK pathway and Akt - eNOS pathway, including production of cGMP.

Objective - Accelerated coronary arteriosclerosis remains a major problem in the long-term survival of cardiac transplant recipients. However, the pathogenesis of graft vasculopathy is poorly understood, and there is no effective therapy. Transplant arteriosclerosis is characterized by early mononuclear cell attachment on the transplanted vessel followed by development of concentric neointimal hyperplasia. Early and persistent expression of monocyte chemoattractant protein-1 (MCP-1) in cardiac allografts has been implicated for the pathogenesis of transplant arteriosclerosis. Methods and Results - We investigated whether anti-MCP-1 gene therapy can inhibit the development of intima hyperplasia in a mouse model of cardiac transplantation. Either the dominant-negative form of MCP-1 (7ND) or control vector was transfected into the skeletal muscles of B10.D2 mice. Cardiac allografts from DBA/2 mice were transplanted heterotopically into B10.D2 mice. 7ND gene transfer was associated with a significant reduction of the number of mononuclear cells accumulating in the lumen of the graft coronary arteries at 1 week and an attenuation of the development of the lesion at 8 weeks (intima/media ratio 0.79 +/- 0.05 versus 0.48 +/- 0.04). Conclusions - The MCP-1/chemokine receptor 2 (CCR2) signaling pathway plays a critical role in the pathogenesis of graft vasculopathy. This new anti-MCP-1 gene therapy might be useful to treat graft vascular disease.

We previously demonstrated that insulin receptor substrate 2 (Irs2) KO mice develop diabetes associated with hepatic insulin resistance, lack of compensatory beta cell hyperplasia, and leptin resistance. To more precisely determine the roles of Irs2 in beta cells and the hypothalamus, we generated beta cell-specific Irs2 KO and hypothalamus-specific Irs2 knockdown (betaHT-IRS2) mice. Expression of Irs2 mRNA was reduced by approximately 90% in pancreatic islets and was markedly reduced in the arcuate nucleus of the hypothalamus. By contrast, Irs2 expression in liver, muscle, and adipose tissue of betaHT-IRS2 mice was indistinguishable from that of control mice. The betaHT-IRS2 mice displayed obesity and leptin resistance. At 4 weeks of age, the betaHT-IRS2 mice showed normal insulin sensitivity, but at 8 and 12 weeks, they were insulin resistant with progressive obesity. Despite their normal insulin sensitivity at 8 weeks with caloric restriction, the betaHT-IRS2 mice exhibited glucose intolerance and impaired glucose-induced insulin secretion. beta Cell mass and beta cell proliferation in the betaHT-IRS2 mice were reduced significantly at 8 and 12 weeks but not at 10 days. Insulin secretion, normalized by cell number per islet, was significantly increased at high glucose concentrations in the betaHT-IRS2 mice. We conclude that, in beta cells and the hypothalamus, Irs2 is crucially involved in the regulation of beta cell mass and leptin sensitivity.

Cardiac hypertrophy is formed in response to hemodynamic overload. Although a variety of factors such as catecholamines, angiotensin II (AngII), and endothelin-1 (ET-1) have been reported to induce cardiac hypertrophy, little is known regarding the factors that inhibit the development of cardiac hypertrophy. Production of atrial natriuretic peptide (ANP) is increased in the hypertrophied heart and ANP has recently been reported to inhibit the growth of various cell types. We therefore examined whether ANP inhibits the development of cardiac hypertrophy. Pretreatment of cultured cardiomyocytes with ANP inhibited the AngII- or ET-1-induced increase in the cell size and the protein synthesis. ANP also inhibited the AngII- or ET-1-induced hypertrophic responses such as activation of mitogen-activated protein kinase (MAPK) and induction of immediate early response genes and fetal type genes. To determine how ANP inhibits cardiomyocyte hypertrophy, we examined the mechanism of ANP-induced suppression of the MAPK activation. ANP strongly induced expression of MAPK phosphatase-1 (MKP-1) and overexpression of MKP-1 inhibited AngII- or ET-1-induced hypertrophic responses. These growth-inhibitory actions of ANP were mimicked by a cyclic GMP analog 8-bromo-cyclic GMP. Taken together, ANP directly inhibits the growth factor-induced cardiomyocyte hypertrophy at least partly via induction of MKP-1. Our present study suggests that the formation of cardiac hypertrophy is regulated not only by positive but by negative factors in response to hemodynamic load.

BACKGROUND: Endothelial production of nitric oxide (NO) is attenuated in patients with essential hypertension. We investigated whether treatment with amlodipine increased exhaled NO output (VNO) at rest and during exercise in patients with essential hypertension. METHODS: We studied the effect of amlodipine in seven untreated hypertensive patients. Cardiopulmonary exercise testing and NO measurement of exhaled air were performed on these patients before and after 2 months of amlodipine treatment. RESULTS: Amlodipine decreased blood pressure (BP) both at rest and during exercise (at rest: 147.1 +/- 6.4 [SEM]/89.9 +/- 4.4 v 133.6 +/- 5.4/82.7 +/- 3.9 mm Hg, P <.05; at peak exercise: 224.9 +/- 8.0/113.1 +/- 5.3 v 207.0 +/- 6.0/100.7 +/- 5.0 mm Hg, P <.05) without affecting heart rate (at rest: 67.6 +/- 3.9 v 70.4 +/- 4.5 beats/min, P =.33; peak exercise: 146.4 +/- 7.4 v 144.0 +/- 7.2 beats/min, P =.49). Amlodipine did not affect minute ventilation (VE) at rest or during exercise. It did not alter anaerobic threshold, peak oxygen uptake (peak VO(2)), or peak workload. However, after amlodipine treatment, VNO was significantly greater both at rest (130.8 +/- 19.4 v 180.4 +/- 24.8 nL/min, P <.05) and at peak exercise (380.0 +/- 47.5 v 582.6 +/- 74.3 nL/min, P <.05). CONCLUSIONS: Amlodipine increased NO production, at least in the pulmonary circulation, in patients with essential hypertension. In addition to its antihypertensive effect, the enhancement of NO production by amlodipine in the vasculature of other organs may contribute to its beneficial effects on the cardiovascular system.

Adrenomedullin (AM) is a novel vasodilating peptide involved in the regulation of circulatory homeostasis and implicated in the pathophysiology of cardiovascular disease. We tested the hypothesis that AM also possesses angiogenic properties. Using laser Doppler perfusion imaging, we found that AM stimulated recovery of blood flow to the affected limb in the mouse hind-limb ischemia model. AM exerted this effect in part by promoting expression of vascular endothelial growth factor (VEGF) in the ischemic limb, and immunostaining for CD31 showed the enhanced flow to reflect increased collateral capillary density. By enhancing tumor angiogenesis, AM also promoted the growth of subcutaneously transplanted sarcoma 180 tumor cells. However, heterozygotic AM knockout mice (AM+/-) showed significantly less blood flow recovery with less collateral capillary development and VEGF expression than their wild-type littermates. Similarly, mice treated with AM22-52, a competitive inhibitor of AM, showed reduced capillary development, and growth of sarcoma 180 tumors was inhibited in AM+/- and AM22-52-treated mice. Notably, administration of VEGF or AM rescued blood flow recovery and capillary formation in AM+/- and AM22-52-treated mice. In cocultures of endothelial cells and fibroblasts, AM enhanced VEGF-induced capillary formation, whereas in cultures of endothelial cells AM enhanced VEGF-induced Akt activation. These results show that AM possesses novel angiogenic properties mediated by its ability to enhance VEGF expression and Akt activity. This may make AM a useful therapeutic tool for relieving ischemia; conversely, inhibitors of AM could be useful for clinical management of tumor growth.

Dec2, a member of the basic helix-loop-helix superfamily, is a recently confirmed regulatory protein for the clockwork system. Transcripts of Dec2, as well as those of its related gene Dec1, exhibit a striking circadian oscillation in the suprachiasmatic nucleus, and Dec2 inhibits transcription from the Per1 promoter induced by Clock/Bmal1 [Honma, Kawamoto, Takagi, Fujimoto, Sato, Noshiro, Kato and Honma (2002) Nature (London) 419, 841-844]. It is known that mammalian circadian rhythms are controlled by molecular clockwork systems based on negative-feedback loop(s), but the molecular mechanisms for the circadian regulation of Dec2 gene expression have not been clarified. We show here that transcription of the Dec2 gene is regulated by several clock molecules and a negative-feedback loop. Luciferase and gel retardation assays showed that expression of Dec2 was negatively regulated by binding of Dec2 or Dec1 to two CACGTG E-boxes in the Dec2 promoter. Forced expression of Clock/Bmal1 and Clock/Bmal2 markedly increased Dec2 mRNA levels, and upregulated the transcription of the Dec2 gene through the CACGTG E-boxes. Like Dec, Cry and Per also suppressed Clock/Bmal-induced transcription from the Dec2 promoter. Moreover, the circadian expression of Dec2 transcripts was abolished in the kidney of Clock/Clock mutant mice. These findings suggest that the Clock/Bmal heterodimer enhances Dec2 transcription via the CACGTG E-boxes, whereas the induced transcription is suppressed by Dec2, which therefore must contribute to its own rhythmic expression. In addition, Cry and Per may also modulate Dec2 transcription.

Triglyceride (TG) accumulation in pancreatic beta-cells is thought to be associated with impaired insulin secretory response to glucose (lipotoxicity). To better understand the mechanism of the impaired insulin secretory response to glucose in beta-cell lipotoxicity, we overexpressed a constitutively active form of the sterol regulatory element-binding protein- 1c (SREBP-1c), a master transcriptional factor of lipogenesis, in INS-1 cells with an adenoviral vector. This treatment was associated with strong activation of transcription of the genes involved in fatty acid biosynthesis, increased cellular TG content, severely blunted glucose-stimulated insulin secretion, and enhanced expression of the uncoupling protein-2 (UCP-2), which supposedly dissipates the mitochondrial electrochemical potential. To decrease the up-regulated UCP-2 expression, small interfering RNA for UCP-2 was used. Introduction of the small interfering RNA increased the ATP/ADP ratio and partially rescued the glucose-stimulated insulin secretion in the cells overexpressing SREBP-1c, but did not affect the cellular TG content. Next, the effect of the AMP-activated protein kinase (AMPK) agonist, 5-amino-4-imidazolecarboxamide riboside, was examined in the lipotoxicity model. Exposure of the cells with lipotoxicity to 5-amino-4-imidazolecarboxamide riboside increased free fatty acid oxidation, partially reversed the TG accumulation, phosphorylated AMPK and acetyl-coenzyme A carboxylase, and improved the impaired glucose-stimulated insulin secretion. These results suggest that UCP-2 down-regulation and AMPK activation could be candidate targets for releasing beta-cells from lipotoxicity.

We have previously shown that abnormal iron metabolism might be one underlying mechanism of the renal damage observed in the angiotensin II-infused rat. Transforming growth factor-beta1 (TGF-beta1) is known to play a crucial role in the development of renal damage induced by activation of the renin-angiotensin-aldosterone system. The purpose of the present study was to examine the effects of an iron chelator and a free radical scavenger on the angiotensin II-induced upregulation of TGF-beta1 in the kidney. Rats were given angiotensin II (0.7 mg/kg/day) via osmotic minipumps for 7 days. The expressions of the mRNAs of TGF-beta1 and collagen types I and IV were significantly increased in response to angiotensin II treatment. Histologic analysis showed that TGF-beta1 expression was upregulated mainly in tubular epithelial cells, and occasionally in glomerular and perivascular cells, some of which were identified as monocytes and/or macrophages. Although tubular cells that overexpressed TGF-beta1 did not contain iron particles, angiotensin II-induced TGF-beta1 upregulation was suppressed by the iron chelator and the free radical scavenger. The free radical scavenger also suppressed angiotensin II-induced upregulation of heme oxygenase-1, an oxidative-stress sensitive gene. By contrast, administration of iron dextran to rats induced upregulation of TGF-beta1 mRNA. Collectively, these data suggest that the renal iron overload and presumed subsequent increase in oxidative stress play a role in angiotensin II-induced upregulation of the mRNAs of TGF-beta1 and collagen types I and IV in the kidney.

Endothelial PAS domain protein 1 (EPAS1) is a basic-helix-loop-helix/PAS domain transcription factor that is expressed preferentially in vascular endothelial cells. EPAS1 shares high homology with hypoxia-inducible factor-1alpha (HIF-1alpha) and is reported to transactivate vascular endothelial growth factor (VEGF), fetal liver kinase-1 (Flk-1), and Tie2 promoters. In this study, we analyzed the role of EPAS1 in the process of angiogenesis. Using microarray technology, we looked for target genes regulated by EPAS1 in vascular endothelial cells. A total of 130 genes were upregulated by EPAS1, including fms-like tyrosine kinase-1 (Flt-1). Reporter analysis using human Flt-1 promoter and gel mobility shift assays showed that the heterodimer of EPAS1 and aryl hydrocarbon receptor nuclear translocator binds directly to HIF-1-binding site upstream of Flt-1 promoter and transactivates it. Small interfering RNA targeted to EPAS1 but not HIF-1alpha attenuated desferrioxamine-induced Flt-1 mRNA expression, thus EPAS1 is thought to play an essential role in hypoxic induction of Flt-1 gene. Furthermore, using mouse wound healing models, we demonstrated that adenovirus-mediated delivery of EPAS1 gene significantly induced the expression of VEGF, Flt-1, Flk-1, and Tie2 mRNA at the wound site and promoted mature angiogenesis. The proportion of the number of mural cells in newly formed vessels was significantly higher in EPAS1-treated wound area than VEGF-treated area. In conclusion, EPAS1 promotes Flt-1 gene expression and induces mRNA expression of VEGF, Flk-1, and Tie2, leading to enhancement of mature angiogenesis in vivo. Thus, EPAS1 may contribute to the construction of mature vessels by modulating the coordinated expressions of VEGF, Flt-1, Flk-1, and Tie2.

Growth factors, cell-surface receptors, adhesion molecules, and extracellular matrix proteins play critical roles in vascular pathophysiology by affecting growth, migration, differentiation, and survival of vascular cells. In a search for secreted and cell-surface molecules expressed in the cardiovascular system, by using a retrovirus-mediated signal sequence trap method, we isolated a cell-surface protein named vasorin. Vasorin is a typical type I membrane protein, containing tandem arrays of a characteristic leucine-rich repeat motif, an epidermal growth factor-like motif, and a fibronectin type III-like motif at the extracellular domain. Expression analyses demonstrated that vasorin is predominantly expressed in vascular smooth muscle cells, and that its expression is developmentally regulated. To clarify biological functions of vasorin, we searched for its binding partners and found that vasorin directly binds to transforming growth factor (TGF)-beta and attenuates TGF-beta signaling in vitro. Vasorin expression was down-regulated during vessel repair after arterial injury, and reversal of vasorin down-regulation, by using adenovirus-mediated in vivo gene transfer, significantly diminished injury-induced vascular lesion formation, at least in part, by inhibiting TGF-beta signaling in vivo. These results suggest that down-regulation of vasorin expression contributes to neointimal formation after vascular injury and that vasorin modulates cellular responses to pathological stimuli in the vessel wall. Thus, vasorin is a potential therapeutic target for vascular fibroproliferative disorders.

Adiponectin/Acrp30 is a hormone secreted by adipocytes, which acts as an antidiabetic and antiatherogenic adipokine. We reported previously that AdipoR1 and -R2 serve as receptors for adiponectin and mediate increased fatty acid oxidation and glucose uptake by adiponectin. In the present study, we examined the expression levels and roles of AdipoR1/R2 in several physiological and pathophysiological states such as fasting/refeeding, obesity, and insulin resistance. Here we show that the expression of AdipoR1/R2 in insulin target organs, such as skeletal muscle and liver, is significantly increased in fasted mice and decreased in refed mice. Insulin deficiency induced by streptozotocin increased and insulin replenishment reduced the expression of AdipoR1/R2 in vivo. Thus, the expression of AdipoR1/R2 appears to be inversely correlated with plasma insulin levels in vivo. Interestingly, the incubation of hepatocytes or myocytes with insulin reduced the expression of AdipoR1/R2 via the phosphoinositide 3-kinase/Foxo1-dependent pathway in vitro. Moreover, the expressions of AdipoR1/R2 in ob/ob mice were significantly decreased in skeletal muscle and adipose tissue, which was correlated with decreased adiponectin binding to membrane fractions of skeletal muscle and decreased AMP kinase activation by adiponectin. This adiponectin resistance in turn may play a role in worsening insulin resistance in ob/ob mice. In conclusion, the expression of AdipoR1/R2 appears to be inversely regulated by insulin in physiological and pathophysiological states such as fasting/refeeding, insulin deficiency, and hyper-insulinemia models via the insulin/phosphoinositide 3-kinase/Foxo1 pathway and is correlated with adiponectin sensitivity.