研究者詳細 - 廣畑　聡

論文 - 廣畑　聡

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ADAMTS9 activation by interleukin 1 beta via NFATc1 in OUMS-27 chondrosarcoma cells and in human chondrocytes. 査読国際誌

Kursat Oguz Yaykasli, Toshitaka Oohashi, Satoshi Hirohata, Omer Faruk Hatipoglu, Kiichi Inagawa, Kadir Demircan, Yoshifumi Ninomiya

Molecular and cellular biochemistry 323 ( 1-2 ) 69 - 79 2009年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SPRINGER

ADAMTS9 is a member of the disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) genes, with aggrecan-degrading activity. It has also been characterized to be reactive and highly activated ADAMTS by IL-1 beta in both chondrosarcoma cells and human chondrocytes (Demircan et al. Arthritis Rheum 52:1451-1460, 2005). In order to understand the regulation of ADAMTS9 gene expression a functional 3.0 kb human ADAMTS9 promoter has been cloned and characterized. A sequence analysis of the promoter revealed the presence of putative binding sites for Nuclear Factor of Activated T cells (NFAT), which is commonly found in the ADAMTS4 and ADAMTS5 promoters. NFATc1 was up-regulated in an activated form by IL-1 beta in human chondrocytes. The IL-1 beta inducible ADAMTS9 expression was inhibited by NFAT inhibitors, FK506 and 11Arg (11R)-VIVIT. Furthermore, direct binding of NFATc1 on distal and proximal promoters of ADAMTS9 was demonstrated by a chromatin immunoprecipitation assay. Promoter-reporter assays supported those results. These findings may provide a better understanding of the regulation of ADAMTS9 expression induced by inflammatory cytokines.

DOI： 10.1007/s11010-008-9965-4

siRNA silencing reveals role of vascular cell adhesion molecule-1 in vascular smooth muscle cell migration 査読国際誌

Erik J. Petersen, Toru Miyoshi, Zuobiao Yuan, Satoshi Hirohata, Jin Zhong Li, Weibin Shi, John F. Angle

ATHEROSCLEROSIS 198 ( 2 ) 301 - 306 2008年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER IRELAND LTD

Vascular cell adhesion molecule-1 (VCAM-1) is an adhesion molecule expressed by endothelial cells for recruitment of leukocytes during inflammation. It is also abundantly expressed by smooth muscle cells in atherosclerotic lesions and in injured arteries. In this study, we examined the role of VCAM-1 in smooth muscle cell migration. Smooth muscle cells were isolated from the aorta of C57BL/6 mice and transfected with short interfering RNAs (siRNAs) targeting VCAM-1. Inhibition on VCAM-1 expression by siRNAs was assessed by Western blot analysis, RT-PCR and by measuring soluble VCAM-1 concentrations in the incubation medium. One siRNA that showed greater suppression on VCAM-1 expression was used for migration assay. A single scratch wound was made on 70% confluent cells and cells migrated from wounded monolayer were counted 24 and 48 h after injury. Treatment with VCAM-1 siRNA resulted in a significant reduction in the number of migrated cells. This siRNA also exhibited a minor effect on smooth muscle cell proliferation. Thus, our findings indicate that VCAM-1 is necessary for the migration of smooth muscle cells and interfering VCAM-1 expression could be an effective approach to prevention and treatment of atherosclerosis and restenosis. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.atherosclerosis.2007.10.015

Trichostatin A, a histone deacetylase inhibitor, suppresses synovial inflammation and subsequent cartilage destruction in a collagen anti body-induced arthritis mouse model 査読

Y. Nasu, K. Nishida, S. Miyazawa, T. Komiyama, Y. Kadota, N. Abe, A. Yoshida, S. Hirohata, A. Ohtsuka, T. Ozaki

OSTEOARTHRITIS AND CARTILAGE 16 ( 6 ) 723 - 732 2008年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCI LTD

Objective: To investigate the effect of the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), on joint inflammation and cartilage degeneration in a collagen antibody-induced arthritis (CAIA) mouse model.
Methods: CAIA mice were given daily subcutaneous injections of various concentrations of TSA (0, 0.5, 1.0, and 2.0 mg/kg) and various parameters were monitored for 14 days. On Day 15, the hind paws were examined histologically. To investigate the effects of TSA on the expressions of matrix metal lop roteinase (MMP)-3, MMP-13, tissue inhibitor of MMP-1 (TIMP-1), and acetyl-H4 by chondrocytes, another group of mice was sacrificed on Day 6. In vitro direct effect of TSA was examined by real-time PCR for mRNA of type II collagen, aggrecan, MMP-3, and MMP-13 in murine chondrogenic ATDC5 cells after pro-inflammatory cytokine stimulation.
Results: In the TSA-treated group, clinical arthritis was significantly ameliorated in a dose-dependent manner. The severity of synovial inflammation and the cartilage destruction score were significantly lower in the TSA 2.0 mg/kg group compared to the other TSA-treated groups. On immunohistochemistry, the number of MMP-3 and MMP-13-positive chondrocytes was significantly lower in the TSA 2.0 mg/kg group than in the control group. In contrast, the number of TIMP-II-positive cells and acetyl-histone H4-positive cells was significantly higher in the TSA 2.0 mg/kg group than in the control group. TSA suppressed interleukin 1-beta and tumor necrosis factor-a-stimulated up-regulation of MMP-3, but not MMP-13 mRNA expression by ATDC5.
Conclusion: The systemic administration of TSA ameliorated synovial inflammation in CAIA mice. Subsequently cartilage destruction was also suppressed by TSA, at least in part, by modulating chondrocyte gene expression. (C) 2007 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.joca.2007.10.014

Association of augmentation index of radial pressure wave form with diurnal variation pattern of blood pressure in untreated patients with essential hypertension 査読国際誌

Ryoko Shinohata, Takaaki Nakatsu, Yoko Yuki, Aya Nishitani, Keiichi Mashima, Shinji Toyonaga, Hiroko Ogawa, Satoshi Hirohata, Shinichi Usui, Tomoki Kitawaki, Shozo Kusachi

JOURNAL OF HYPERTENSION 26 ( 3 ) 535 - 543 2008年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：LIPPINCOTT WILLIAMS & WILKINS

Objectives The augmentation index of the radial pulse wave has been reported to be a sensitive aortic stiffness marker in relatively young but not in older individuals. We studied the relationship between augmentation index and the diurnal blood pressure profiles.
Patients and methods Twenty-four-hour ambulatory blood pressure monitoring was performed in 90 untreated patients with uncomplicated essential hypertension. The patients were classified into four groups: dippers, extreme dippers, nondippers, and risers. Augmentation index was calculated as the percentage of the second systolic peak relative to the first systolic peak.
Results No significant differences in the averaged whole 24-h systolic or diastolic blood pressure were observed in the whole set of patients or in subgroup patients with age 60 years or under. In the whole set of patients (58.7 +/- 12.9 years), there were significant differences in augmentation index between patients with abnormal (other than dippers) and normal diurnal blood pressure profiles (dippers). In subgroup patients with age 60 years or below (49.1 +/- 9.1 years, n = 48), the abnormal diurnal blood pressure profile group showed significantly higher augmentation index (89.6 +/- 10.3%) than dippers (80.5 +/- 11.8%). The area under the curve in the receiver operating characteristics curve for distinguishing between dippers than other dippers was 0.73 (P < 0.01). Multivariate analysis demonstrated that abnormal diurnal blood pressure profile was independently associated with increase in augmentation index. In contrast, these relationships were not significant in the over 60 years subgroup patients (69.8 +/- 5.6 years old, n = 42).
Conclusions The present study revealed that augmentation index was associated with dipping blood pressure patterns in untreated hypertensive patients aged 60 years or younger. Augmentation index determination would be useful for initial assessment in connection with possible abnormal diurnal blood pressure variability in patients with age 60 years or younger.

DOI： 10.1097/HJH.0b013e3282f2fdb6

Serum interferon-gamma-inducible protein 10 level was increased in myocardial infarction patients, and negatively correlated with infarct size 査読国際誌

Kazuya Koten, Satoshi Hirohata, Toru Miyoshi, Hiroko Ogawa, Shinichi Usui, Ryoko Shinohata, Mutsumi Iwamoto, Tomoki Kitawaki, Shozo Kusachi, Kosaku Sakaguchi, Tohru Ohe

CLINICAL BIOCHEMISTRY 41 ( 1-2 ) 30 - 37 2008年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD

Objectives: We examined the serum levels of interferon-gamma-inducible protein 10 (IP-10), an inflammation-induced chemokine, in acute myocardial infarction (AMI).
Design and methods: The subjects were 33 AMI patients, 20 stable angina pectoris patients (AP) and 20 normal subjects. In AMI patients, blood samples were collected before percutaneous coronary intervention (PCI) and on days 3, 7 and 28.
Results: Patients with AMI showed significantly higher serum IP-10 levels (137.5 +/- 79.8 pg/mL) than control subjects (91.2 +/- 40.1 pg/mL) and patients with AP (93.3 +/- 41.1 pg/mL). The serum IP-10 level before PCI was negatively correlated with infarct size, as indicated by cumulative release of creatine kinase (CK) and peak CK and its isoenzyme CK-MB Stepwise multiple regression analysis revealed that the serum IP-10 level before PCI was an independent predictor of cumulative CK release.
Conclusions: The serum IP-10 level was increased in AMI, and a higher level of serum IP- 10 before PCI may be informative regarding infarct size. (c) 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

DOI： 10.1016/j.clinbiochem.2007.10.001

Antibodies against heat shock protein 60 derived from Helicobacter pylori: Diagnostic implications in cardiovascular disease 査読

Tomoyuki Okada, Kiyoshi Ayada, Shinichi Usui, Kenji Yokata, Jinhua Cui, Yoshiro Kawahara, Tomoki Inaba, Satoshi Hirohata, Motowo Mizuno, Daisuke Yamamoto, Shozo Kusachi, Eiji Matsuura, Keiji Oguma

JOURNAL OF AUTOIMMUNITY 29 ( 2-3 ) 106 - 115 2007年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD

Immune responses against heat shock protein 60 (HSP60) of pathogen-origin are thought to be defensive events which, due to molecular mimicry, misdirect to a human counterpart. Therefore, atherosclerosis may be serologically predicted by anti-HSP60 antibodies (Abs). In the present study, we analyzed the clinical prevalence of the serum IgG Abs against Helicobacter pylori (Hp)-derived HSP60 (Hp-HSP60) or its peptide fragments in patients with cardiovascular disease (CVD; n = 250), as compared to those in age- and gender-matched non-CVD patients (n = 293). Anti-Hp cell lysate Abs frequently appeared in Hp-infected patients who were not associated with CVD. In contrast, Abs against the particular amino acid sequence Hp-HSP60(II3) (II3 region, Glu(141)-Leu(160), in Hp-HSP60) predominantly appeared in CVD patients, as well as IgG anti-human HSP60 (Hu-HSP60(w)). Furthermore, neither titer of anti-Hp-HSP60(113) nor anti-Hu-HSP60(w) Abs was correlated with the levels of high sensitivity C-reactive protein (hsCRP). This data strongly suggested that IgG anti-Hp-HSP60(II3) Abs cross-reacted with Hu-HSP60(w) were independent diagnostic markers relevant to CVD. Further, the 20 amino acid residues (Glu(141)-Leu(160)) might be predominant CVD-associated epitopes that induce anti-Hu-HSP60 auto-Abs, whose location was predicted in the tertiary structure of Hu-HSP60. (c) 2007 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jaut.2007.05.004

Use of plasma B-Type natriuretic peptide level to identify asymptomatic hypertensive patients with abnormal diurnal blood pressure variation profiles: nondippers, extreme dippers, and risers 査読国際誌

Takaaki Nakatsu, Ryoko Shinohata, Keiichi Mashima, Yoko Yuki, Aya Nishitani, Shinji Toyonaga, Hiroko Ogawa, Satoshi Hirohata, Shinichi Usui, Shozo Kusachi

HYPERTENSION RESEARCH 30 ( 7 ) 651 - 658 2007年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：NATURE PUBLISHING GROUP

We examined the relationship between plasma B-type natriuretic peptide (BNP) level and diurnal variability pattern of blood pressure (BP). Twenty-four-hour ambulatory BP monitoring was performed in 98 patients with asymptomatic essential hypertension, and the patients were classified into four groups according to their circadian BP variation profiles: dippers (n=29), nondippers (n=36), extreme dippers (n=19), and risers (n=14). Plasma BNP was measured by enzyme immunoassay. Based on the distribution pattern of BNP values, the values were analyzed after logarithmic transformation. Significant differences in plasma BNP levels among the types of circadian BP variations were demonstrated by analysis of variance (p < 0.0005). Nondippers and risers showed significantly higher plasma BNP levels (mean [range: -1 SD and +1 SD]: 16.1 [6.3, 41.6) pg/mL and 29.2 [15.9, 53.4] pg/mL, respectively) than dippers (8.4 [3.7, 19.1] pg/mL). The area under the receiver operating characteristics curve for distinguishing patients with abnormal circadian BP variation from those with normal variation was 0.72, indicating that plasma BNP levels were useful for distinguishing between these patients. Specificity of 69% and sensitivity of 72% were obtained with a cut-off value of 10.5 pg/mL (log plasma BNP, 1.02) for distinguishing the abnormal diurnal BP profile group from the normal group. In conclusion, hypertensive patients with abnormal diurnal BP variation patterns (nondippers, extreme dippers, and risers) showed higher plasma BNP levels than those with normal circadian BP variation (dippers). Plasma BNP level is clinically useful for the identification of hypertensive patients who have abnormal circadian BP variability, which increases the risk of cardiovascular events.

DOI： 10.1291/hypres.30.651

Thrombospondin-1 is induced in rat myocardial infarction and its induction is accelerated by ischemia/reperfusion 査読国際誌

S Sezaki, S Hirohata, A Iwabu, K Nakamura, K Toeda, T Miyoshi, H Yamawaki, K Demircan, S Kusachi, Y Shiratori, Y Ninomiya

EXPERIMENTAL BIOLOGY AND MEDICINE 230 ( 9 ) 621 - 630 2005年10月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOC EXPERIMENTAL BIOLOGY MEDICINE

Thrombospondin-1 (TSP-1) is a multifunctional, rapid-turnover matricellular protein. Recent studies demonstrated that TSP-1 has a role in regulating inflammatory reactions. Myocardial infarction (MI) is associated with an inflammatory response, ultimately leading to healing and scar formation. In particular, an enhanced inflammatory reaction and a massive accumulation of monocytes/macrophages is seen with reperfusion after MI. To examine the role of TSP-1 in MI, we isolated rat TSP-1 complementary DNA (cDNA) and analyzed the level and distribution of the mRNA expression. In infarcted rat hearts, TSP-1 mRNA increased markedly at 6 and 12 hrs after coronary artery ligation (27.97 +/- 3.40-fold and 22.77 +/- 1.83-fold, respectively, compared with sham-operated hearts). Western blot analysis revealed that TSP-1 protein was transiently induced in the infarcted heart. Using in situ hybridization analysis, TSP-1 mRNA signals were observed in the infiltrating cells at the border area of infarction. We then examined the effect of ischemia/reperfusion (I/R) on TSP-1 mRNA induction in the rats with infarcted hearts. Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) demonstrated that I/R enhanced the TSP-1 mRNA expression approximately 4-fold, as compared with the level in the permanently ligated heart. Finally, we examined the effect of TSP-1 on proinflammatory cytokine release in mononuclear cells. The releases of interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) from human mononuclear cells were enhanced by TSP-1 in a dose-dependent manner. Thus, the immediate and marked increase of TSP-1 expression suggests that TSP-1 has an inflammatory-associated role in MI.

DOI： 10.1177/153537020523000904

Therapeutic efficacy of PUMA for malignant glioma cells regardless of p53 status 査読国際誌

H Ito, T Kanzawa, T Miyoshi, S Hirohata, S Kyo, A Iwamaru, H Aoki, Y Kondo, S Kondo

HUMAN GENE THERAPY 16 ( 6 ) 685 - 698 2005年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：MARY ANN LIEBERT INC

Replacement of the p53 tumor suppressor gene is a rational approach to the management of malignant gliomas because p53 is frequently mutated or inactivated in these cancers. Major weaknesses of this approach are that malignant gliomas are mixtures of cells with wild- type and mutant p53, and that tumor cells exhibiting wildtype p53 are resistant to p53 gene transfer. An effective alternative is needed to overcome these difficulties. p53-upregulated modulator of apoptosis ( PUMA) was identified as a p53- inducible proapoptotic molecule. Our purpose was to elucidate a role for PUMA in p53 gene therapy and to investigate whether PUMA is an efficient substitute for p53 in cancer therapy. We demonstrated that PUMA was upregulated in mutant p53 malignant glioma cells (U373-MG and T98G) undergoing apoptosis but was not upregulated in apoptosis-resistant wild- type p53 malignant glioma cells (U87-MG and D54) after adenoviral transfer of p53. Overexpression of PUMA resulted in massive apoptosis associated with mitochondrial damage and caspase-3 activation in all tumor cells tested. Use of the human telomerase reverse transcriptase ( hTERT) promoter system induced apoptosis only in malignant glioma cells with telomerase activity, while sparing normal cells lacking telomerase. The ability of PUMA to induce apoptosis was greater than that of caspase-6 or caspase-8 transfer, using the same system. Moreover, exogenous expression of PUMA under the hTERT promoter system significantly suppressed the growth of subcutaneous U87-MG tumors in nude mice and did not induce apoptosis in surrounding nontumor tissues. These results indicate that PUMA, which is regulated under a tumor-specific expression system such as the hTERT promoter, may be better than p53 as a therapeutic tool for malignant gliomas.

DOI： 10.1089/hum.2005.16.685

ADAMTS-9 is synergistically induced by interleukin-1 beta and tumor necrosis factor alpha in OUMS-27 chondrosarcoma cells and in human chondrocytes 査読国際誌

K Demircan, S Hirohata, K Nishida, OF Hatipoglu, T Oohashi, T Yonezawa, SS Apte, Y Ninomiya

ARTHRITIS AND RHEUMATISM 52 ( 5 ) 1451 - 1460 2005年5月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY-LISS

Objective. To compare induction of the aggrecanases (ADAMTS-1, ADAMTS-4, ADAMTS-5, ADAMTS-8, ADAMTS-9, and ADAMTS-15) by interleukin-1β (IL-1β) and tumor necrosis factor a (TNFα) in chondrocyte-like OUMS-27 cells and human chondrocytes, and to determine the mechanism of induction of the most responsive aggrecanase gene.
Methods. OUMS-27 cells were stimulated for different periods of time and with various concentrations of IL-1β and/or TNFa. Human chondrocytes obtained from osteoarthritic joints and human skin fibroblasts were also stimulated with IL-1β and/or TNFα. Total RNA was extracted, reverse transcribed, and analyzed by quantitative real-time polymerase chain reaction and Northern blotting. ADAMTS-9 protein was examined by Western blotting, and the role of the MAPK signaling pathway for ADAMTS9 induction in IL-1β-stimulated OUMS-27 cells was investigated.
Results. IL-1β increased messenger RNA (mRNA) levels of ADAMTS4, ADAMTS5, and ADAMTS9 but not ADAMTS1 and ADAMTS8. The fold increase for ADAMTS9 mRNA was greater than that for mRNA of the other aggrecanase genes. The increase of ADAMTS9 mRNA by IL-1β stimulation was greater in chondrocytes than in fibroblasts. The combination of IL-1β and TNFα had a synergistic effect, resulting in a considerable elevation in the level of ADAMTS9 mRNA. ADAMTS-9 protein was also induced in IL-1β-stimulated OUMS-27 cells. The MAPK inhibitors SB203580 and PD98059 decreased ADAMTS9 upregulation in OUMS-27 cells.
Conclusion. ADAMTS9 is an IL-1β- and TNFα-inducible gene that appears to be more responsive to these proinflammatory cytokines than are other aggrecanase genes. Furthermore, these cytokines had a synergistic effect on ADAMTS9. Together with the known ability of ADAMTS-9 to proteolytically degrade aggrecan and its potential to cleave other cartilage molecules, the data suggest that ADAMTS-9 may have a pathologic role in arthritis.

DOI： 10.1002/art.21010

Suppression of chondrosarcoma cells by 15-deoxy-Delta (12,14)-prostaglandin J(2) is associated with altered expression of Bax/Bcl-xL and p21 査読国際誌

ZN Shen, K Nishida, H Doi, T Oohashi, S Hirohata, T Ozaki, A Yoshida, Y Ninomiya, H Inoue

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 328 ( 2 ) 375 - 382 2005年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE

We previously reported that 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), the most potent agonist for peroxisome proliferator-activated receptor gamma (PPARgamma), induces apoptosis of human chondrosarcoma cell line OUMS-27. The current study aimed to explore the mechanism of 15d-PGJ(2)-induced apoptosis and inhibition of cell proliferation in OUMS-27 cells. The preliminary results of cDNA microarray analysis showed the down-regulation of anti-apoptotic Bcl-xL and up-regulation of pro-apoptotic Bax in the process of 15d-PGJ2-induced apoptosis. These changes were further confirmed at mRNA and protein levels by RT-PCR and Western blot analysis, respectively. Among cyclin-dependent kinase inhibitors, p21 was induced and up-regulated by 15d-PGJ2, but p 16 and p27 were not changed, suggesting that the involvement of p21 in inhibition of cell proliferation. Activation of caspase-3 by 15d-PGJ2 was partly, but not completely, blocked by PPARgamma antagonist (GW9662) suggesting the 15d-PGJ2 exerted its effect by PPARgamma-dependent and -independent pathways. Interestingly, immunohistochemical study on human chondrosarcoma samples revealed that Bcl-xL is frequently expressed by tumor cells. The results of the current study suggest that the potential ability of 15d-PGJ2 in regulation of cell cycle and inhibition of Bcl-xL expression might be beneficial in the development of novel pharmacological agents for chondrosarcoma. (C) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2004.12.186

Time-dependent changes in plasma osteopontin levels in patients with anterior-wall acute myocardial infarction after successful reperfusion: Correlation with left-ventricular volume and function 国際誌

C Suezawa, S Kusachi, T Murakami, K Toeda, S Hirohata, K Nakamura, K Yamamoto, K Koten, T Miyoshi, Y Shiratori

JOURNAL OF LABORATORY AND CLINICAL MEDICINE 145 ( 1 ) 33 - 40 2005年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：MOSBY, INC

Osteopontin Is a secreted extracellular-matrix glycoprotein that plays a role in the healing of remodeling tissue. We examined the relationship of plasma osteopontin levels with left-ventricular (LV) volume and function In 18 consecutive patients who underwent successful reperfusion after anterior-wall acute myocardial Infarction (AMI). The plasma osteopontin level was within the control range at admission (mean +/- SD 420 +/- 195 ng/mL), began to Increase on day 2 (935 +/- 464 ng/mL), and reached a maximum around day 3 (1139 482 ng/mL). The level remained high on days 4, 5, and 7 (similar to1000 ng/mL) and then decreased on day 14. Maximal plasma osteopontin levels and the difference between maximal and minimal levels were positively correlated with LV end-systolic volume index (r = .58, P < .05; and r = .65, P < .01, respectively) and negatively correlated with LV election fraction (r = -.52, P < .05; and r = -.60, P < .01, respectively). The area under the curve of plasma osteopontin levels for 14 days after AMI was significantly correlated with LV end-systolic volume index (r = .66, P < .01), LV end-diastolic volume index (r = .50, P < .05), and LV ejection fraction (r = -.55, P < .05). In subgroup patients with the same area of risk for myocardial infarction (ie, responsible lesions located at the same proximal left anterior descending coronary artery), essentially the some or a closer relationship between plasma osteopontin level and LV volume and function was noted. Plasma osteopontin levels were correlated substantially with plasma levels of high-sensitivity C-reactive protein (hsCRP) and weakly with serum creatine kinase release. In conclusion, the plasma level of osteopontin changes In a time-dependent fashion and Is correlated with LV volumes and function and associated substantially with the extent of the Inflammatory response Indicated by the plasma hsCRP level and weakly with Infarct size estimated on the basis of cardiac-enzyme release.

DOI： 10.1016/j.lab.2004.08.007

Lp3/Hapln3, a novel link protein that co-localizes with versican and is coordinately up-regulated by platelet-derived growth factor in arterial smooth muscle cells 国際誌

H Ogawa, T Oohashi, M Sata, Y Bekku, S Hirohataa, K Nakamura, T Yonezawa, S Kusachi, Y Shiratori, Y Ninomiya

MATRIX BIOLOGY 23 ( 5 ) 287 - 298 2004年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE BV

Link proteins (LPs) belong to the link-module superfamily, which can stabilize and enhance the binding of lecticans to hyaluronan. We report here the identification and characterization of a novel rat link protein gene (Lp3/Hapln3). The deduced protein sequence shares the typical modular elements of link proteins and has an estimated mass of 39 kDa. Examination of the rat genomic DNA sequence revealed that Lp3/ Hapln3 and aggrecan genes were paired on chromosome 1q31. Another LP gene and the lectican gene were also paired at a different locus, as they are in the human and mouse genomes. Immunohistochemical analysis showed the prominent expression of Lp3/Hapln3 in the smooth muscle tissues of the vascular wall and gastrointestinal tract. Further comparative studies revealed that Lp3/Hapln3 was well co-localized with versican around the smooth muscle cells of blood vessels but not around endothelial cells. In vitro experiments using primary cultured rat arterial smooth muscle cells (ASMCs) demonstrated the coordinated up-regulation of Lp3/Hapln3 and versican by platelet-derived growth factor (PDGF). These data were supported by in vivo studies of a mechanical vascular injury model in mice. Altogether, our results suggest that Lp3/Hapln3 is involved, together with versican and hyaluronan, in the formation of the pericellular matrix of vascular smooth muscle cells. (C) 2004 Elsevier B.V./International Society of Matrix Biology. All rights reserved.

DOI： 10.1016/j.matbio.2004.07.001

Increased expression of dermatopontin mRNA in the infarct zone of experimentally induced myocardial infarction in rats: comparison with decorin and type I collagen mRNAs 国際誌

S Takemoto, T Murakami, S Kusachi, A Iwabu, S Hirohata, K Nakamura, S Sezaki, J Hayashi, C Suezawa, Y Ninomiya, T Tsuji

BASIC RESEARCH IN CARDIOLOGY 97 ( 6 ) 461 - 468 2002年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：DR DIETRICH STEINKOPFF VERLAG

Objectives Dermatopontin, a 22 kDa extracellular matrix (ECM) protein, has been shown to interact with other ECM components, especially decorin, and to regulate ECM formation. We examined dermatopontin mRNA expression in the myocardial infarct zone. Methods The cDNA encoding the rat dermatopontin was cloned by RT-PCR based on screening results from the Expressed Sequence Tag(TM) database. The dermatopontin mRNA expression was examined in the infarct zone after experimentally induced myocardial infarction in rats by the methods of Northern blotting and in situ hybridization. The expression of dermatopontin mRNA was compared to that of decorin and type I collagen mRNAs. Results The isolated clone contained a 609 bp cDNA insert containing a complete open reading frame encoding 202 amino acids. The rat dermatopontin cDNA showed high homology to human and mouse counterparts (>96%). Northern blotting demonstrated that dermatopontin mRNA expression did not markedly increase on day 2, but was increased on days 7, 14 and 28 by 2.4-, 4.1- and 4.2-fold, respectively, compared to that in preligation hearts. Dermatopontin mRNA expression was regulated almost in parallel with decorin mRNA expression. In situ hybridization demonstrated mRNA signals for dermatopontin in macrophages and spindle-shaped mesenchymal cells (fibroblasts and myofibroblasts) located in the infarct interior zone around infarcted necrotic tissue on day 7. Coexpression of dermatopontin mRNA with decorin and type I collagen mRNAs was observed in spindle-shaped mesenchymal cells. Conclusions The present results demonstrated the time-dependent increase in the expression of dermatopontin mRNA in parallel with that of decorin mRNA in the infarct zone. Coexpression of dermatopontin mRNA with decorin and type I collagen mRNAs suggests that dermatopontin plays a role in ECM (fibrillar collagen matrix) reformation in the infarct along with decorin and type I collagen.

DOI： 10.1007/s00395-002-0371-x

Caspase-8 gene therapy using the human telomerase reverse transcriptase promoter for malignant glioma cells 国際誌

T Komata, Y Kondo, T Kanzawa, H Ito, S Hirohata, S Koga, H Sumiyoshi, M Takakura, M Inoue, BP Barna, EM Germano, S Kyo, S Kondo

HUMAN GENE THERAPY 13 ( 9 ) 1015 - 1025 2002年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：MARY ANN LIEBERT INC PUBL

Telomerase is a distinctive candidate for targeted gene therapy of malignant gliomas, because the vast majority of malignant gliomas express telomerase activity while normal brain tissues do not. Recently, we developed a telomerase-specific expression system of caspase-8 gene using the promoter of the human telomerase reverse transcriptase (hTERT) gene. However, the transcriptional activity of hTERT-181 promoter (a 181-base pair [bp] region upstream of the transcription start site) was relatively lower in malignant glioma cells than in other tumors such as prostate cancer cells. To establish the hTERT/caspase-8 construct as a novel therapy for malignant gliomas, we need to increase the transcriptional activity of the hTERT promoter in malignant glioma cells. In the present study, we demonstrate that the transcriptional activity of hTERT-378 promoter (a 378-bp region) was 2- to 40-fold higher in hTERT-positive malignant glioma cells (A172, GB-1, T98G, U87-MG, U251-MG, and U373-MG) than that of hTERT-181. We further demonstrate that by using the hTERT-378/caspase-8 construct, apoptosis was restricted to malignant glioma cells, and was not seen in astrocytes or fibroblasts lacking hTERT. Moreover, the growth of subcutaneously established U373-MG tumors in mice was significantly inhibited by seven daily intratumoral injections of hTERT-378/caspase-8 construct and its inhibitory effect persisted during 3 additional weeks without additional treatment. These results suggest that the telomerase-specific expression of caspase-8 under hTERT-378 promoter is a novel targeting approach for the treatment of telomerase-positive malignant gliomas.

DOI： 10.1089/104303402753812421

Punctin, a novel ADAMTS-like molecule, ADAMTSL-1, in extracellular matrix 国際誌

S Hirohata, LW Wang, M Miyagi, L Yan, MF Seldin, DR Keene, JW Crabb, SS Apte

JOURNAL OF BIOLOGICAL CHEMISTRY 277 ( 14 ) 12182 - 12189 2002年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Punctin (ADAMTSL-1) is a secreted molecule resembling members of the ADAMTS family of proteases. Punctin lacks the pro-metalloprotease and the disintegrin-like domain typical of this family but contains other ADAMTS domains in precise order including four thrombospondin type I repeats. Punctin is the product of a distinct gene on human chromosome 9p21-22 and mouse chromosome 4 that is expressed in adult skeletal muscle. His-tagged punctin expressed in stably transfected High-Five(TM) insect cells was purified to apparent homogeneity by Ni-chromatography of conditioned medium. The NH2 terminus is not blocked and has the sequence EEDRD and so forth as determined by Edman degradation, demonstrating signal peptidase processing. Recombinant epitope-tagged punctin has a calculated mass of 59,991 Da but exhibits major molecular species of 61970 +/- 6 Da and 62131 +/- 5 Da as measured by liquid chromatography electrospray mass spectrometry. Punctin is a glycoprotein based on carbohydrate staining and liquid chromatography electrospray mass spectrometry glycopeptide analysis. Glycosylation occurs at a single N-linked site as demonstrated by altered electrophoretic migration of punctin expressed in the presence of tunicamycin A. Punctin contains disulfide bonds based on antibody accessibility and electrophoretic migration under reducing versus nonreducing conditions. Rotary shadowing demonstrates that punctin is hatchet-shaped having a globular region attached to a short stem. In transfected COS-1 cells, punctin is deposited in the cell substratum in a punctate fashion and is excluded from focal contacts. Punctin is the first member of a novel family of ADAMTS-like proteins that may have important functions in the extracellular matrix.

DOI： 10.1074/jbc.M109665200

Procollagen II amino propeptide processing by ADAMTS-3 - Insights on dermatosparaxis

RJ Fernandes, S Hirohata, JM Engle, A Colige, DH Cohn, DR Eyre, SS Apte

JOURNAL OF BIOLOGICAL CHEMISTRY 276 ( 34 ) 31502 - 31509 2001年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

The amino and carboxyl propeptides of procollagens I and Il are removed by specific enzymes as a prerequisite for fibril assembly. Null mutations in procollagen I N-propeptidase (ADAMTS-2) cause dermatosparaxis in cattle and the Ehlers-Danlos syndrome (dermatosparactic type) in humans by preventing proteolytic excision of the N-propeptide of procollagen I. We have found that procollagen II is processed normally in dermatosparactic nasal cartilage, suggesting the existence of another N-propeptidase(s). We investigated such a role for ADAMTS-3 in Swarm rat chondrosarcoma RCS-LTC cells, which fail to process the procollagen II N-propeptide. Stable transfection of RCS-LTC cells with bovine ADAMTS-2 or human ADAMTS-3 partially rescued the, processing defect, suggesting that ADAMTS-3 has procollagen II N-propeptidase activity. Human skin and skin fibroblasts showed 30-fold higher mRNA levels of ADAMTS-2 than ADAMTS-3, whereas ADAMTS-3 mRNA was 5-fold higher than ADAMTS-2 mRNA in human cartilage. We propose that both ADAMTS-2 and ADAMTS-3 process procollagen II, but ADAMTS-3 is physiologically more relevant, given its preferred expression in cartilage. The findings provide an explanation for the sparing of cartilage in dermatosparaxis and, perhaps, for the relative sparing of some procollagen I-containing tissues.

DOI： 10.1074/jbc.M103466200

Treatment of malignant glioma cells with the transfer of constitutively active caspase-6 using the human telomerase catalytic subunit (human telomerase reverse transcriptase) gene promoter

T Komata, Y Kondo, T Kanzawa, S Hirohata, S Koga, H Sumiyoshi, SM Srinivasula, BP Barna, IM Germano, M Takakura, M Inoue, ES Alnemri, JW Shay, S Kyo, S Kondo

CANCER RESEARCH 61 ( 15 ) 5796 - 5802 2001年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER ASSOC CANCER RESEARCH

Because the apoptotic pathway is often disrupted in tumor cells, its genetic restoration is a very attractive approach for the treatment of tumors. To treat malignant gliomas with this approach, it would be preferred to restrict induction of apoptosis to tumor cells by establishing a tumor-specific expression system. Telomerase is an attractive target because the vast majority of malignant gliomas have telomerase activity whereas normal brain cells do not. Activation of telomerase is tightly regulated at the transcriptional level of the telomerase catalytic subunit [human telomerase reverse transcriptase, (hTERT)]. Therefore, we hypothesized that using a hTERT promoter-driven vector system, an apoptosis-inducible gene may be preferentially restricted to telomerase- or hTERT-positive tumor cells. In this study, we constructed an expression vector consisting of the constitutively active caspase-6 (rev-caspase-6) under the hTERT promoter (hTERT/rev-caspase-6) and then investigated its antitumor effect on malignant glioma cells. The rationale for using the rev-caspase-6 gene is because it induces apoptosis independent of the initiator caspases. We demonstrated that the hTERT/rev-caspase-6 construct induced apoptosis in hTERT-positive malignant glioma cells, but not in hTERT-negative astrocytes, fibroblasts, and alternative lengthening of telomeres cells. In addition, the growth of s.c. tumors in nude mice was significantly suppressed by the treatment with hTERT/rev-caspase-6 construct. The present results strongly suggest that the telomerase-specific transfer of the rev-caspase-6 gene under the hTERT promoter is a novel targeting approach for the treatment of malignant gliomas.

FADD gene therapy using the human telomerase catalytic subunit (hTERT) gene promoter to restrict induction of apoptosis to tumors in vitro and in vivo

S Koga, S Hirohata, Y Kondo, T Komata, M Takakura, M Inoue, S Kyo, S Kondo

ANTICANCER RESEARCH 21 ( 3B ) 1937 - 1943 2001年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：INT INST ANTICANCER RESEARCH

Gene transfer vectors will dramatically increase the safety and effectiveness of cancer gene therapy, if they could restrict expression of the therapeutic products to the target tumors. To realize such a tumor-targeting system, telomerase is one of the most promising candidates. It is because telomerase activity is detected in the vast majority of tumors, but not in most normal cells. Activation of telomerase is tightly regulated at the transcriptional level of the telomerase catalytic subunit (hTERT). Therefore, the use of the hTERT promoter-driven vector system could restrict the expression of therapeutic products to telomerase positive tumors. In this study, we constructed the expression vector of FADD gene with death domain afforded by the hTERT promoter (hTERT/FADD) and investigated its effect on tumors in vitro and in vivo. Transient transfection with the hTERT/FADD construct induced apoptosis in telomerase-positive tumor cells of wide range. In contrast, normal fibroblast cells without telomerase did not undergo apoptosis following the hTERT/FADD transfer. Furthermore, the growth of subcutaneous tumors in nude mice was significantly suppressed by the intratumoral injection of the hTERT/FADD construct (every day for one week) compared to the control (P <0.0005). The findings described here indicate the high potentiality of a novel telomerase-specific gene therapy of tumors with telomerase.

ADAMTS family - New extracellular matrix degrading enzyme

S. Hirohata

Seikagaku 73 ( 11 ) 1333 - 1337 2001年

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