Researchers Database

takahashi masafumi

    DivisionofInflammationResearch Professor
Last Updated :2021/12/04

Researcher Information


  • (BLANK)


J-Global ID

Research Interests

  • 炎症反応   再生医学   心血管生物学   Cardiology   

Research Areas

  • Life sciences / Cardiology

Academic & Professional Experience

  • 2009 - Today  Jichi Medical UniversityProfessor
  • 2003 - 2009  Shinshu UniversityGraduate School of Medicine
  • 2001 - 2003  Jichi Medical University
  • 1997 - 2001  Jichi Medical University
  • 1997 - 2001  Instructor, Cardiology, Jichi Medical School


  •        - 1997  Jichi Medical University  医学研究科
  •        - 1997  Jichi Medical University  Graduate School, Division of Medicine
  •        - 1989  Jichi Medical University  医学部
  •        - 1989  Jichi Medical University  Faculty of Medicine

Association Memberships

  • 日本動脈硬化学会   日本循環器学会   日本内科学会   

Published Papers

  • Mishra PK, Adameova A, Hill JA, Baines CP, Kang PM, Downey JM, Narula J, Takahashi M, Abbate A, Piristine HC, Kar S, Su S, Higa JK, Kawasaki NK, Matsui T
    American journal of physiology. Heart and circulatory physiology 317 (5) H891 - H922 0363-6135 2019/11 [Refereed][Not invited]
  • Homare Ito, Ai Sadatomo, Yoshiyuki Inoue, Naoya Yamada, Emi Aizawa, Erika Hishida, Ryo Kamata, Tadayoshi Karasawa, Hiroaki Kimura, Sachiko Watanabe, Takanori Komada, Hisanaga Horie, Joji Kitayama, Naohiro Sata, Masafumi Takahashi
    Biochemical and biophysical research communications 519 (1) 15 - 22 0006-291X 2019/10 [Refereed][Not invited]
    BACKGROUND: Intestinal ischemia/reperfusion (I/R) injury is a life-threatening complication that leads to inflammation and remote organ damage. However, the underlying mechanism is not yet fully understood. Toll-like receptor 5 (TLR5) is highly expressed in mucosa and recognizes flagellin, the main component of the bacterial flagella. Here, we investigated the role of TLR5 in inflammation and tissue damage after intestinal I/R injury using TLR5-deficient mice. METHODS AND RESULTS: Intestinal levels of TLR5 mRNA and flagellin protein were elevated in wild-type mice subjected to intestinal I/R. Although TLR5 deficiency had no effect on intestinal flagellin levels, it significantly attenuated intestinal injury and inflammatory responses after intestinal I/R. TLR5 deficiency also markedly improved survival in mice after intestinal I/R injury. In wild-type mice, intestinal I/R injury induced remote organ damage, particularly in the lung, which was attenuated by TLR5 deficiency. Furthermore, TLR5 deficiency prevented lung inflammatory responses and vascular permeability after intestinal I/R injury. CONCLUSION: These findings demonstrate a novel role of TLR5 and provide new insights into the mechanism underlying inflammation and tissue damage after intestinal I/R injury.
  • Takahashi M
    Journal of cardiovascular pharmacology 74 (3) 188 - 193 0160-2446 2019/09 [Refereed][Not invited]
  • Mizushina Y, Karasawa T, Aizawa K, Kimura H, Watanabe S, Kamata R, Komada T, Mato N, Kasahara T, Koyama S, Bando M, Hagiwara K, Takahashi M
    Journal of immunology (Baltimore, Md. : 1950) 203 (1) 236 - 246 0022-1767 2019/07 [Refereed][Not invited]
    Inflammation plays a pivotal role in the pathophysiology of gastric aspiration-induced acute lung injury (ALI). However, its mechanism remains unclear. In this study, we investigated the role of NLRP3 inflammasome-driven IL-1β production in a mouse model of acid aspiration-induced inflammation and ALI. Acid aspiration-induced inflammatory responses and ALI in wild-type mice were significantly attenuated in IL-1β-/- mice, but not NLRP3-/- mice. In vitro experiments revealed that severe acidic stress (pH 1.75) induced the processing of pro-IL-1β into its 18-kDa mature form (p18-IL-1β), which was different from the caspase-1-processed 17-kDa form (p17-IL-1β), in human THP-1 macrophages and primary murine macrophages. Deficiency of NLRP3 and caspase-1 had no effect on acidic stress-produced IL-1β. The production of IL-1β by severe acidic stress was prevented by inhibitors of serine proteases [4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride], but not of cysteine proteases (E-64), cathepsin G, or inflammasome. The cathepsin D inhibitor pepstatin A inhibited IL-1β production induced by mild acidic stress (pH 6.2) or lactic acid, but not severe acidic stress. Using mass spectrometry and processing-site mutants of pro-IL-1β, we identified D109 as a novel cleavage site of pro-IL-1β in response to severe acidic stress and calculated the theoretical molecular mass of the mature form to be 18.2 kDa. The bioactivity of acidic stress-produced IL-1β was confirmed by its ability to promote p38 phosphorylation and chemokine upregulation in alveolar epithelial cells. These findings demonstrate a novel mechanism of acid-induced IL-1β production and inflammation independent of NLRP3 inflammasome and provide new insights into the therapeutic strategies for aspiration pneumonitis and ALI.
  • Hishida E, Ito H, Komada T, Karasawa T, Kimura H, Watanabe S, Kamata R, Aizawa E, Kasahara T, Morishita Y, Akimoto T, Nagata D, Takahashi M
    Scientific reports 9 (1) 10363 - 10363 2019/07 [Refereed][Not invited]
    Long-term peritoneal dialysis (PD) therapy leads to peritoneal inflammation and fibrosis. However, the mechanism underlying PD-related peritoneal inflammation and fibrosis remains unclear. NLRP3 inflammasome regulates the caspase-1-dependent release of interleukin-1β and mediates inflammation in various diseases. Here, we investigated the role of NLRP3 inflammasome in a murine model of PD-related peritoneal fibrosis induced by methylglyoxal (MGO). Inflammasome-related proteins were upregulated in the peritoneum of MGO-treated mice. MGO induced parietal and visceral peritoneal fibrosis in wild-type mice, which was significantly reduced in mice deficient in NLRP3, ASC, and interleukin-1β (IL-1β). ASC deficiency reduced the expression of inflammatory cytokines and fibrotic factors, and the infiltration of macrophages. However, myeloid cell-specific ASC deficiency failed to inhibit MGO-induced peritoneal fibrosis. MGO caused hemorrhagic ascites, fibrin deposition, and plasminogen activator inhibitor-1 upregulation, but all of these manifestations were inhibited by ASC deficiency. Furthermore, in vitro experiments showed that MGO induced cell death via the generation of reactive oxygen species in vascular endothelial cells, which was inhibited by ASC deficiency. Our results showed that endothelial NLRP3 inflammasome contributes to PD-related peritoneal inflammation and fibrosis, and provide new insights into the mechanisms underlying the pathogenesis of this disorder.
  • Zuurbier CJ, Abbate A, Cabrera-Fuentes HA, Cohen MV, Collino M, De Kleijn DPV, Downey JM, Pagliaro P, Preissner KT, Takahashi M, Davidson SM
    Cardiovascular research 115 (7) 1131 - 1142 0008-6363 2019/06 [Refereed][Not invited]
  • Shirasuna K, Karasawa T, Takahashi M
    Journal of cellular physiology 234 (5) 5436 - 5450 0021-9541 2019/05 [Refereed][Not invited]
  • Murakami T, Ruengsinpinya L, Nakamura E, Takahata Y, Hata K, Okae H, Taniguchi S, Takahashi M, Nishimura R
    Journal of immunology (Baltimore, Md. : 1950) 202 (7) 1942 - 1947 0022-1767 2019/04 [Refereed][Not invited]
  • Karasawa T, Takahashi M
    Aging 11 (6) 1613 - 1614 2019/03 [Refereed][Not invited]
  • Yamada N, Katano T, Hirata Y, Okada N, Sanada Y, Ihara Y, Urahashi T, Ushijima K, Karasawa T, Takahashi M, Mizuta K
    Journal of gastroenterology and hepatology 34 (2) 418 - 424 0815-9319 2019/02 [Refereed][Not invited]
    BACKGROUND AND AIM: Serum Mac-2 binding protein glycosylation isomer (M2BPGi) is a novel fibrosis marker for various chronic liver diseases. We investigated the ability of M2BPGi to predict liver fibrosis in liver transplant (LT) recipients. METHODS: A total of 116 liver biopsies were performed in 113 LT recipients. The serum level of M2BPGi was also measured on the same day. The median age at LT and liver biopsy was 1.1 and 11.8 years, respectively. Serum M2BPGi levels and liver fibrosis status using METAVIR fibrosis score were compared. Immunohistological evaluation by anti-α-smooth-muscle actin (αSMA) was performed, and the relationship between αSMA positive rate and serum M2BPGi levels was investigated. RESULTS: The median M2BPGi level was 0.78 (range, 0.22-9.50), and 65, 29, 16, 5, and 1 patient(s) had METAVIR fibrosis scores of F0, F1, F2, F3, and F4, respectively. In patients with F0 fibrosis, median M2BPGi level was 0.69 and was significantly lower than in patients with F1 (median 0.99, P < 0.01), F2 (median 1.00, P = 0.01), and F3 fibrosis (median 1.53, P < 0.01). Area-under-the-curve analysis of the ability of M2BPGi level to predict liver fibrosis grade were > F1: 0.716, > F2: 0.720, and > F3: 0.900. Three patients with acute cellular rejection showed high levels of M2BPGi, which decreased after the treatment. A positive correlation existed between M2BPGi levels and αSMA positive rate (r2  = 0.715, P < 0.01). CONCLUSION: Mac-2 binding protein glycosylation isomer is a novel liver fibrosis marker in LT recipients and is also increased in patients with acute liver injuries, especially acute cellular rejection, even when fibrosis is absent.
  • Takahashi M
    Biological & pharmaceutical bulletin 42 (4) 518 - 523 0918-6158 2019 [Refereed][Not invited]
  • Naoko Matsushita, Nanae Ishida, Miho Ibi, Maki Saito, Masafumi Takahashi, Shunichiro Taniguchi, Yoichiro Iwakura, Yoshihiro Morino, Eiichi Taira, Yohei Sawa, Masamichi Hirose
    Biological & pharmaceutical bulletin 42 (4) 543 - 546 0918-6158 2019 [Refereed][Not invited]
    Hypertension is one risk for atrial fibrillation (AF) and induces cardiac inflammation. Recent evidence indicates that pressure overload-induced ventricular structural remodeling is associated with the activation of nucleotide binding-oligomerization domain (NOD)-like receptor P3 (NLRP3) inflammasomes, including an apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC). We hypothesized that NLRP3 inflammasomes are an initial sensor for danger signals in pressure overload-induced atrial remodeling, leading to AF. Transverse aortic constriction (TAC) or a sham procedure was performed in mice deficient for ASC-/- and interleukin-1β (IL-1β-/-). One week after the procedure, electrical left atrial burst pacing from the esophagus was performed for 30 s to induce AF. IL-1β, monocyte chemotactic protein 1 (MCP-1), connective tissue growth factor (CTGF), and collagen 1 gene expression were also examined. The electrical burst pacing induced AF in TAC-operated wild-type (WT) (p < 0.001) and ASC-/- (p < 0.05) mice, compared to no AF in the sham-operated WT and ASC-/- mice, respectively. In contrast, the number of mice in which sustained AF was induced was similar between TAC-operated IL-1β-/- and sham-operated IL-1β-/- mice (p > 0.05). The expression of all genes tested was increased in TAC-operated WT and ASC-/- mice compared with sham-operated WT and ASC-/- mouse atria, respectively. CTGF and collagen 1, but not MCP-1, gene expressions were increased in TAC-operated IL-1β-/- mouse atria compared with sham-operated WT and IL-1β-/- mouse atria. In contrast, the IL-1β gene was not detected in either TAC-operated or sham-operated IL-1β-/- mouse atria. These results suggest that an IL-1β activation pathway, different from NLRP3 inflammasomes, plays an important role in pressure overload-induced sustained AF.
  • Wakabayashi T, Takahashi M, Yamamuro D, Karasawa T, Takei A, Takei S, Yamazaki H, Nagashima S, Ebihara K, Takahashi M, Ishibashi S
    Arteriosclerosis, thrombosis, and vascular biology 38 (11) 2576 - 2589 1079-5642 2018/11 [Refereed][Not invited]
  • Sakai K, Nagashima S, Wakabayashi T, Tumenbayar B, Hayakawa H, Hayakawa M, Karasawa T, Ohashi K, Yamazaki H, Takei A, Takei S, Yamamuro D, Takahashi M, Yagyu H, Osuga JI, Takahashi M, Tominaga SI, Ishibashi S
    Arteriosclerosis, thrombosis, and vascular biology 38 (11) 2590 - 2600 1079-5642 2018/11 [Refereed][Not invited]
    Objective- Inhibition of HMGCR (3-hydroxy-3-methylglutaryl-coenzyme A reductase) is atheroprotective primarily by decreasing plasma LDL (low-density lipoprotein)-cholesterol. However, it is unknown whether inhibition of HMGCR in myeloid cells contributes to this atheroprotection. We sought to determine the role of myeloid HMGCR in the development of atherosclerosis. Approach and Results- We generated mice with genetically reduced Hmgcr in myeloid cells ( Hmgcr m-/m-) using LysM (Cre) and compared various functions of their macrophages to those of Hmgcr fl/fl control mice. We further compared the extent of atherosclerosis in Hmgcr m-/ m- and Hmgcr fl/fl mice in the absence of Ldlr (LDL receptor). Hmgcr m-/ m- macrophages and granulocytes had significantly lower Hmgcr mRNA expression and cholesterol biosynthesis than Hmgcr fl/fl cells. In vitro, Hmgcr m-/ m- monocytes/macrophages had reduced ability to migrate, proliferate, and survive compared with Hmgcr fl/fl monocytes/macrophages. However, there was no difference in ability to adhere, phagocytose, store lipids, or polarize to M1 macrophages between the 2 types of macrophages. The amounts of plasma membrane-associated small GTPase proteins, such as RhoA (RAS homolog family member A), were increased in Hmgcr m-/ m- macrophages. In the setting of Ldlr deficiency, Hmgcr m-/ m- mice developed significantly smaller atherosclerotic lesions than Hmgcr fl/fl mice. However, there were no differences between the 2 types of mice either in plasma lipoprotein profiles or in the numbers of proliferating or apoptotic cells in the lesions in vivo. The in vivo migration of Hmgcr m-/ m- macrophages to the lesions was reduced compared with Hmgcr fl/fl macrophages. Conclusions- Genetic reduction of HMGCR in myeloid cells may exert atheroprotective effects primarily by decreasing the migratory activity of monocytes/macrophages to the lesions.
  • Nakamura J, Watanabe S, Kimura H, Kobayashi M, Karasawa T, Kamata R, Usui-Kawanishi F, Sadatomo A, Mizukami H, Nagi-Miura N, Ohno N, Kasahara T, Minota S, Takahashi M
    Scientific reports 8 (1) 7601  2018/05 [Refereed][Not invited]
  • Tadayoshi Karasawa, Akira Kawashima, Fumitake Usui-Kawanishi, Sachiko Watanabe, Hiroaki Kimura, Ryo Kamata, Koumei Shirasuna, Yutaro Koyama, Ayana Sato-Tomita, Takashi Matsuzaka, Hiroshi Tomoda, Sam-Yong Park, Naoya Shibayama, Hitoshi Shimano, Tadashi Kasahara, Masafumi Takahashi
    Arteriosclerosis, Thrombosis, and Vascular Biology 38 (4) 744 - 756 1524-4636 2018/04 [Refereed][Not invited]
    Objective - Inflammation provoked by the imbalance of fatty acid composition, such as excess saturated fatty acids (SFAs), is implicated in the development of metabolic diseases. Recent investigations suggest the possible role of the NLRP3 (nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3) inflammasome, which regulates IL-1β (interleukin 1β) release and leads to inflammation, in this process. Therefore, we investigated the underlying mechanism by which SFAs trigger NLRP3 inflammasome activation. Approach and Results - The treatment with SFAs, such as palmitic acid and stearic acid, promoted IL-1β release in murine primary macrophages while treatment with oleic acid inhibited SFA-induced IL-1β release in a dose-dependent manner. Analyses using polarized light microscopy revealed that intracellular crystallization was provoked in SFA-treated macrophages. As well as IL-1β release, the intracellular crystallization and lysosomal dysfunction were inhibited in the presence of oleic acid. These results suggest that SFAs activate NLRP3 inflammasome through intracellular crystallization. Indeed, SFA-derived crystals activated NLRP3 inflammasome and subsequent IL-1β release via lysosomal dysfunction. Excess SFAs also induced crystallization and IL-1β release in vivo. Furthermore, SFA-derived crystals provoked acute inflammation, which was impaired in IL-1β-deficient mice. Conclusions - These findings demonstrate that excess SFAs cause intracellular crystallization and subsequent lysosomal dysfunction, leading to the activation of the NLRP3 inflammasome, and provide novel insights into the pathogenesis of metabolic diseases.
  • Sogawa Y, Nagasu H, Itano S, Kidokoro K, Taniguchi S, Takahashi M, Kadoya H, Satoh M, Sasaki T, Kashihara N
    PloS one 13 (10) e0203823  2018 [Refereed][Not invited]
  • Akira Kawashima, Tadayoshi Karasawa, Kenji Tago, Hiroaki Kimura, Ryo Kamata, Fumitake Usui-Kawanishi, Sachiko Watanabe, Satoshi Ohta, Megumi Funakoshi-Tago, Ken Yanagisawa, Tadashi Kasahara, Koichi Suzuki, Masafumi Takahashi
    JOURNAL OF IMMUNOLOGY 199 (10) 3614 - 3622 0022-1767 2017/11 [Refereed][Not invited]
    The nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is a molecular platform that induces caspase-1 activation and subsequent IL-1b maturation, and is implicated in inflammatory diseases; however, little is known about the negative regulation of NLRP3 inflammasome activation. In this article, we identified an E3 ligase, Ariadne homolog 2 (ARIH2), as a posttranslational negative regulator of NLRP3 inflammasome activity in macrophages. ARIH2 interacted with NLRP3 via its NACHT domain (aa 220-575) in the NLRP3 inflammasome complex. In particular, we found that while using mutants of ARIH2 and ubiquitin, the really interesting new gene 2 domain of ARIH2 was required for NLRP3 ubiquitination linked through K48 and K63. Deletion of endogenous ARIH2 using CRISPR/Cas9 genome editing inhibited NLRP3 ubiquitination and promoted NLRP3 inflammasome activation, resulting in apoptosis-associated speck-like protein containing a caspase recruitment domain oligomerization, pro-IL-1b processing, and IL-1b production. Conversely, ARIH2 overexpression promoted NLRP3 ubiquitination and inhibited NLRP3 inflammasome activation. Our findings reveal a novel mechanism of ubiquitination-dependent negative regulation of the NLRP3 inflammasome by ARIH2 and highlight ARIH2 as a potential therapeutic target for inflammatory diseases.
  • Ai Sadatomo, Yoshiyuki Inoue, Homare Ito, Tadayoshi Karasawa, Hiroaki Kimura, Sachiko Watanabe, Yoshiko Mizushina, Jun Nakamura, Ryo Kamata, Tadashi Kasahara, Hisanaga Horie, Naohiro Sata, Masafumi Takahashi
    JOURNAL OF IMMUNOLOGY 199 (9) 3306 - 3315 0022-1767 2017/11 [Refereed][Not invited]
    Accumulating evidence suggests that IL-1 beta plays a pivotal role in the pathophysiology of hepatic ischemia-reperfusion (I/R) injury; however, the mechanism by which I/R triggers IL-1 beta production in the liver remains unclear. Recent data have shown that neutrophils contribute to hepatic I/R injury independently of the inflammasomes regulating IL-1 beta maturation. Thus, we investigated the role of neutrophils in IL-1 beta maturation and tissue injury in a murine model of hepatic I/R. IL-1 beta was released from the I/R liver and its deficiency reduced reactive oxygen species generation, apoptosis, and inflammatory responses, such as inflammatory cell infiltration and cytokine expression, thereby resulting in reduced tissue injury. Depletion of either macrophages or neutrophils also attenuated IL-1 beta release and hepatic I/R injury. In vitro experiments revealed that neutrophil-derived proteinases process pro-IL-1 beta derived from macrophages into its mature form independently of caspase-1. Furthermore, pharmacological inhibition of serine proteases attenuated IL-1 beta release and hepatic I/R injury in vivo. Taken together, the interaction between neutrophils and macrophages promotes IL-1 beta maturation and causes IL-1 beta-driven inflammation in the I/R liver. Both neutrophils and macrophages are indispensable in this process. These findings suggest that neutrophil-macrophage interaction is a therapeutic target for hepatic I/R injury and may also provide new insights into the inflammasome-independent mechanism of IL-1 beta maturation in the liver.
  • Yuji Sogawa, Hajime Nagasu, Shigeki Iwase, Chieko Ihoriya, Seiji Itano, Atsushi Uchida, Kengo Kidokoro, Shun'ichiro Taniguchi, Masafumi Takahashi, Minoru Satoh, Tamaki Sasaki, Takafumi Suzuki, Masayuki Yamamoto, Tiffany Horng, Naoki Kashihara
    SCIENTIFIC REPORTS 7 (1) 8801  2045-2322 2017/08 [Refereed][Not invited]
    Chronic inflammation can be a major driver of the failure of a variety of organs, including chronic kidney disease (CKD). The NLR family pyrin domain-containing 3 (NLRP3) inflammasome has been shown to play a pivotal role in inflammation in a mouse kidney disease model. Nuclear factor erythroid 2-related factor 2 (Nrf2), the master transcription factor for anti-oxidant responses, has also been implicated in inflammasome activation under physiological conditions. However, the mechanism underlying inflammasome activation in CKD remains elusive. Here, we show that the loss of Nrf2 suppresses fibrosis and inflammation in a unilateral ureter obstruction (UUO) model of CKD in mice. We consistently observed decreased expression of inflammation-related genes NLRP3 and IL-1 beta in Nrf2-deficient kidneys after UUO. Increased infiltration of M1, but not M2, macrophages appears to mediate the suppression of UUO-induced CKD symptoms. Furthermore, we found that activation of the NLRP3 inflammasome is attenuated in Nrf2-deficient bone marrow-derived macrophages. These results demonstrate that Nrf2-related inflammasome activation can promote CKD symptoms via infiltration of M1 macrophages. Thus, we have identified the Nrf2 pathway as a promising therapeutic target for CKD.
  • Motoi Kobayashi, Fumitake Usui-Kawanishi, Tadayoshi Karasawa, Hiroaki Kimura, Sachiko Watanabe, Nathan Mise, Fujio Kayama, Tadashi Kasahara, Naoyuki Hasebe, Masafumi Takahashi
    PLOS ONE 12 (5) e0176676  1932-6203 2017/05 [Refereed][Not invited]
    Cardiac glycosides such as digoxin are Na+/K+ -ATPase inhibitors that are widely used for the treatment of chronic heart failure and cardiac arrhythmias; however, recent epidemiological studies have suggested a relationship between digoxin treatment and increased mortality. We previously showed that nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasomes, which regulate caspase-1-dependent interleukin (IL)-1 beta release, mediate the sterile cardiovascular inflammation. Because the Na+/K+ -ATPase is involved in inflammatory responses, we investigated the role of NLRP3 inflammasomes in the pathophysiology of cardiac glycoside-induced cardiac inflammation and dysfunction. The cardiac glycoside ouabain induced cardiac dysfunction and injury in wild-type mice primed with a low dose of lipopolysaccharide (LPS), although no cardiac dysfunction was observed in mice treated with either ouabain or LPS alone. Ouabain also induced cardiac inflammatory responses, such as macrophage infiltration and IL-d1 beta release, when mice were primed with LPS. These cardiac manifestations were all significantly attenuated in mice deficient in IL-1 beta. Furthermore, deficiency of NLRP3 inflammasome components, NLRP3 and caspase-1, also attenuated ouabain-induced cardiac dysfunction and inflammation. In vitro experiments revealed that ouabain induced NLRP3 inflammasome activation as well as subsequent IL-1 beta release from macrophages, and this activation was mediated by K+ efflux. Our findings demonstrate that cardiac glycosides promote cardiac inflammation and dysfunction through NLRP3 inflammasomes and provide new insights into the mechanisms underlying the adverse effects of cardiac glycosides.
  • Shinya Yamasaki, Yusuke Hashimoto, Junsei Takigami, Shozaburo Terai, Masafumi Takahashi, Shigeyuki Wakitani, Hiroaki Nakamura
    JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE 11 (3) 609 - 617 1932-6254 2017/03 [Refereed][Not invited]
    The purpose of this study was to assess how peripheral blood cells (PBCs) contribute to meniscus repair, using a parabiotic rat model. Wild-type (WT) and green fluorescent protein (GFP) transgenic rats were conjoined at the torso. After 4 weeks, the anterior part of the medial meniscus of both groups of rats was removed. At 1, 2, 4, 8 and 12 weeks post-meniscectomy, repaired tissue was evaluated using stereomicroscopy, histology with toluidine blue staining, and immunofluorescence microscopy. Stereomicroscopic observations and confocal laser microscopy revealed that a high number of GFP-positive cells were present in the repaired meniscus of WT rats 1 week post-meniscectomy, and the number of GFP- positive cells decreased over time. Based on blood chimerism, the ratios of PBCs in the repaired meniscus were 20.5 +/- 2.3% at 1 week, 8.3 +/- 0.9% at 2 weeks, 4.4 +/- 0.9% at 4 weeks, 2.1 +/- 0.9% at 8 weeks, and 0.5 +/- 0.4% at 12 weeks, post-meniscectomy. Histologically, fibrochondrocytes were observed in the repaired meniscus of WT rats after 4 weeks, some of which were GFP- positive. The chondrogenic marker, type II collagen, was merged within the PBCs in the repaired tissue. However, type-II-collagen-positive cell ratio and metachromasia in the repaired meniscus were not equivalent in normal meniscaltissue. This indicated that PBCs were present within the repaired meniscus at an early phase, replacing the excised meniscalcells, suggesting PBCs contributed to meniscalhealing. The tissue repair contribution by these cells decreased at later phases. Copyright (C) 2014 John Wiley & Sons, Ltd.
  • Shozaburo Terai, Yusuke Hashimoto, Kumi Orita, Shinya Yamasaki, Junsei Takigami, Takafumi Shinkuma, Takanori Teraoka, Yohei Nishida, Masafumi Takahashi, Hiroaki Nakamura
    CONNECTIVE TISSUE RESEARCH 58 (6) 562 - 572 0300-8207 2017 [Refereed][Not invited]
    We previously reported that circulating peripheral blood-borne cells (PBCs) contribute to early-phase meniscal reparative change. Because macrophages and myofibroblasts are important contributors of tissue regeneration, we examined their origin and distribution in the reparative meniscus. Reparative menisci were evaluated at 1, 2, and 4 weeks post-meniscectomy by immunohistochemistry to locate monocytes and macrophages (stained positive for CD68 and CD163), and myofibroblasts (stained positive for SMA). Of the total number of cells, 13% were CD68(+) at 1 week post-meniscectomy, which decreased to 1% by 4 weeks post-meniscectomy; of these, almost half of CD68(+) cells (49.4%: 98.8% as PBCs) were green fluorescent protein (GFP)-positive post-meniscectomy (1, 2, and 4 weeks), indicating that the majority of CD68(+) cells were derived from PBCs. Of the total cells, 6% were CD163(+) at 1 week post-meniscectomy, which decreased to 1% by week 4. Of the CD163(+) cells, the majority were GFP-positive (42.5%: 85.0% as PBCs) after 1 week; however, this decreased significantly over time, which indicates that the majority of CD163(+) cells are derived from PBCs during the early phase of meniscal reparative change, but are derived from resident cells at later time points. Of the total cells, 38% were SMA(+) at 1 week post-meniscectomy, which decreased to 3% by 4 weeks. The proportion of GFP-positive SMA(+) cells was 2.8% after 1 week, with no significant change over time, which indicates that the majority of SMA(+) cells originated from resident cells. Here, we describe the origin and distribution of macrophages and myofibroblasts during meniscal reparative change.
  • Tadayoshi Karasawa, Masafumi Takahashi
    JOURNAL OF ATHEROSCLEROSIS AND THROMBOSIS 24 (5) 443 - 451 1340-3478 2017 [Refereed][Not invited]
    Inflammation with macrophage infiltration is a key feature of atherosclerosis. Although the mechanisms had been unclear, emerging evidence unveiled that NLRP3 inflammasomes, which regulate caspase-1 activation and subsequent processing of pro-IL-1 beta, trigger vascular wall inflammatory responses and lead to progression of atherosclerosis. NLRP3 inflammasomes are activated by various danger signals, such as cholesterol crystals, calcium phosphate crystals, and oxidized low-density lipoprotein in macrophages, to initiate inflammatory responses in the atherosclerotic lesion. Recent studies have further clarified the regulatory mechanisms and the potential therapeutic agents that target NLRP3 inflammasomes. In this study, we reviewed the present state of knowledge on the role of NLRP3 inflammasomes in the pathogenesis of atherosclerosis and discussed the therapeutic approaches that target NLRP3 inflammasomes.
  • Karasawa T, Takahashi M
    Inflammation and regeneration 37 18  1880-9693 2017 [Refereed][Not invited]
  • Hiroaki Kimura, Tadayoshi Karasawa, Fumitake Usui, Akira Kawashima, Yuka Endo, Motoi Kobayashi, Ai Sadatomo, Jun Nakamura, Yusaku Iwasaki, Toshihiko Yada, Hiroko Tsutsui, Tadashi Kasahara, Masafumi Takahashi
    AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM 311 (5) E881 - E890 0193-1849 2016/11 [Refereed][Not invited]
    Caspase-1 is a cysteine protease responsible for the processing of the proinflammatory cytokine interleukin-1 beta and activated by the formation of inflammasome complexes. Although several investigations have found a link between diet-induced obesity and caspase-1, the relationship remains controversial. Here, we found that mice deficient in caspase-1 were susceptible to high-fat dietinduced obesity with increased adiposity as well as normal lipid and glucose metabolism. Caspase-1 deficiency clearly promoted the infiltration of inflammatory macrophages and increased the production of C-C motif chemokine ligand 2 (CCL2) in the adipose tissue. The dominant cellular source of CCL2 was stromal vascular fraction rather than adipocytes in the adipose tissue. These findings demonstrate a critical role of caspase-1 in macrophage-driven inflammation in the adipose tissue and the development of obesity. These data provide novel insights into the mechanisms underlying inflammation in the pathophysiology of obesity.
  • Keiko Omori, Eiji Kobayashi, Jeffrey Rawson, Masafumi Takahashi, Yoko Mullen
    CRYOBIOLOGY 73 (2) 126 - 134 0011-2240 2016/10 [Refereed][Not invited]
    Prolonged pancreas cold ischemia is known to negatively correlate with islet isolation outcomes, hindering successful islet transplantation to treat Type-1 Diabetes. Due to poor islet isolation outcome, pancreata with over 16 h cold ischemia are currently not considered for islet transplantation. Mechanisms involved in pancreas cold ischemia/rewarming mediated islet damage during islet isolation and culture are not well understood. Using an en bloc cold preserved rat pancreas preparation, we attempted to clarify possible mechanisms of islet death associated with prolonged pancreas cold ischemia and subsequent rewarming. Cold ischemia lasting 16 h decreased post-isolation islet yield and increased islet death during the initial 6 h of culture. Electron micrographs revealed swelling and severe disruption of cellular and mitochondrial membranes, as well as an enlarged endoplasmic reticulum (ER) in beta-cells isolated from cold preserved pancreata. Prolonged cold ischemia of the pancreas transiently activated mitogen-activated protein kinases (MAPKs) in isolated islets and increased lipid peroxidation products 4-hydroxynonenal (HNE) and heat shock protein (Hsp) 70 after culture, indicating the activation of oxidative stress signaling pathways. The islet isolation process, irrespective of pancreas cold ischemia, activated unfolded protein response (UPR), while the ER protective chaperon BiP was further upregulated by pancreas cold ischemia/rewarming. During the first 6 h of culture following islet isolation, p53 upregulated modulator of apoptosis (Puma) and caspase-3 activation were also upregulated. Our study indicates the involvement of both apoptosis and necrosis in islet death, and suggests oxidative stress and disruption of membranes are critical mechanisms mediated by pancreas cold ischemia/rewarming. (C) 2016 Elsevier Inc. All rights reserved.
  • Koumei Shirasuna, Hiroki Takano, Kotomi Seno, Ayaka Ohtsu, Tadayoshi Karasawa, Masafumi Takahashi, Akihide Ohkuchi, Hirotada Suzuki, Shigeki Matsubara, Hisataka Iwata, Takehito Kuwayama
    JOURNAL OF REPRODUCTIVE IMMUNOLOGY 116 104 - 112 0165-0378 2016/08 [Refereed][Not invited]
    Maternal obesity, a major risk factor for adverse pregnancy complications, results in inflammatory cytokine release in the placenta. Levels of free fatty acids are elevated in the plasma of obese human. These fatty acids include obesity-related palmitic acids, which is a major saturated fatty acid, that promotes inflammatory responses. Increasing evidence indicates that nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3) inflammasomes mediate inflammatory responses induced by endogenous danger signals. We hypothesized that inflammatory responses associated with gestational obesity cause inflammation. To test this hypothesis, we investigated the effect of palmitic acid on the activation of NLRP3 inflammasomes and inflammatory responses in a human Sw.71 trophoblast cell line. Palmitic acid stimulated caspase-1 activation and markedly increased interleukin (IL)-1 beta secretion in Sw.71 cells. Treatment with a caspase-1 inhibitor diminished palmitic acid-induced IL-1 beta release. In addition, NLRP3 and caspase-1 genome editing using a CRISPR/Cas9 system in Sw.71 cells suppressed IL-1 beta secretion, which was stimulated by palmitic acid. Moreover, palmitic acid stimulated caspase-3 activation and inflammatory cytokine secretion (e.g., IL-6 and IL-8). Palmitic acid-induced cytokine secretion were dependent on caspase-3 activation. In addition, palmitic acid-induced IL-1 beta, IL-6, and IL-8 secretion was depended on reactive oxygen species (ROS) generation. In conclusion, palmitic acid caused activation of NLRP3 inflammasomes and inflammatory responses, inducing IL-1 beta, IL-6, and IL-8 secretion, which is associated with ROS generation, in human Sw.71 placental cells. We suggest that obesity-related palmitic acid induces placental inflammation, resulting in association with pregnancy complications. (C) 2016 Elsevier Ireland Ltd. All rights reserved.
  • Keiko Omori, Eiji Kobayashi, Hirotake Komatsu, Jeffrey Rawson, Garima Agrawal, Mounika Parimi, Alina R. Oancea, Luis Valiente, Kevin Ferreri, Ismail H. Al-Abdullah, Fouad Kandeel, Masafumi Takahashi, Yoko Mullen
    AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM 310 (11) E1016 - E1026 0193-1849 2016/06 [Refereed][Not invited]
    Longterm pancreatic cold ischemia contributes to decreased islet number and viability after isolation and culture, leading to poor islet transplantation outcome in patients with type 1 diabetes. In this study, we examined mechanisms of pancreatic cold preservation and rewarming-induced injury by interrogating the proapoptotic gene BBC3/Bbc3, also known as Puma (p53 upregulated modulator of apoptosis), using three experimental models: 1) bioluminescence imaging of isolated luciferase-transgenic ("Firefly") Lewis rat islets, 2) cold preservation of en bloc-harvested pancreata from Bbc3-knockout (KO) mice, and 3) cold preservation and rewarming of human pancreata and isolated islets. Cold preservation-mediated islet injury occurred during rewarming in "Firefly" islets. Silencing Bbc3 by transfecting Bbc3 siRNA into islets in vitro prior to cold preservation improved postpreservation mitochondrial viability. Cold preservation resulted in decreased postisolation islet yield in both wild-type and Bbc3 KO pancreata. However, after culture, the islet viability was significantly higher in Bbc3-KO islets, suggesting that different mechanisms are involved in islet damage/loss during isolation and culture. Furthermore, Bbc3-KO islets from cold-preserved pancreata showed reduced HMGB1 (high-mobility group box 1 protein) expression and decreased levels of 4-hydroxynonenal (4-HNE) protein adducts, which was indicative of reduced oxidative stress. During human islet isolation, BBC3 protein was upregulated in digested tissue from cold-preserved pancreata. Hypoxia in cold preservation increased BBC3 mRNA and protein in isolated human islets after rewarming in culture and reduced islet viability. These results demonstrated the involvement of BBC3/Bbc3 in cold preservation/rewarming-mediated islet injury, possibly through modulating HMGB1- and oxidative stress-mediated injury to islets.
  • Motoi Kobayashi, Fumitake Usui, Tadayoshi Karasawa, Akira Kawashima, Hiroaki Kimura, Yoshiko Mizushina, Koumei Shirasuna, Hiroaki Mizukami, Tadashi Kasahara, Naoyuki Hasebe, Masafumi Takahashi
    SCIENTIFIC REPORTS 6 26489  2045-2322 2016/05 [Refereed][Not invited]
    NLRP3 inflammasomes recognize non-microbial danger signals and induce release of proinflammatory cytokine interleukin (IL)-1 beta, leading to sterile inflammation in cardiovascular disease. Because sterile inflammation is involved in doxorubicin (Dox)-induced cardiotoxicity, we investigated the role of NLRP3 inflammasomes in Dox-induced cardiotoxicity. Cardiac dysfunction and injury were induced by low-dose Dox (15 mg/kg) administration in NLRP3-deficient (NLRP3(-/-)) mice but not in wild-type (WT) and IL-1 beta(-/-) mice, indicating that NLRP3 deficiency enhanced the susceptibility to Dox-induced cardiotoxicity independent of IL-1 beta. Although the hearts of WT and NLRP3(-/-) mice showed no significant difference in inflammatory cell infiltration, macrophages were the predominant inflammatory cells in the hearts, and cardiac IL-10 production was decreased in Dox-treated NLRP3(-/-) mice. Bone marrow transplantation experiments showed that bone marrow-derived cells contributed to the exacerbation of Dox-induced cardiotoxicity in NLRP3(-/-) mice. In vitro experiments revealed that NLRP3 deficiency decreased IL-10 production in macrophages. Furthermore, adeno-associated virus-mediated IL-10 overexpression restored the exacerbation of cardiotoxicity in the NLRP3(-/-) mice. These results demonstrated that NLRP3 regulates macrophage IL-10 production and contributes to the pathophysiology of Dox-induced cardiotoxicity, which is independent of IL-1 beta. Our findings identify a novel role of NLRP3 and provided new insights into the mechanisms underlying Dox-induced cardiotoxicity.
  • Ayako Okada, Yuichiro Kashima, Takeshi Tomita, Takahiro Takeuchi, Kazunori Aizawa, Masafumi Takahashi, Uichi Ikeda
    HEART AND VESSELS 31 (1) 80 - 87 0910-8327 2016/01 [Refereed][Not invited]
    Atrial fibrillation (AF) is associated with oxidative stress and elevated brain natriuretic peptide (BNP) levels. However, the exact cardiac origin of oxidative stress and its association with BNP levels in AF patients remain unclear. Therefore, we investigated the chamber-specific plasma oxidative stress levels in patients with paroxysmal AF (PAF) and persistent AF (PSAF). Diacron-reactive oxygen metabolite (dROM) levels were measured in patients with PAF (n = 50) and PSAF (n = 35) at different cardiac sites before ablation and in peripheral vein 3 months after ablation. For all sites, dROM levels were higher in PSAF patients than in PAF patients; the levels were the highest in the coronary sinus at 429.0 (interquartile range: 392.0-449.0) vs. 374.0 (357.0-397.8) Carratelli units (P < 0.05). dROM levels in the coronary sinus were related to the BNP levels (r = 0.436, P < 0.001). Furthermore, the reduction in the peripheral dROM levels was related to that in the peripheral BNP levels in patients with symptomatic improvement (r = 0.473, P < 0.001). Cardiac oxidative stress may either be a cause or consequence of prolonged AF, and cardiac oxidative stress levels correlated with BNP levels, though a possible source of oxidative stress in AF patients may be systemic circulation.
  • Koumei Shirasuna, Tadayoshi Karasawa, Fumitake Usui, Motoi Kobayashi, Tadanori Komada, Hiroaki Kimura, Akira Kawashima, Akihide Ohkuchi, Shun'ichiro Taniguchi, Masafumi Takahashi
    ENDOCRINOLOGY 156 (11) 4281 - 4292 0013-7227 2015/11 [Refereed][Not invited]
    Preeclampsia is a pregnancy-specific syndrome characterized by elevated blood pressure, proteinuria, and intrauterine growth restriction (IUGR). Although sterile inflammation appears to be involved, its pathogenesis remains unclear. Recent evidence indicates that sterile inflammation is mediated through the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasomes, composed of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and caspase-1. Here we investigated the role of the NLRP3 inflammasomes in the pathogenesis of preeclampsia using Nlrp3(-/-) and Asc(-/-) (Nlrp3 and Asc deficient) pregnant mice. During pregnancy in mice, continuous infusion of high-dose angiotensin II (AngII) induced hypertension, proteinuria, and IUGR, whereas infusion of low-dose AngII caused hypertension alone. AngII-induced hypertension was prevented in Nlrp3(-/-) mice but not in Asc(-/-), indicating that NLRP3 contributes to gestational hypertension independently of ASC-mediated inflammasomes. Although NLRP3 deficiency had no effect on IUGR, it restored the IL-6 up-regulation in the placenta and kidney of AngII-infused mice. Furthermore, treatment with hydralazine prevented the development of gestational hypertension but not IUGR or IL-6 expression in the placenta and kidney. These findings demonstrate that NLRP3 contributes to the development of gestational hypertension independently of the inflammasomes and that IUGR and kidney injury can occur independent of blood pressure elevation during pregnancy.
  • Shiho Nagayama, Akihide Ohkuchi, Koumei Shirasuna, Kayo Takahashi, Hirotada Suzuki, Chikako Hirashima, Asuka Sakata, Satoshi Nishimura, Masafumi Takahashi, Shigeki Matsubara
    HYPERTENSION IN PREGNANCY 34 (4) 443 - 455 1064-1955 2015/10 [Refereed][Not invited]
    Objective: We compared the frequency of peripheral blood Treg cells in women with pre-eclampsia (PE) and in those without, and investigated whether the frequency of Treg cells in women with high-risk factor for PE changed during pregnancy. Methods: We examined the frequency of CD4(+)FoxP3(+) Treg cells in the peripheral blood using flow cytometry. Eleven women with PE and 10 women without PE (controls) were included. Every control had any risk factors for PE, such as high blood pressure, bilateral notching or a past history of PE or gestational hypertension. Blood sampling was conducted 1-3 times in the controls. Results: No significant differences were observed in the frequency of Treg cells between women with PE and the controls [mean +/- SE (%): 5.74 +/- 0.91 versus 5.48 +/- 0.94, p=0.843]. In five controls with serial sampling, the frequency of Treg cells significantly decreased from 5.83 +/- 1.20 to 2.99 +/- 0.54 (p=0.046) (week of the first sampling to that of the last sampling [mean +/- SD]: 21.5 +/- 1.6 weeks to 31.2 +/- 2.5 weeks). Conclusion: The frequency of Treg cells in women with PE was almost identical to that in the controls. The frequency of Treg cells in the controls was reduced by half from the second trimester to the third trimester. These results suggested that the levels of Treg cells in a high-risk pregnant cohort were decreased to those in women with PE in the third trimester irrespective of the occurrence of PE.
  • Hiroaki Kimura, Fumitake Usui, Tadayoshi Karasawa, Akira Kawashima, Koumei Shirasuna, Yoshiyuki Inoue, Takanori Komada, Motoi Kobayashi, Yoshiko Mizushina, Tadashi Kasahara, Koichi Suzuki, Yusaku Iwasaki, Toshihiko Yada, Patrizio Caturegli, Masafumi Takahashi
    SCIENTIFIC REPORTS 5 15883  2045-2322 2015/10 [Refereed][Not invited]
    Inflammation plays an important role in the development of obesity and metabolic disorders; however, it has not been fully understood how inflammation occurs and is regulated in their pathogenesis. Low-molecular mass protein-7 (LMP7) is a proteolytic subunit of the immunoproteasome that shapes the repertoire of antigenic peptides on major histocompatibility complex class I molecule. In this study, we investigated the role of LMP7 in the development of obesity and metabolic disorders using LMP7-deficient mice. LMP7 deficiency conveyed resistant to obesity, and improved glucose intolerance and insulin sensitivity in mice fed with high-fat diet (HFD). LMP7 deficiency decreased pancreatic lipase expression, increased fecal lipid contents, and inhibited the increase of plasma triglyceride levels upon oral oil administration or HFD feeding. Using bone marrow-transferred chimeric mice, we found that LMP7 in both bone marrow-and non-bone marrow-derived cells contributes to the development of HFD-induced obesity. LMP7 deficiency decreased inflammatory responses such as macrophage infiltration and chemokine expression while it increased serum adiponection levels. These findings demonstrate a novel role for LMP7 and provide new insights into the mechanisms underlying inflammation in the pathophysiology of obesity and metabolic disorders.
  • Hiroyuki Kadoya, Minoru Satoh, Tamaki Sasaki, Shun'ichiro Taniguchi, Masafumi Takahashi, Naoki Kashihara
    FASEB JOURNAL 29 (9) 3899 - 3910 0892-6638 2015/09 [Refereed][Not invited]
    High levels of aldosterone impair renal function by activating proinflammatory and profibrotic pathways. However, the molecular mechanism underlying aldosterone-induced inflammation and fibrosis is unknown. Inflammasome activation contributes to chronic kidney disease. We hypothesized that aldosterone induces renal tubulointerstitial inflammation and fibrosis by activating the inflammasome. Infusing wild-type mice with aldosterone (0.25 mg/kg/d) caused tubulointerstitial damage, increased expression of inflammasome components, caspase 1 activation, and overproduction of IL-1 beta and IL-18. These changes were suppressed by eplerenone treatment (100 mg/kg/d) in wild-type mice or in mice deficient in apoptosis-associated speck-like protein with a caspase-recruitment domain (ASC). Caspase 1-positive and F4/80-positive cells colocalized in the interstitium. Bone marrow transplantation using ASC-deficient mice indicated that inflammasome activation in macrophages mediated aldosterone-induced renal fibrosis. IL-18 was detected in culture supernatants of macrophages treated with aldosterone, and mitochondria-derived reactive oxygen species activated the inflammasome in these macrophages. Our results indicate that exposure of macrophages to high levels of aldosterone resulted in the activation of inflammasomes via the mitochondria-derived reactive oxygen species. Thus, inflammasome activation in macrophages may serve as a new therapeutic target for chronic kidney disease.
  • Inoue Y, Takahashi M
    Journal of immunology (Baltimore, Md. : 1950) 194 (11) 5039  0022-1767 2015/06 [Refereed][Not invited]
  • Takanori Komada, Fumitake Usui, Akira Kawashima, Hiroaki Kimura, Tadayoshi Karasawa, Yoshiyuki Inoue, Motoi Kobayashi, Yoshiko Mizushina, Tadashi Kasahara, Shun'ichiro Taniguchi, Shigeaki Muto, Daisuke Nagata, Masafumi Takahashi
    SCIENTIFIC REPORTS 5 10901  2045-2322 2015/06 [Refereed][Not invited]
    Rhabdomyolysis is one of the main causes of community-acquired acute kidney injury (AKI). Although inflammation is involved in the pathogenesis of rhabdomyolysis-induced AKI (RIAKI), little is known about the mechanism that triggers inflammation during RIAKI. Recent evidence has indicated that sterile inflammation triggered by tissue injury can be mediated through multiprotein complexes called the inflammasomes. Therefore, we investigated the role of NLRP3 inflammasomes in the pathogenesis of RIAKI using a glycerol-induced murine rhabdomyolysis model. Inflammasomerelated molecules were upregulated in the kidney of RIAKI. Renal tubular injury and dysfunction preceded leukocyte infiltration into the kidney during the early phase of RIAKI, and they were markedly attenuated in mice deficient in NLRP3, ASC, caspase-1, and interleukin (IL)-1 beta compared with those in wild-type mice. No difference in leukocyte infiltration was observed between wild-type and NLRP3-deficient mice. Furthermore, NLRP3 deficiency strikingly suppressed the expression of renal injury markers and inflammatory cytokines and apoptosis of renal tubular cells. These results demonstrated that NLRP3 inflammasomes contribute to inflammation, apoptosis, and tissue injury during the early phase of RIAKI and provide new insights into the mechanism underlying the pathogenesis of RIAKI.
  • Tadayoshi Karasawa, Masafumi Takahashi
    JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY 81 136 - 138 0022-2828 2015/04 [Refereed][Not invited]
  • Milan Gautam, Daiki Fujita, Kazuhiro Kimura, Hinako Ichikawa, Atsushi Izawa, Masamichi Hirose, Toshihide Kashihara, Mitsuhiko Yamada, Masafumi Takahashi, Uichi Ikeda, Yuji Shiba
    JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY 81 139 - 149 0022-2828 2015/04 [Refereed][Not invited]
    The transplantation of adipose tissue-derived stem cells (ADSCs) improves cardiac contractility after myocardial infarction (MI); however, little is known about the electrophysiological consequences of transplantation. The purpose of this study was to clarify whether the transplantation of ADSCs increases or decreases the incidence of ventricular tachyarrhythmias (VT) in a rat model of MI. MI was induced experimentally by permanent occlusion of the left anterior descending artery of Lewis rats. ADSCs were harvested from GFP-transgenic rats, and were cultured until passage four. ADSCs (10 x 10(6)) resuspended in 100 mu L saline or pro-survival cocktail (PSC), which enhances cardiac graft survival, were injected directly into syngeneic rat hearts 1 week after MI. The recipients of ADSCs suspended in PSC had a larger graft area compared with those receiving ASDCs suspended in saline at 1 week post-transplantation (number of graft cells/section: 148.7 +/- 10.6 vs. 22.4 +/- 3.4, p < 0.05, n = 5/group). Thereafter, all ADSC recipients were transplanted with ASDCs in PSC. ADSCs were transplanted into infarcted hearts, and the mechanical and electrophysiological functions were assessed. Echocardiography revealed that ADSC recipients had improved contractile function compared with those receiving PSC vehicle (fractional shortening: 21.1 +/- 0.9 vs. 14.1 +/- 1.2, p <0.05, n >= 12/group). Four weeks post-transplantation, VT was induced via in vivo programmed electrical stimulation. The recipients of ADSCs showed a significantly lower incidence of induced VT compared with the control (31.3% vs. 83.3%, p < 0.05, n >= 12/group). To understand the electrical activity following transplantation, we performed ex vivo optical mapping using a voltage sensitive dye, and found that ADSC transplantation decreased conduction velocity and its dispersion in the pen-infarct area. These results suggest that ADSC transplantation improved cardiac mechanical and electrophysiological functions in subacute MI. (C) 2015 Elsevier Ltd. All rights reserved.
  • Koichi Yano, Hiroyuki Yasuda, Kunio Takaoka, Masafumi Takahashi, Hiroaki Nakamura, Yuuki Imai, Shigeyuki Wakitani
    JOURNAL OF ORTHOPAEDIC SCIENCE 20 (2) 390 - 396 0949-2658 2015/03 [Refereed][Not invited]
    The efficacy of autologous bone grafting in repairing nonunion fractures, large bone defects and spinal instability is widely accepted. However, the cellular and molecular mechanisms underlying new bone formation in bone grafting have yet to be fully elucidated. The purpose of this study was to clarify the fate, origin and the contribution of the cells within the grafted bone. This study was designed to investigate the role and fate of cells contained in the grafted bone and their contribution to new bone formation in the graft in an animal model. Middiaphyseal cylindrical bone samples obtained from green fluorescent protein (GFP) transgenic and wild-type rats were transplanted into the back muscle of wild-type and GFP rats, respectively. The transplanted bones were evaluated by immunohistochemistry, in situ hybridization and quantitative reverse transcription polymerase chain reaction. Immunohistochemical analyses showed that all the cells in the newly formed bone originated from the grafted bone, and osteoblasts were gradually replaced by host cells. Conversely, osteoclasts were immediately replaced by host cells 2 weeks after the bone graft. In addition, expression of bone morphogenetic protein (Bmp)-4, Bmp receptors and Noggin in the grafted bone was significantly upregulated before new bone formation occurred, indicating that the grafted cells might contribute to the recruitment of mesenchymal cells into the graft bed. This study revealed the possible molecular mechanisms of the contribution of cells contained in grafted bone to facilitate new bone formation.
  • Yoshiko Mizushina, Koumei Shirasuna, Fumitake Usui, Tadayoshi Karasawa, Akira Kawashima, Hiroaki Kimura, Motoi Kobayashi, Takanori Komada, Yoshiyuki Inoue, Naoko Mato, Hideaki Yamasawa, Eicke Latz, Yoichiro Iwakura, Tadashi Kasahara, Masashi Bando, Yukihiko Sugiyama, Masafumi Takahashi
    JOURNAL OF BIOLOGICAL CHEMISTRY 290 (8) 5065 - 5077 0021-9258 2015/02 [Refereed][Not invited]
    Supplemental oxygen inhalation is frequently used to treat severe respiratory failure; however, prolonged exposure to hyperoxia causes hyperoxic acute lung injury (HALI), which induces acute respiratory distress syndrome and leads to high mortality rates. Recent investigations suggest the possible role of NLRP3 inflammasomes, which regulate IL-1 beta production and lead to inflammatory responses, in the pathophysiology of HALI; however, their role is not fully understood. In this study, we investigated the role of NLRP3 inflammasomes in mice with HALI. Under hyperoxic conditions, NLRP3(-/-) mice died at a higher rate compared with wild-type and IL-1 beta(-/-) mice, and there was no difference in IL-1 beta production in their lungs. Under hyperoxic conditions, the lungs of NLRP3(-/-) mice exhibited reduced inflammatory responses, such as inflammatory cell infiltration and cytokine expression, as well as increased and decreased expression of MMP-9 and Bcl-2, respectively. NLRP3(-/-) mice exhibited diminished expression and activation of Stat3, which regulatesMMP-9 and Bcl-2, in addition to increased numbers of apoptotic alveolar epithelial cells. In vitro experiments revealed that alveolar macrophages and neutrophils promoted Stat3 activation in alveolar epithelial cells. Furthermore, NLRP3 deficiency impaired the migration of neutrophils and chemokine expression by macrophages. These findings demonstrate that NLRP3 regulates Stat3 signaling in alveolar epithelial cells by affecting macrophage and neutrophil function independent of IL-1 beta production and contributes to the pathophysiology of HALI.
  • Koumei Shirasuna, Fumitake Usui, Tadayoshi Karasawa, Hiroaki Kimura, Akira Kawashima, Hiroaki Mizukami, Akihide Ohkuchi, Satoshi Nishimura, Junji Sagara, Tetsuo Noda, Keiya Ozawa, Shun'ichiro Taniguchi, Masafumi Takahashi
    NANOTOXICOLOGY 9 (5) 554 - 567 1743-5390 2015 [Refereed][Not invited]
    Despite the increasing commercial use of nanoparticles, little is known about their effects on placental inflammation and pregnancy complications. In this study, nanosilica (NS) particles upregulated the inflammasome component nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) and induced placental inflammation and reactive oxygen species (ROS) generation, resulting in pregnancy complications. Furthermore, NS-induced pregnancy complications were markedly improved in N mice but not in component apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)-deficient (Asc) mice, indicating the independence of NLRP3 inflammasomes. Pregnancy complications in Nlrp3(-/-) and Asc(-/-) mice phenotypes were dependent on the balance between interleukin (IL)-1 a and IL-10. NS-induced pregnancy complications were completely prevented by either inhibition of ROS generation or forced expression of IL-10. Our findings provide important information about NS-induced placental inflammation and pregnancy complications and the novel pathophysiological roles of NLRP3 and ASC in pregnancy.
  • Fumitake Usui, Koumei Shirasuna, Hiroaki Kimura, Kazuki Tatsumi, Akira Kawashima, Tadayoshi Karasawa, Koichi Yoshimura, Hiroki Aoki, Hiroko Tsutsui, Tetsuo Noda, Junji Sagara, Shun'ichiro Taniguchi, Masafumi Takahashi
    ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY 35 (1) 127 - 136 1079-5642 2015/01 [Refereed][Not invited]
    Objective-Abdominal aortic aneurysm (AAA) is considered a chronic inflammatory disease; however, the molecular basis underlying the sterile inflammatory response involved in the process of AAA remains unclear. We previously showed that the inflammasome, which regulates the caspase-1-dependent interleukin-1 beta production, mediates the sterile cardiovascular inflammatory responses. Therefore, we hypothesized that the inflammasome is a key mediator of initial inflammation in AAA formation. Approach and Results-Apoptosis-associated speck-like protein containing a caspase recruitment domain is highly expressed in adventitial macrophages in human and murine AAA tissues. Using an established mouse model of AAA induced by continuous infusion of angiotensin II in Apoe(-/-) mice, NLR family pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase recruitment domain, and caspase-1 deficiency in Apoe(-/-) mice were shown to decrease the incidence, maximal diameter, and severity of AAA along with adventitial fibrosis and inflammatory responses significantly, such as inflammatory cell infiltration and cytokine expression in the vessel wall. NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain, and caspase-1 deficiency in Apoe(-/-) mice also reduced elastic lamina degradation and metalloproteinase activation in the early phase of AAA formation. Furthermore, angiotensin II stimulated generation of mitochondria-derived reactive oxygen species in the adventitial macrophages, and this mitochondria-derived reactive oxygen species generation was inhibited by NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain, and caspase-1 deficiency. In vitro experiments revealed that angiotensin II stimulated the NLRP3 inflammasome activation and subsequent interleukin-1 beta release in macrophages, and this activation was mediated through an angiotensin type I receptor/mitochondria-derived reactive oxygen species-dependent pathway. Conclusions-Our results demonstrate the importance of the NLRP3 inflammasome in the initial inflammatory responses in AAA formation, indicating its potential as a novel therapeutic target for preventing AAA progression.
  • Masafumi Takahashi
    JOURNAL OF ATHEROSCLEROSIS AND THROMBOSIS 22 (6) 553 - 554 1340-3478 2015 [Refereed][Not invited]
  • Tadayoshi Karasawa, Akira Kawashima, Fumitake Usui, Hiroaki Kimura, Koumei Shirasuna, Yoshiyuki Inoue, Takanori Komada, Motoi Kobayashi, Yoshiko Mizushina, Junji Sagara, Masafumi Takahashi
    FEBS OPEN BIO 5 348 - 356 2211-5463 2015 [Refereed][Not invited]
    Increasing evidence indicates that caspase recruitment domain (CARD)-mediated caspase-1 (CASP1) assembly is an essential process for its activation and subsequent interleukin (IL)-1 beta release, leading to the initiation of inflammation. Both CARD16 and CARD17 were previously reported as inhibitory homologs of CASP1; however, their molecular function remains unclear. Here, we identified that oligomerization activity allows CARD16 to function as a CASP1 activator. We investigated the molecular characteristics of CARD16 and CARD17 in transiently transfected HeLa cells. Although both CARD16 and CARD17 interacted with CASP1CARD, only CARD16 formed a homo-oligomer. Oligomerized CARD16 formed a filament-like structure with CASP1CARD and a speck with apoptosis-associated speck-like protein containing a CARD. A filament-like structure formed by CARD16 promoted CASP1 filament assembly and IL-1 beta release. In contrast, CARD17 did not form a homo-oligomer or filaments and inhibited CASP1-dependent IL-1 beta release. Mutated CARD16(D27G), mimicking the CARD17 amino acid sequence, formed a homo-oligomer but failed to form a filament-like structure. Consequently, CARD16D27G weakly promoted CASP1 filament assembly and subsequent IL-1 beta release. These results suggest that oligomerized CARD16 promotes CARD-mediated molecular assembly and CASP1 activation. (C) 2015 The Authors. Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. This is an open access article under the CC BY-NC-ND license.
  • Hiroaki Kimura, Patrizio Caturegli, Masafumi Takahashi, Koichi Suzuki
    JOURNAL OF IMMUNOLOGY RESEARCH 2015 541984  2314-8861 2015 [Refereed][Not invited]
    The immunoproteasome is a highly efficient proteolytic machinery derived from the constitutive proteasome and is abundantly expressed in immune cells. The immunoproteasome plays a critical role in the immune system because it degrades intracellular proteins, for example, those of viral origin, into small proteins. They are further digested into short peptides to be presented by major histocompatibility complex (MHC) class I molecules. In addition, the immunoproteasome influences inflammatory disease pathogenesis through its ability to regulate T cell polarization. The immunoproteasome is also expressed in nonimmune cell types during inflammation or neoplastic transformation, supporting a role in the pathogenesis of autoimmune diseases and neoplasms. Following the success of inhibitors of the constitutive proteasome, which is now an established treatment modality for multiple myeloma, compounds that selectively inhibit the immunoproteasome are currently under active investigation. This paper will review the functions of the immunoproteasome, highlighting areas where novel pharmacological treatments that regulate immunoproteasome activity could be developed.
  • Kyoko Hara, Koumei Shirasuna, Fumitake Usul, Tadayoshi Karasawa, Yoshiko Mizushina, Hiroaki Kimura, Akira Kawashima, Akihide Ohkuchi, Shuichi Matsuyama, Koji Kimura, Masafumi Takahashi
    PLOS ONE 9 (12) e113974  1932-6203 2014/12 [Refereed][Not invited]
    Background: Type I interferons (IFNs), including IFN-alpha (IFNA) and IFN-beta (IFNB), have anti-inflammatory properties and are used to treat patients with autoimmune and inflammatory disorders. However, little is known of the role of IFN-tau (IFNT), a type I IFN produced by ruminant animals for inflammation. Because IFNB has recently been shown to inhibit nucleotide-binding oligomerization domain-like receptor, pyrin domain-containing 3 (NLRP3) inflammasome activation and subsequent secretion of the potent inflammatory cytokine interleukin (IL)-1 beta, we examined the effects of ruminant IFNT on NLRP3 inflammasome-mediated IL-1 beta secretion in human THP-1 macrophages. Methods and Results: IFNT dose-dependently inhibited IL-1 beta secretion induced by nano-silica, a well-known activators of NLRP3 inflammasomes, in human macrophages primed with lipopolysaccharide (LPS, TLR4 agonist) and Pam3CSK4 (TLR1/2 agonist). IFNT also suppressed phagocytosis of nano-silica and reactive oxygen species (ROS) generation. Western blot analysis showed that IFNT inhibited both pro-IL-1 beta and mature IL-1 beta. In addition, real-time RT-PCR analysis showed that IFNT suppressed IL-1 beta mRNA expression induced by LPS and Pam3CSK4. Although nano-silica particles did not induce IL-10 secretion, IFNT induced IL-10 secretion in a dose-dependent manner. Furthermore, IFNT-suppressed IL-1 beta secretion was restored by anti-IL-10 neutralizing antibody. Conclusions: Ruminant IFNT inhibits NLRP3 inflammasome-driven IL-1 beta secretion in human macrophages via multiple pathways, including the uptake of nano-silica particles, generation of ROS, and IL-10-mediated inhibition of pro-IL-1 beta induction. It may be a therapeutic alternative to IFNA and IFNB.
  • Hirohiko Motoki, Jun Koyama, Atsushi Izawa, Takeshi Tomita, Yusuke Miyashita, Masafumi Takahashi, Uichi Ikeda
    ECHOCARDIOGRAPHY-A JOURNAL OF CARDIOVASCULAR ULTRASOUND AND ALLIED TECHNIQUES 31 (10) 1230 - 1238 0742-2822 2014/11 [Refereed][Not invited]
    BackgroundThe impact of long-acting calcium channel blocker (CCB) administration on serial changes in left ventricular (LV) function and morphology in hypertensive patients with LV hypertrophy remains unclear. This study attempted to clarify this impact by comparing the effects of administration of azelnidipine with that of amlodipine using conventional and speckle tracking echocardiography. MethodsAn equal number (16) of 32 hypertensive patients was prospectively assigned to a group administered 5mg of amlodipine/day or a group administered 16mg of azelnidipine/day. LV function and morphology was examined by conventional and speckle tracking echocardiography at baseline and at 1, 3, 6, and 12months after treatment initiation. ResultsBoth groups were found to have experienced a significant decrease in systolic blood pressure by 1month after treatment initiation; a significant reduction in septal thickness and LV mass index at 6 and 12months. Transmitral flow E/A ratio and early diastolic mitral annular velocity at lateral wall significantly improved at 12months. On the other hand, a significant improvement of global longitudinal strain was observed earlier than the above indexes at 3, 6, and 12months. Ar-A duration difference was significantly decreased at 3months. The global circumferential strain improved significantly at 3months, but there were no significant changes in mid-/apical circumferential and radial strains throughout the study period. ConclusionAzelnidipine has beneficial effects on LV mass regression, transmitral flow, tissue Doppler, and LV longitudinal strain that are comparable to those of amlodipine on the same parameters.
  • Takahashi M
    Circulation 130 (7) e62  0009-7322 2014/08 [Refereed][Not invited]
  • Tadashi Okano, Shigeyuki Wakitani, Takahiro Okabe, Masafumi Takahashi, Tatsuya Koike, Hiroaki Nakamura
    JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE 8 (5) 414 - 420 1932-6254 2014/05 [Refereed][Not invited]
    The role of cells circulating in the peripheral blood to participate in the natural repair process of osteochondral defects was evaluated in a green fluorescent protein (GFP) transgenic and wild rat parabiosis model. Two weeks after the parabiosis operation, vascular communication between the conjoined rats was confirmed by flow-cytometry analysis. A 1.5 mm diameter and 1.0 mm depth osteochondral defect was made in the patellar groove of each rat femoral bone. Histological examination was performed at 1, 2, 4 and 24 weeks following surgery. In the early postoperative phase (1-4 weeks) there were GFP-negative and -positive cells in the defects of both parabiotic rats. GFP-positive chondrocytes were confirmed partly in the repair tissue of the wild parabiotic rat. In the late postoperative phase (24 weeks), the repaired defects were occupied by cells originating from the adjacent tissue and not from the peripheral blood. The ratio of cells originating from the peripheral blood was approximately 30-40% in the repair tissue at 1 week after surgery, reduced to 0-7% at 24 weeks. From these results it is confirmed that cells circulating in the peripheral blood contributed to the repair of the osteochondral defects, particularly in the early phase of healing. Thus, peripheral blood not only supplies the factors needed for repair but also provides a cell population involved in the wound-healing process. Copyright (c) 2012 John Wiley & Sons, Ltd.
  • Takanori Komada, Fumitake Usui, Koumei Shirasuna, Akira Kawashima, Hiroaki Kimura, Tadayoshi Karasawa, Satoshi Nishimura, Junji Sagara, Tetsuo Noda, Shun'ichiro Taniguchi, Shigeaki Muto, Daisuke Nagata, Eiji Kusano, Masafumi Takahashi
    AMERICAN JOURNAL OF PATHOLOGY 184 (5) 1287 - 1298 0002-9440 2014/05 [Refereed][Not invited]
    Inflammation plays a crucial role in the pathophysiologicat characteristics of chronic kidney disease; however, the inflammatory mechanisms underlying the chronic kidney disease process remain unclear. Recent evidence indicates that sterile inflammation triggered by tissue injury is mediated through a muttiprotein complex called the inflammasome. Therefore, we investigated the role of the inflammasome in the development of chronic kidney disease using a murine unilateral ureteral obstruction (UUO) model. Inflammasome-related molecules were up-regulated in the kidney after UUO. Apoptosis-associated speck-like protein containing a caspase recruitment domain deficiency significantly reduced inflammatory responses, such as inflammatory cell infiltration and cytokine expression, and improved subsequent renal injury and fibrosis. Furthermore, apoptosis-associated speck-like protein containing a caspase recruitment domain was specifically up-regulated in collecting duct (CD) epithelial cells of the UUO-treated kidney. In vitro experiments showed that extracellular adenosine triphosphate (ATP) induced inflammasome activation in CD epithelial cells through P2X(7)-potassium efflux and reactive oxygen species dependent pathways. These results demonstrate the molecular basis for the inflammatory response in the process of chronic kidney disease and suggest the CD inflammasome as a potential therapeutic target for preventing chronic kidney disease progression.
  • Yoshiyuki Inoue, Koumei Shirasuna, Hiroaki Kimura, Fumitake Usui, Akira Kawashima, Tadayoshi Karasawa, Kenji Tago, Katsuya Dezaki, Satoshi Nishimura, Junji Sagara, Tetsuo Noda, Yoichiro Iwakura, Hiroko Tsutsui, Shun'ichiro Taniguchi, Ken Yanagisawa, Toshihiko Yada, Yoshikazu Yasuda, Masafumi Takahashi
    JOURNAL OF IMMUNOLOGY 192 (9) 4342 - 4351 0022-1767 2014/05 [Refereed][Not invited]
    Inflammation plays a key role in the pathophysiology of hepatic ischemia-reperfusion (I/R) injury. However, the mechanism by which hepatic I/R induces inflammatory responses remains unclear. Recent evidence indicates that a sterile inflammatory response triggered by I/R is mediated through a multiple-protein complex called the inflammasome. Therefore, we investigated the role of the inflammasome in hepatic I/R injury and found that hepatic I/R stimuli upregulated the inflammasome-component molecule, nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain-containing 3 (NLRP3), but not apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). NLRP3(-/-) mice, but not ASC(-/-) and caspase-1(-/-) mice, had significantly less liver injury after hepatic I/R. NLRP3(-/-) mice showed reduced inflammatory responses, reactive oxygen species production, and apoptosis in I/R liver. Notably, infiltration of neutrophils, but not macrophages, was markedly inhibited in the I/R liver of NLRP3(-/-) mice. Bone marrow transplantation experiments showed that NLRP3 not only in bone marrow-derived cells, but also in non-bone marrow-derived cells contributed to liver injury after I/R. In vitro experiments revealed that keratinocyte-derived chemokine-induced activation of heterotrimeric G proteins was markedly diminished. Furthermore, NLRP3(-/-) neutrophils decreased keratinocyte-derived chemokine-induced concentrations of intracellular calcium elevation, Rac activation, and actin assembly formation, thereby resulting in impaired migration activity. Taken together, NLRP3 regulates chemokine-mediated functions and recruitment of neutrophils, and thereby contributes to hepatic I/R injury independently of inflammasomes. These findings identify a novel role of NLRP3 in the pathophysiology of hepatic I/R injury.
  • Yuumi Ishizuka, Kazuhiro Nakayama, Ayumi Ogawa, Saho Makishima, Supichaya Boonvisut, Atsushi Hirao, Yusaku Iwasaki, Toshihiko Yada, Yoshiko Yanagisawa, Hiroshi Miyashita, Masafumi Takahashi, Sadahiko Iwamoto
    JOURNAL OF MOLECULAR ENDOCRINOLOGY 52 (2) 145 - 158 0952-5041 2014/04 [Refereed][Not invited]
    Mammalian tribbles homolog 1 (TRIB1) regulates hepatic lipogenesis and is genetically associated with plasma triglyceride (TG) levels and cholesterol, but the molecular mechanisms remain obscure. We explored these mechanisms in mouse livers transfected with a TRIB1 overexpression, a shRNA template or a control (LacZ) adenovirus vector. The overexpression of TRIB1 reduced, whereas induction of the shRNA template increased, plasma glucose, TG, and cholesterol and simultaneously hepatic TG and glycogen levels. The involvement of TRIB1 in hepatic lipid accumulation was supported by the findings of a human SNP association study. A TRIB1 SNP, rs6982502, was identified in an enhancer sequence, modulated enhancer activity in reporter gene assays, and was significantly (P = 9.39 x 10(-7)) associated with ultrasonographically diagnosed nonalcoholic fatty liver disease in a population of 5570 individuals. Transcriptome analyses of mouse livers revealed significant modulation of the gene sets involved in glycogenolysis and lipogenesis. Enforced TRIB1 expression abolished CCAAT/enhancer binding protein A (CEBPA), CEBPB, and MLXIPL proteins, whereas knockdown increased the protein level. Levels of TRIB1 expression simultaneously affected MKK4 (MAP2K4), MEK1 (MAP2K1), and ERK1/2 (MAPK1/3) protein levels and the phosphorylation of JNK, but not of ERK1/2. Pull-down and mammalian two-hybrid analyses revealed novel molecular interaction between TRIB1 and a hepatic lipogenic master regulator, MLXIPL. Co-expression of TRIB1 and CEBPA or MLXIPL reduced their protein levels and proteasome inhibitors attenuated the reduction. These data suggested that the modulation of TRIB1 expression affects hepatic lipogenesis and glycogenesis through multiple molecular interactions.
  • Masafumi Takahashi
    INTERNATIONAL HEART JOURNAL 55 (2) 101 - 105 1349-2365 2014/03 [Refereed][Not invited]
    Inflammasomes are multiple protein complexes that serve as molecular platforms to activate caspase-1 and regulate maturation of a potent proinflammatory cytokine, interleukin (IL)-1 beta, as well as proinflammatory cell death, pyroptosis. Although several types of inflammasomes have been reported so far, recent investigations indicate that the NLRP3 in-flammasome recognizes non-microbial danger signals and leads to sterile inflammatory responses in various disease conditions. Sterile inflammatory responses are also implicated in the development of myocardial infarction (MI). In particular, IL-1 beta is an early and prominent mediator of inflammatory responses in MI, suggesting the pathophysiologic role of NLRP3 inflammasomes in MI. This review highlights the current state of knowledge regarding the role of NLRP3 inflammasomes in MI.
  • Tadashi Ninomiya, Toru Hiraga, Akihiro Hosoya, Kiyoshi Ohnuma, Yuzuru Ito, Masafumi Takahashi, Susumu Ito, Makoto Asashima, Hiroaki Nakamura
    CELL TRANSPLANTATION 23 (6) 691 - 701 0963-6897 2014 [Refereed][Not invited]
    Regeneration of alveolar bone is critical for the successful treatment of periodontal diseases. The periodontal ligament (PDL) has been widely investigated as a source of cells for the regeneration of periodontal tissues. In the present study where we attempted to develop an effective strategy for alveolar bone regeneration, we examined the osteogenic potential of side population (SP) cells, a stem cell-containing population that has been shown to be highly abundant in several kinds of tissues, in PDL cells. Isolated SP cells from the rat PDL exhibited a superior ability to differentiate into osteoblastic cells compared with non-SP (NSP) and unsorted PDL cells in vitro. The mRNA expressions of osteoblast markers and bone morphogenetic protein (BMP) 2 were significantly upregulated in SP cells and were further increased by osteogenic induction. To examine the bone-forming activity of SP cells in vivo, PDL SP cells isolated from green fluorescent protein (GFP)-transgenic rats were transplanted with hydroxyapatite (HA) disks into wild-type animals SP cells exhibited a high ability to induce the mineralized matrix compared with NSP and unsorted PDL cells. At 12 weeks after the implantation, some of the pores in the HA disks with SP cells were filled with mineralized matrices, which were positive for bone matrix proteins, such as osteopontin, bone sialoprotein, and osteocalcin. Furthermore, osteoblast- and osteocyte-like cells on and in the bone-like mineralized matrices were GFP positive, suggesting that the matrices were directly formed by the transplanted cells. These results suggest that PDL SP cells possess enhanced osteogenic potential and could be a potential source for cell-based regenerative therapy for alveolar bone.
  • Takahashi M
    International heart journal 55 (4) 380  1349-2365 2014 [Refereed][Not invited]
  • Yoshiyuki Inoue, Yoshikazu Yasuda, Masafumi Takahashi
    HEPATOLOGY 58 (6) 2212 - 2212 0270-9139 2013/12 [Refereed][Not invited]
  • Jun Koyama, Ayako Kozuka, Masatoshi Minamisawa, Hirohiko Motoki, Atsushi Izawa, Takeshi Tomita, Yusuke Miyashita, Masafumi Takahashi, Uichi Ikeda
    International Journal of Cardiology 168 (6) 5462 - 5464 0167-5273 2013/10 [Refereed][Not invited]
  • Masafumi Takahashi
    Cardiovascular Research 99 (1) 4 - 5 0008-6363 2013/07 [Refereed][Not invited]
  • Takahashi M
    Current vascular pharmacology 1570-1611 2013/04 [Refereed][Not invited]
  • Yuichiro Kashima, Masafumi Takahashi, Yuji Shiba, Naoki Itano, Atsushi Izawa, Jun Koyama, Jun Nakayama, Shun'ichiro Taniguchi, Koji Kimata, Uichi Ikeda
    PLoS ONE 8 (3) e58760  1932-6203 2013/03 [Refereed][Not invited]
    Background: Hyaluronan (HA) is a primary component of the extracellular matrix of cells, and it is involved in the pathogenesis of atherosclerosis. The purpose of this study was to investigate the role of HA in neointimal formation after vascular injury and determine its tissue-specific role in vascular smooth muscle cells (VSMCs) by using a cre-lox conditional transgenic (cTg) strategy. Methods and Results: HA was found to be expressed in neointimal lesions in humans with atherosclerosis and after wire-mediated vascular injury in mice. Inhibition of HA synthesis using 4-methylumbelliferone markedly inhibited neointimal formation after injury. In vitro experiments revealed that low-molecular-weight HA (LMW-HA) induced VSMC activation, including migration, proliferation, and production of inflammatory cytokines, and reactive oxygen species (ROS). The migration and proliferation of VSMCs were mediated by the CD44/RhoA and CD44/ERK1/2 pathways, respectively. Because HA synthase 2 (HAS2) is predominantly expressed in injured arteries, we generated cTg mice that overexpress the murine HAS2 gene specifically in VSMCs (cHAS2/CreSM22α mice) and showed that HA overexpression markedly enhanced neointimal formation after cuff-mediated vascular injury. Further, HA-overexpressing VSMCs isolated from cHAS2/CreSM22α mice showed augmented migration, proliferation, and production of inflammatory cytokines and ROS. Conclusion: VSMC-derived HA promotes neointimal formation after vascular injury, and HA may be a potential therapeutic target for cardiovascular disease. © 2013 Kashima et al.
  • Yasuhiro Tezuka, Shunsuke Endo, Aya Matsui, Atsuko Sato, Katsuyo Saito, Kentaro Semba, Masafumi Takahashi, Takashi Murakami
    LUNG CANCER 78 (3) 185 - 192 0169-5002 2012/12 [Refereed][Not invited]
    Interferon (IFN)-lambda s (IL-28A/IL-28B/IL-29) classified as type III IFNs, are the latest members identified of the interferon family. As with type I IFNs such as IFN-alpha, type III IFNs share antiviral and antitumor activity and may have fewer side effects due to a more selective receptor distribution. Therefore, type III IFNs may be clinically useful for human viral and malignant diseases. Here we demonstrate the potential anti-tumor effect of IFN-lambda 2 (IL-28A) against human lung cancer cells. All lung cancer cell lines that we examined expressed both IFN-lambda receptors (IL-28R1 and IL-10R2). Lung cancer cells with epidermal growth factor receptor (EGFR) mutations were more sensitive to IFN-lambda 2 treatment compared with cells with KRAS mutations. HCC827 cells with an EGFR mutation treated with IFN-lambda 2 underwent growth suppression and apoptotic cell death by STAT1 phosphorylation. Administration of neutralizing antibodies to IFN-lambda inhibited caspase-3/7 activity induced by IFN-lambda 2. Finally, in vivo luminescent imaging also demonstrated the anti-tumor effect of IFN-lambda 2 in a cancer cell transplant animal model. Taken together, IFN-lambda 2 would be a new therapeutic agent for clinical lung cancers with EGFR mutations. (c) 2012 Elsevier Ireland Ltd. All rights reserved.
  • Akihiro Hosoya, Akira Yukita, Kunihiko Yoshiba, Nagako Yoshiba, Masafumi Takahashi, Hiroaki Nakamura
    JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY 60 (11) 861 - 873 0022-1554 2012/11 [Refereed][Not invited]
    Dental pulp is involved in the formation of bone-like tissue in response to external stimuli. However, the origin of osteoblast-like cells constructing this tissue and the mechanism of their induction remain unknown. We therefore evaluated pulp mineralization induced by transplantation of a green fluorescent protein (GFP)-labeled tooth into a GFP-negative hypodermis of host rats. Five days after the transplantation, the upper pulp cavity became necrotic; however, cell-rich hard tissue was observed adjacent to dentin at the root apex. At 10 days, woven bone-like tissue was formed apart from the dentin in the upper pulp. After 20 days, these hard tissues expanded and became histologically similar to bone. GFP immunoreactivity was detected in the hard tissue-forming cells within the root apex as well as in the upper pulp. Furthermore, immunohistochemical observation of alpha-smooth muscle actin, a marker for undifferentiated cells, showed a positive reaction in cells surrounding this bone-like tissue within the upper pulp but not in those within the root apex. Immunoreactivities of Smad4, Runx2, and Osterix were detected in the hard tissue-forming cells within both areas. These results collectively suggest that the dental pulp contains various types of osteoblast progenitors and that these cells might thus induce bone-like tissue in severely injured pulp. (J Histochem Cytochem 60:861-873, 2012)
  • Fumitake Usui, Koumei Shirasuna, Hiroaki Kimura, Kazuki Tatsumi, Akira Kawashima, Tadayoshi Karasawa, Shigeaki Hida, Junji Sagara, Shun'ichiro Taniguchi, Masafumi Takahashi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 425 (2) 162 - 168 0006-291X 2012/08 [Refereed][Not invited]
    Objective: Recent investigations have suggested that the inflammasome plays a role in the development of vascular inflammation and atherosclerosis; however, its precise role remains controversial. We produced double-deficient mice for apolipoprotien E (Apoe) and caspase-1 (Casp1), a key component molecule of the inflammasome, and investigated the effect of caspase-1 deficiency on vascular inflammation and atherosclerosis. Methods and results: Atherosclerotic plaque areas in whole aortas and aortic root of Western diet (WD)-fed Apoe(-/-)Casp1(-/-) mice were significantly reduced compared to those in Apoe(-/-) mice. The amount of macrophages and vascular smooth muscle cells in the plaques was also reduced in Apoe(-/-)Casp1(-/-) mice. No significant differences in plasma lipid profiles and body weight change were observed between these mice. Expression of interleukin (IL)-1 beta in the plaques as well as plasma levels of IL-1 beta, IL-1 alpha, IL-6, CCL2, and TNF-alpha, in Apoe(-/-)Casp1(-/-) mice were lower than those in Apoe(-/-) mice. In vitro experiments showed that calcium phosphate crystals induced caspase-1 activation and secretion of IL-1 beta and IL-1 alpha, in macrophages. Conclusion: Our findings suggest that caspase-1 plays a critical role in vascular inflammation and atherosclerosis, and that modulation of caspase-1 could be a potential target for prevention and treatment of atherosclerosis. (c) 2012 Elsevier Inc. All rights reserved.
  • Aya Matsui, Hideaki Yokoo, Yoichi Negishi, Yoko Endo-Takahashi, Nicole A. L. Chun, Ichiro Kadouchi, Ryo Suzuki, Kazuo Maruyama, Yukihiko Aramaki, Kentaro Semba, Eiji Kobayashi, Masafumi Takahashi, Takashi Murakami
    PLOS ONE 7 (8) e44080  1932-6203 2012/08 [Refereed][Not invited]
    Background: Chemokines are involved in multiple aspects of pathogenesis and cellular trafficking in tumorigenesis. In this study, we report that the latest member of the C-X-C-type chemokines, CXCL17 (DMC/VCC-1), recruits immature myeloid-derived cells and enhances early tumor progression. Methodology/Principal Findings: CXCL17 was preferentially expressed in some aggressive types of gastrointestinal, breast, and lung cancer cells. CXCL17 expression did not impart NIH3T3 cells with oncogenic potential in vitro, but CXCL17-expressing NIH3T3 cells could form vasculature-rich tumors in immunodeficient mice. Our data showed that CXCL17-expressing tumor cells increased immature CD11b(+)Gr1(+) myeloid-derived cells at tumor sites in mice and promoted CD31(+) tumor angiogenesis. Extensive chemotactic assays proved that CXCL17-responding cells were CD11b(+)Gr1(high)F4/80(-) cells (similar to 90%) with a neutrophil-like morphology in vitro. Although CXCL17 expression could not increase the number of CD11b(+)Gr1(+) cells in tumor-burdened SCID mice or promote metastases of low metastatic colon cancer cells, the existence of CXCL17-responding myeloid-derived cells caused a striking enhancement of xenograft tumor formation. Conclusions/Significance: These results suggest that aberrant expression of CXCL17 in tumor cells recruits immature myeloid-derived cells and promotes tumor progression through angiogenesis.
  • Masafumi Takahashi
    CIRCULATION JOURNAL 76 (7) 1597 - 1598 1346-9843 2012/07 [Refereed][Not invited]
  • Akihiro Hosoya, Toru Hiraga, Tadashi Ninomiya, Akira Yukita, Kunihiko Yoshiba, Nagako Yoshiba, Masafumi Takahashi, Susumu Ito, Hiroaki Nakamura
    HISTOCHEMISTRY AND CELL BIOLOGY 137 (6) 733 - 742 0948-6143 2012/06 [Refereed][Not invited]
    The cells of the subodontoblastic cell-rich layer in dental pulp are speculated to contain odontoblast progenitor cells because of their positional relationship with odontoblasts as well as their high alkaline phosphatase (ALP) activity. However, it has yet to be determined whether these cells have the ability to differentiate into odontoblastic cells. In the present study, we firstly found that the majority of cells in the subodontoblastic layer expressed Thy-1, a cell-surface marker of stem and progenitor cells. Then, we evaluated the capacity of Thy-1 high- and low-expressing (Thy-1(high) and Thy-1(low)) cells separated from rat dental pulp cells by use of a fluorescence-activated cell sorter to differentiate into hard tissue-forming cells in vitro and in vivo. Following stimulation with bone morphogenetic protein-2, Thy-1(high) cells in vitro showed accelerated induction of ALP activity and formation of alizarin red-positive mineralized matrix compared with Thy-1(low) cells. Furthermore, subcutaneous implantation of Thy-1(high) cells efficiently induced the formation of bone-like matrix. These results collectively suggest that Thy-1-positive dental pulp cells localized in the subodontoblastic layer had the ability to differentiate into hard tissue-forming cells, and thus these cells may serve as a source of odontoblastic cells.
  • Shin-ichi Takeda, Junko Chinda, Takashi Murakami, Akihiko Numata, Yoshitaka Iwazu, Tetsu Akimoto, Yoshitomo Hamano, Shigeaki Muto, Masafumi Takahashi, Eiji Kusano
    NEPHROLOGY DIALYSIS TRANSPLANTATION 27 (5) 1786 - 1792 0931-0509 2012/05 [Refereed][Not invited]
    Background. It has been well-recognized that cancer patients occasionally develop renal disorders independently of direct tumor invasion. However, the clinical entity of paraneoplastic glomerulopathy remains poorly understood, in part due to the lack of an animal model for basic research. In the present study, we investigated whether cancer-bearing rats develop features of glomerulopathy. Methods. RCN-9 rat colon cancer cells (1 x 10(7)) were injected into F344 rats (n = 13) and T cell-deficient F344 rats (nude rats; n = 7) via the portal system. Urinalysis and histological examinations were performed in comparison with control rats (n = 6) that received a vehicle injection. Results. Metastatic growth of RCN-9 cells exclusively in the liver was observed in the cancer-injected F344 rats, whereas direct invasion into the kidney was not evident even microscopically. Two of the cancer-injected F344 rats died within 2 days, but 9 of the 11 that avoided early death showed elevation of urinary protein (up to 158.0 mg/day) compared to controls (mean values: 60.8 +/- 12.9 versus 17.8 +/- 2.1 mg/day, P = 0.0291). Although morphological changes were not evident in light microscopy, abundant IgG in the glomerular tufts of the proteinuric rats was shown immunohistochemically. Ultrastructure analysis revealed electron-dense deposits in the glomerular basement membrane zone and effacement of the podocytic foot process. Interestingly, none of the nude rats showed proteinuria despite of cancer growth, suggesting that a specific immune response was involved. Conclusions. The tumor-bearing rats developed features of glomerulopathy, as expected from the clinical perspective, and this animal model may provide new insights into the development of paraneoplastic glomerulopathies.
  • Minoru Kobayashi, Tatsuo Morita, Nicole A. L. Chun, Aya Matsui, Masafumi Takahashi, Takashi Murakami
    TUMOR BIOLOGY 33 (2) 551 - 559 1010-4283 2012/04 [Refereed][Not invited]
    There has been little information about metastatic behavior of renal cell carcinoma (RCC) cells because human cancers metastasize only rarely in immunodeficient mice. Moreover, it is difficult to know the effect of host immunity on RCC metastasis due to lack of such RCC cells as transplantable in not only xenograft models but also counterparts with intact immunity. Therefore, we scrutinized in vivo metastasis of RCC cells to seek for the optimal preclinical model to study metastatic behavior. The luciferase-expressing three representative human RCC cell lines (Caki-1, A498, and 786-O) and rat ACI-RCC cell which were established in our laboratory were transplanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice or immunocompetent ACI rats by intracardiac injection as well as orthotopic inoculation. Metastasis was monitored using a bioluminescent imaging technique. Metastasis was rare in the three human RCC cells even when they were directly disseminated into systemic circulation under the condition least susceptible to host immune attack in NOD/SCID mice. In contrast, ACI-RCC cells spontaneously metastasized to pulmonary tissue from orthotopic tumor sites and systemically spread via intracardiac route. Metastases were more extensive when the cells were inoculated into an immunodeficient host, implying suppressive effect of host immunity on colonization of RCC cells. These results suggest that the representative human RCC cells are not adequate resource to study metastasis but that the luciferase-labeled ACI-RCC cell characterized by its luminescent stability, enhanced tumorigenicity, and widespread metastatic potential provides a useful in vivo model for preclinical assessment of cancer progression and potential therapies against RCC.
  • Shingo Akita, Koji Kubota, Akira Kobayashi, Ryosuke Misawa, Akira Shimizu, Takenari Nakata, Takahide Yokoyama, Masafumi Takahashi, Shinichi Miyagawa
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 420 (4) 743 - 749 0006-291X 2012/04 [Refereed][Not invited]
    Bone marrow cell (BMC)-derived myofibroblast-like cells have been reported in various organs, including the pancreas. However, the contribution of these cells to pancreatic fibrosis has not been fully discussed. The present study examined the possible involvement of pancreatic stellate cells (PSCs) originating from BMCs in the development of pancreatic fibrosis in a clinically relevant rat model of acute pancreatitis induced by a choline-deficient/ethionine-supplemented (CDE) diet. BMCs from female transgenic mice ubiquitously expressing green fluorescent protein (GFP) were transplanted into lethally irradiated male rats. Once chimerism was established, acute pancreatitis was induced by a CDE diet. Chronological changes in the number of PSCs originating from the donor BMCs were examined using double immunofluorescence for GFP and markers for PSCs, such as desmin and alpha smooth muscle actin (alpha SMA), 1, 3 and 8 weeks after the initiation of CDE feeding. We also used immunohistochemical staining to evaluate whether the PSCs from the BMCs produce growth factors, such as platelet-derived growth factor (PDGF) and transforming growth factor (TGF) beta 1. The percentage of BMC-derived activated PSCs increased significantly, peaking after 1 week of CDE treatment (accounting for 23.3 +/- 0.9% of the total population of activated PSCs) and then decreasing. These cells produced both PDGF and TGF beta 1 during the early stage of pancreatic fibrosis. Our results suggest that PSCs originating from BMCs contribute mainly to the early stage of pancreatic injury, at least in part, by producing growth factors in a rat CDE diet-induced pancreatitis model. (C) 2012 Elsevier Inc. All rights reserved.
  • Fumitake Usui, Hiroaki Kimura, Taichi Ohshiro, Kazuki Tatsumi, Akira Kawashima, Akiyo Nishiyama, Yo-ichiro Iwakura, Shun Ishibashi, Masafumi Takahashi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 420 (1) 72 - 77 0006-291X 2012/03 [Refereed][Not invited]
    Objective: Several reports describe the role of interleukin (IL)-17 in the development of atherosclerosis; however, its precise role remains controversial. We generated double-deficient mice for apolipoprotein E (apoE) and IL-17 (apoE(-/-)IL-17(-/-) mice) and investigated the effect of IL-17 deficiency on vascular inflammation and atherosclerosis. Methods and results: Atherosclerotic plaque areas in apoE(-/-)IL-17(-/-) mice fed a Western diet (WD) were significantly reduced compared with those in apoE(-/-) mice. No significant differences in plasma lipid profiles were observed between apoE(-/-) and apoE(-/-)IL-17(-/-) mice. The number of infiltrated macrophages in the plaques was significantly decreased in WD-fed apoE(-/-)IL-17(-/-) mice compared with WD-fed apoE(-/-) mice, whereas vascular smooth muscle cell content was not altered by IL-17 deficiency. Expression of inflammatory cytokines (MCP-1, IL-1 beta, IL-6, IFN-gamma, and IL-12 p40) and scavenger receptors (Msr-1, Scarb1, and Olr1) in the plaques was inhibited in WD-fed apoE(-/-)IL-17(-/-) mice. Furthermore, expression of inducible nitric oxide (M1 marker) and arginase-1 (M2 marker) was inhibited in WD-fed apoE(-/-)IL-17(-/-) mice. Conclusion: Our results indicate that IL-17 deficiency reduces vascular inflammation and atherosclerosis and that modulation of IL-17 could be a potential target for prevention and treatment of atherosclerosis. (C) 2012 Elsevier Inc. All rights reserved.
  • Hirohiko Motoki, Jun Koyama, Hideyuki Nakazawa, Kazunori Aizawa, Hiroki Kasai, Atsushi Izawa, Takeshi Tomita, Yusuke Miyashita, Setsuo Kumazaki, Masafumi Takahashi, Uichi Ikeda
    EUROPEAN HEART JOURNAL-CARDIOVASCULAR IMAGING 13 (1) 95 - 103 2047-2404 2012/01 [Refereed][Not invited]
    Aims Anthracyclines have profound consequences on the structure and function of the heart, which over time cause a cardiomyopathy that leads to congestive heart failure. Early detection of subclinical left ventricular (LV) dysfunction following a low dose of anthracyclines may be a preventive strategy. The aim of this study was to determine torsion analysis using two-dimensional speckle-tracking imaging (STI), useful for detecting early anthracycline-mediated cardiotoxicity. Methods and results Conventional and Doppler echocardiography images were obtained from 25 patients (mean age 58 +/- 11 years) before chemotherapy and 1 and 3 months after treatment. The cumulative anthracycline doses were 98 +/- 59 and 170 +/- 87 g/m(2) at 1 and 3 months, respectively. After standard echocardiography, LV torsion and twisting velocity profiles from apical and basal short-axis images were analysed using STI. LV dimensions and ejection fraction did not change throughout follow-up. Although isovolumic relaxation time showed prolongation 3 months after chemotherapy, other Doppler indices did not show significant changes. However, significant deteriorations in torsion (P < 0.0001 by ANOVA), twisting rate (P < 0.0001 by ANOVA), and untwisting rate (P < 0.001 by ANOVA) were found 1 month after chemotherapy. A significant negative correlation was observed between cumulative anthracycline doses and torsion (r = 20.524, P < 0.0001). Conclusion LV torsion analysis could be a useful non-invasive approach for early detection of subclinical anthracycline cardiotoxicity.
  • Mamiko Ise, Hirohiko Ise, Yuji Shiba, Satoshi Kobayashi, Mitsuaki Goto, Masafumi Takahashi, Toshihiro Akaike, Uichi Ikeda
    JOURNAL OF ARTIFICIAL ORGANS 14 (4) 301 - 309 1434-7229 2011/12 [Refereed][Not invited]
    The targeted delivery of anti-inflammatory agents has great therapeutic potential for treating restenosis following percutaneous coronary intervention. To develop a drug delivery system targeted to injured blood vessels, we examined whether N-acetylglucosamine (GlcNAc)-bearing polymer-coated liposomes (GlcNAc-Ls) are specifically taken up by vascular smooth muscle cells (VSMCs). Flow cytometric analysis revealed that GlcNAc-Ls were taken up by VSMCs in vitro. Furthermore, GlcNAc-Ls were intravenously administered to mice that had undergone wire-mediated vascular injury. GlcNAc-Ls markedly accumulated at the intramural site of the injured vessel walls but not at the contralateral (uninjured) vessel walls. These results demonstrated that GlcNAc-Ls can be specifically taken up by VSMCs both in vitro and in vivo. We propose a novel strategy of using GlcNAc-Ls that has potential for application in drug delivery targeted to injured blood vessels.
  • Satoshi Kinugasa, Akihiro Tojo, Tatsuo Sakai, Harukuni Tsumura, Masafumi Takahashi, Yasunobu Hirata, Toshiro Fujita
    KIDNEY INTERNATIONAL 80 (12) 1328 - 1338 0085-2538 2011/12 [Refereed][Not invited]
    The mechanism of selective albuminuria in minimal change nephrotic syndrome, in which glomerular capillaries are diffusely covered by effaced podocyte foot processes with reduced slit diaphragms, is unknown. Podocyte injury is due, in part, to NADPH-induced oxidative stress. Here we studied mechanism of selective albuminuria in puromycin aminonucleoside (PAN) nephrotic rats, a model of minimal change nephrotic syndrome. In these rats, Evans Blue-labeled human albumin was taken up by podocytes and its urinary excretion markedly increased, with retained selectivity for albumin. Immunogold scanning electron micrographic images found increased human albumin in podocyte vesicles and on the apical membrane in nephrotic compared with control rats. Apocynin, an inhibitor of NADPH oxidase, decreased superoxide production in podocytes, and inhibited endocytosis and urinary albumin excretion. Real-time confocal microscopy found an initial delay in the appearance of Evans Blue-labeled human albumin in the tubular lumen, reflecting the time needed for transcellular transport. Immunoprecipitation analysis indicated that FcRn, a receptor for albumin transport, mediated podocyte albumin transport, and treatment with anti-FcRn antibody reduced proteinuria in these nephrotic rats. Thus, podocyte albumin transport was enhanced in PAN nephrotic rats by means of FcRn, which may explain the mechanism of selective proteinuria. This was blocked by apocynin, suggesting a new therapeutic approach.
  • Hidekazu Sekine, Tatsuya Shimizu, Izumi Dobashi, Katsuhisa Matsuura, Nobuhisa Hagiwara, Masafumi Takahashi, Eiji Kobayashi, Masayuki Yamato, Teruo Okano
    Tissue engineering. Part A 17 (23-24) 2973 - 80 1937-3341 2011/12 [Refereed][Not invited]
    Regenerative therapies have currently emerged as one of the most promising treatments for repair of the damaged heart. Recently, numerous researchers reported that isolated cell injection treatments can improve heart function in myocardial infarction models. However, significant cell loss due to primary hypoxia or cell wash-out and difficulty to control the location of the grafted cells remains problem. As an attempt to overcome these limitations, we have proposed cell sheet-based tissue engineering, which involves stacking confluently cultured cells (two-dimensional), cell sheets, to construct three-dimensional cell-dense tissues. Cell sheet transplantation has been able to recover damaged heart function. However, no detailed analysis for transplanted cell survival has been previously performed. The present study compared the survival of cardiac cell sheet transplantation to direct cell injection in a rat myocardial infarction model. Luciferase-expressing neonatal rat cardiac cells were harvested as cell sheets from temperature-responsive culture dishes. The transplantation of cell sheets was compared to the direct injection of isolated cells dissociated with trypsin-ethylenediaminetetraacetic acid. These grafts were transplanted to infarcted rat hearts and cardiac function was assessed by echocardiography at 2 and 4 weeks after transplantation. In vivo bioluminescence and histological analyses were performed to examine cell survival. Cell sheet transplantation consistently yielded greater cell survival than cell injection. Immunohistochemistry revealed that cardiac cell sheets existed over the infarcted area as an intact layer. In contrast, the injected cells were scattered with relatively few cardiomyocytes in the infarcted areas. Four weeks after transplantation, cardiac function was also significantly improved in the cell sheet transplantation group compared with the cell injection. Twenty-four hours after cell grafting, significantly greater numbers of mature capillaries were also observed in the cardiac cell sheet transplantation. Additionally, the numbers of apoptotic cells with deterioration of integrin-mediated attachment were significantly lower after cardiac cell sheet transplantation. In accordance with increased cell survival, cardiac function was significantly improved after cardiac cell sheet transplantation in comparison to cell injection. Cell sheet transplantation can repair damaged hearts through improved cell survival and should become a promising therapy in cardiovascular regenerative medicine.
  • Takeki Hata, Masafumi Takahashi, Shigeaki Hida, Masanori Kawaguchi, Yuichiro Kashima, Fumitake Usui, Hajime Morimoto, Akiyo Nishiyama, Atsushi Izawa, Jun Koyama, Yoichiro Iwakura, Shinsuke Taki, Uichi Ikeda
    CARDIOVASCULAR RESEARCH 90 (2) 364 - 372 0008-6363 2011/05 [Refereed][Not invited]
    Aims Increasing evidence suggests that CD4(+) T cells contribute to neovascularization in ischaemic tissue. However, the T cell subset responsible for neovascularization after ischaemia remains to be determined. Here, we investigated the role of Th17 cells secreting interleukin (IL)-17, a newly identified subset of CD4(+) T cells, in the neovascularization after murine hindlimb ischaemia. Methods and results Unilateral hindlimb ischaemia was produced in wild-type (WT) C57BL/6 mice. Depletion of CD4(+) T cells resulted in significantly reduced blood flow perfusion in the ischaemic limbs. The expression of IL-17 and retinoic acid receptor-related orphan receptor gamma t (ROR gamma t) was up-regulated in the ischaemic limbs. IL-17-deficient mice showed a significant reduction in blood flow perfusion, inflammatory cell infiltration, and production of angiogenic cytokines in the ischaemic limbs compared with WT mice. In bone marrow transplantation experiments, the absence of IL-17 specifically in bone marrow cells diminished the neovascularization after ischaemia. Furthermore, IL-17-deficient CD4(+) T cells transferred into the ischaemic limbs of T cell-deficient athymic nude mice evoked a significantly limited neovascularization compared with WT CD4(+) T cells. Conclusion These findings identify Th17 cells as a new angiogenic T cell subset and provide new insight into the mechanism by which T cells promote neovascularization after ischaemia.
  • Sadao Takahashi, Takashi Ito, Yasuo Zenimaru, Jinya Suzuki, Isamu Miyamori, Masao Takahashi, Masafumi Takahashi, Takafumi Ishida, Tatsuro Ishida, Ken-ichi Hirata, Tokuo T. Yamamoto, Tadao Iwasaki, Hiroaki Hattori, Masashi Shiomi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 407 (4) 656 - 662 0006-291X 2011/04 [Refereed][Not invited]
    Triglyceride-rich lipoproteins (TGRLs) and low-density-lipoprotein (LDL) cholesterol are independent risk factors for coronary artery disease. We have previously proposed that the very low-density-lipoprotein (VLDL) receptor is one of the receptors required for foam cell formation by TGRLs in human macrophages. However, the VLDL receptor proteins have not been detected in atherosclerotic lesions of several animal models. Here we showed no VLDL receptor protein was detected in mouse macrophage cell lines (Raw264.7 and J774.2) or in mouse peritoneal macrophages in vitro. Furthermore, no VLDL receptor protein was detected in macrophages in atherosclerotic lesions of chow-fed apolipoprotein E-deficient or cholesterol-fed LDL receptor-deficient mice in vivo. In contrast, macrophage VLDL receptor protein was clearly detected in human macrophages in vitro and in atherosclerotic lesions in myocardial infarction-prone Watanabe-heritable hyperlipidemic (WHHLMI) rabbits in vivo. There are species differences in the localization of VLDL receptor protein in vitro and in vivo. Since VLDL receptor is expressed on macrophages in atheromatous plaques of both rabbit and human but not in mouse models, the mechanisms of atherogenesis and/or growth of atherosclerotic lesions in mouse models may be partly different from those of humans and rabbits. (C) 2011 Elsevier Inc. All rights reserved.
  • Hirohiko Motoki, Jun Koyama, Takeshi Tomita, Kazunori Aizawa, Hiroki Kasai, Megumi Koshikawa, Atsushi Izawa, Setsuo Kumazaki, Masafumi Takahashi, Uichi Ikeda
    Background: Early diastolic velocity of the mitral annulus and transmitral flow propagation velocity are reported as more reliable determinants of left ventricular diastolic function in patients with atrial fibrillation than are transmitral Doppler indices. This study aimed to test the hypothesis that transmitral flow curve shows pseudorestrictive pattern during rate-controlled atrial fibrillation. Methods: Thirteen paroxysmal atrial fibrillation patients were monitored for three phases: before atrial fibrillation, during atrial fibrillation, and after the recovery of atrial fibrillation to sinus rhythm. Standard two-dimensional, color flow, and tissue Doppler echocardiography were performed. We compared the indices of left ventricular diastolic function among the three phases. Results: The early diastolic velocity of transmitral flow increased significantly during atrial fibrillation (before, 0.76 +/- 0.19 m/sec; during, 0.86 +/- 0.20 m/sec; after recovery to sinus rhythm, 0.73 +/- 0.16 m/sec; P < 0.01). The deceleration time of early transmitral diastolic wave decreased during atrial fibrillation (182.5 +/- 39.6 ms; 149.1 +/- 38.7 ms; 184.0 +/- 44.5 ms, respectively, P < 0.01). The early diastolic velocity of the mitral annulus increased during atrial fibrillation (5.37 +/- 1.31 cm/sec; 7.29 +/- 1.25 cm/sec; 5.37 +/- 1.32 cm/sec; respectively, P < 0.01). The transmitral propagation velocity did not change significantly during atrial fibrillation. Conclusion: Although conventional Doppler indices showed abnormal relaxation pattern, left ventricular diastolic function was preserved during rate-controlled atrial fibrillation, as determined from early diastolic velocity of the mitral annulus and transmitral flow propagation velocity. (Echocardiography 2011;28:289-297).
  • Masafumi Takahashi
    TRENDS IN CARDIOVASCULAR MEDICINE 21 (2) 37 - 41 1050-1738 2011/02 [Refereed][Not invited]
    Inflammation plays a crucial role in the pathophysiology of myocardial infarction (MI). In particular, reperfusion caused by increased thrombolytic activity or revascularization therapy may restore the coronary blood flow and reduce the infarct size, but it also simultaneously enhances the inflammatory response and causes harmful effects on the myocardium a process termed ischemia-reperfusion (I/R) injury. The inflammasome is a large multiprotein complex that is formed in the cytosol in response to danger signals; it drives the proinflammatory cytokine interleukin(IL)-1 beta.Increasing evidence indicates that the inflammasome is a key player in the disease processes of sterile inflammation. In particular, IL-1 beta is a prominent and early mediator of inflammation in I/R injury, suggesting the importance of the inflammasome in myocardial l/R injury. This article reviews the role of the inflammasome in the development of myocardial I/R injury and discusses the potential of the inflammasome as a novel therapeutic target for the treatment of myocardial I/R injury. (Trends Cardiovasc Med 2011;21:37-41) (C) 2011 Elsevier Inc. All rights reserved.
  • Masanori Kawaguchi, Masafumi Takahashi, Takeki Hata, Yuichiro Kashima, Fumitake Usui, Hajime Morimoto, Atsushi Izawa, Yasuko Takahashi, Junya Masumoto, Jun Koyama, Minoru Hongo, Tetsuo Noda, Jun Nakayama, Junji Sagara, Shun'ichiro Taniguchi, Uichi Ikeda
    CIRCULATION 123 (6) 594 - + 0009-7322 2011/02 [Refereed][Not invited]
    Background-Inflammation plays a key role in the pathophysiology of myocardial ischemia/reperfusion (I/R) injury; however, the mechanism by which myocardial I/R induces inflammation remains unclear. Recent evidence indicates that a sterile inflammatory response triggered by tissue damage is mediated through a multiple-protein complex called the inflammasome. Therefore, we hypothesized that the inflammasome is an initial sensor for danger signal(s) in myocardial I/R injury. Methods and Results-We demonstrate that inflammasome activation in cardiac fibroblasts, but not in cardiomyocytes, is crucially involved in the initial inflammatory response after myocardial I/R injury. We found that inflammasomes are formed by I/R and that its subsequent activation of inflammasomes leads to interleukin-1 beta production, resulting in inflammatory responses such as inflammatory cell infiltration and cytokine expression in the heart. In mice deficient for apoptosis-associated speck-like adaptor protein and caspase-1, these inflammatory responses and subsequent injuries, including infarct development and myocardial fibrosis and dysfunction, were markedly diminished. Bone marrow transplantation experiments with apoptosis-associated speck-like adaptor protein-deficient mice revealed that inflammasome activation in bone marrow cells and myocardial resident cells such as cardiomyocytes or cardiac fibroblasts plays an important role in myocardial I/R injury. In vitro experiments revealed that hypoxia/reoxygenation stimulated inflammasome activation in cardiac fibroblasts, but not in cardiomyocytes, and that hypoxia/reoxygenation-induced activation was mediated through reactive oxygen species production and potassium efflux. Conclusions-Our results demonstrate the molecular basis for the initial inflammatory response after I/R and suggest that the inflammasome is a potential novel therapeutic target for preventing myocardial I/R injury. (Circulation. 2011;123:594-604.)
  • Asako Yamaguchi, Takashi Murakami, Masafumi Takahashi, Eiji Kobayashi, Yasushi Sugawara
    PLASTIC AND RECONSTRUCTIVE SURGERY 127 (1) 78 - 87 0032-1052 2011/01 [Refereed][Not invited]
    Background: Bone transplantation is an important procedure often used for bone defect reconstruction after trauma and malignancies. However, the kinetics of free bone graft-derived cells remains unclarified. The authors examined the kinetics of graft-derived cells using transgenic rats systemically expressing firefly luciferase. Methods: Free iliac bone grafts (5 x 5 x 2 mm, n = 10) derived from luciferase transgenic rats were transplanted into the subcutaneous space of the back of wild-type Lewis rats, and the kinetics of graft-derived cells were evaluated over time by determining the level of luminescence emission. Results: Although the luminescence level emitted by luciferase decreased after transplantation, a substantial luminescence level (mean, 1.6 x 10(7) photons/second) was emitted from donor-derived cells even at 180 days after transplantation, suggesting a long-term survival of graft-derived cells. In a computed tomographic image analysis of bone grafts retrieved 180 days after transplantation, high-luminescence grafts with a sufficient number of viable graft-derived cells (mean, 2.6 x 10(7) photons/second; n = 4) showed significantly higher bone graft volume (3.1-fold) and polar moment of inertia of area (7.2-fold) than low-luminescence grafts (mean, 1.0 x 10(7) photons/second; n = 4), indicating that high-luminescence grafts maintain better conditions. Conclusion: These results suggest that bone graft-derived cells can survive for a long time and that the presence of a sufficient number of viable graft-derived cells is essential for graft engraftment and remodeling. (Plast. Reconstr. Surg. 127: 78, 2011.)
  • Setsuo Kumazaki, Jun Koyama, Kazunori Aizawa, Hiroki Kasai, Megumi Koshikawa, Atsushi Izawa, Takeshi Tomita, Masafumi Takahashi, Uichi Ikeda
    HEART AND VESSELS 25 (6) 515 - 521 0910-8327 2010/11 [Refereed][Not invited]
    Left internal mammary artery (LIMA) bypass conduits undergo gradual longitudinal flow transition from the proximal to distal segments, and the diastolic/systolic (D/S) ratios of the flow indices can diagnose graft patency. However, the influence of graft adaptation on this has not been studied. We examined 46 patients with LIMA graft to the left anterior descending artery using a Doppler-tipped guidewire in the proximal, middle, and distal segments; 34 had patent LIMAs (group A: new LIMAs; <1 month postoperatively; n = 22 and group B: old LIMAs; >= 1 month postoperatively; n = 12), and 12 had new LIMAs with distal stenosis (group C). In diastole, the time-averaged peak velocities, maximum peak velocities, and velocity time integrals in each segment were significantly greater in group A than in groups B or C; however, in systole, they did not differ significantly among the three groups. The D/S ratios of the indices in all segments in group A were significantly greater than those in groups B or C; however, they did not differ between groups B and C in any of the segments. Graft adaptation of a patent LIMA, itself, affects the longitudinal flow transition pattern. The D/S ratio of the three indices in the patent old LIMAs did not differ from those in the LIMAs with distal stenosis early after surgery. The timing of LIMA flow assessment must be considered during assessment of the graft patency from the flow velocity patterns.
  • Masafumi Takahashi
    JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY 49 (3) 341 - 342 0022-2828 2010/09 [Refereed][Not invited]
  • Hirohiko Ise, Satoshi Kobayashi, Mitsuaki Goto, Takao Sato, Masatomo Kawakubo, Masafumi Takahashi, Uichi Ikeda, Toshihiro Akaike
    GLYCOBIOLOGY 20 (7) 843 - 864 0959-6658 2010/07 [Refereed][Not invited]
    Vimentin and desmin are intermediate filament proteins found in various mesenchymal and skeletal muscle cells, respectively. These proteins play an important role in the stabilization of the cytoplasmic architecture. Here, we found, using artificial biomimicking glycopolymers, that vimentin and desmin possess N-acetylglucosamine (GlcNAc)-binding lectin-like properties on the cell surfaces of various vimentin- and desmin-expressing cells such as cardiomyocytes and vascular smooth muscle cells. The rod II domain of these proteins was demonstrated to be localized to the cell surface and to directly bind to the artificial biomimicking GlcNAc-bearing polymer, by confocal laser microscopy and surface plasmon resonance analysis. These glycopolymers strongly interact with lectins and are useful tools for the analysis of lectin-carbohydrate interactions, since glycopolymers binding to lectins can induce the clustering of lectins due to multivalent glycoside ligand binding. Moreover, immunocytochemistry and pull-down assay with His-tagged vimentin-rod II domain protein showed that the vimentin-rod II domain interacts with O-GlcNAc proteins. These results suggest that O-GlcNAc proteins might be one candidate for physiological GlcNAc-bearing ligands with which vimentin and desmin interact. These findings demonstrate a novel function of vimentin and desmin that does not involve stabilization of the cytoplasmic architecture by which these proteins interact with physiological GlcNAc-bearing ligands such as O-GlcNAc proteins on the cell surface through their GlcNAc-binding lectin-like properties.
  • Masafumi Takahashi
    CIRCULATION JOURNAL 74 (3) 418 - 423 1346-9843 2010/03 [Refereed][Not invited]
    Myocardial infarction (MI) is accompanied by an inflammatory response, leading to the recruitment of leukocytes and subsequent myocardial injury and healing. Chemokines are potent chemoattractant cytokines that regulate leukocyte trafficking in inflammatory processes. Recent evidence indicates that chemokines play a role not only in leukocyte trafficking but also in angiogenesis and cardioprotection. In particular, stromal cell-derived factor-la (SDF-1 alpha) has generated considerable, interest for its role in the pathophysiology of MI. This review will focus on the role of SDF-1 and its receptor CXC chemokine receptor 4 (CXCR4; ie, the SDF-1/CXCR4 system) in the pathophysiology of MI and discuss their potential as therapeutic targets for MI. (Circ J 2010; 74: 418-423)
  • Hidetoshi Abe, Masafumi Takahashi, Hironobu Yaegashi, Seiichiro Eda, Hideo Tsunemoto, Mamoru Kamikozawa, Jun Koyama, Kyohei Yamazaki, Uichi Ikeda
    HEART AND VESSELS 25 (1) 63 - 69 0910-8327 2010/01 [Refereed][Not invited]
    The purpose of this study was to determine the relationship between obstructive sleep apnea (OSA) and cardiovascular disorders in a large Japanese population, and to assess the efficacy of continuous positive airway pressure (CPAP) in the treatment of OSA-associated arrhythmias. The study population comprised 1394 Japanese subjects (1086 men and 308 women) who were divided into four groups on the basis of polysomnography (PSG) analysis as follows: the no sleep apnea (N-SA) group (n = 44, apnea-hypopnea index [AHI] < 5), the mild OSA (Mi-OSA) group (n = 197, 5 < AHI < 15), the moderate OSA (Mo) group (n = 368, 15 < AHI < 30), and severe OSA (SOSA) group (n = 785, AHI < 30). The following baseline characteristics were significantly associated with OSA: age (P < 0.001), gender (P < 0.001), body mass index (P < 0.001), hypertension (P < 0.001), diabetes (P = 0.009), and hyperlipidemia (P = 0.013). In the OSA group, PSG revealed the predominance of paroxysmal atrial fibrillation (PAF) (P = 0.051), premature atrial complex short run (P < 0.005), premature ventricular complex (PVC, P = 0.004), sinus bradycardia (P = 0.036), and sinus pause (arrest > 2 s, P < 0.001) during the PSG recording. A total of 316 patients from the group underwent CPAP titration and were then re-evaluated. Continuous positive airway pressure therapy significantly reduced the occurrences of PAF (P < 0.001), PVC (P = 0.016), sinus bradycardia (P = 0.001), and sinus pause (P = 0.004). The results of this study demonstrate a significant relationship between OSA and several cardiac disorders, and also demonstrate the efficacy of CPAP in preventing OSA-associated arrhythmias in a large population of Japanese patients.
  • Jinya Suzuki, Masami Ueno, Miyuki Uno, Yoshikazu Hirose, Yasuo Zenimaru, Sadao Takahashi, Jun-ichi Osuga, Shun Ishibashi, Masafumi Takahashi, Masamichi Hirose, Mitsuhiko Yamada, Fredric B. Kraemer, Isamu Miyamori
    AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM 297 (5) E1115 - E1124 0193-1849 2009/11 [Refereed][Not invited]
    Suzuki J, Ueno M, Uno M, Hirose Y, Zenimaru Y, Takahashi S, Osuga J, Ishibashi S, Takahashi M, Hirose M, Yamada M, Kraemer FB, Miyamori I. Effects of hormone-sensitive lipase disruption on cardiac energy metabolism in response to fasting and refeeding. Am J Physiol Endocrinol Metab 297: E1115-E1124, 2009. First published August 25, 2009; doi: 10.1152/ajpendo.91031.2008.-Increased fatty acid (FA) flux and intracellular lipid accumulation (steatosis) give rise to cardiac lipotoxicity in both pathological and physiological conditions. Since hormone-sensitive lipase (HSL) contributes to intracellular lipolysis in adipose tissue and heart, we investigated the impact of HSL disruption on cardiac energy metabolism in response to fasting and refeeding. HSL-knockout (KO) mice and wild-type (WT) littermates were fasted for 24 h, followed by similar to 6 h of refeeding. Plasma FA concentration in WT mice was elevated twofold with fasting, whereas KO mice lacked this elevation, resulting in twofold lower cardiac FA uptake compared with WT mice. Echocardiography showed that fractional shortening was 15% decreased during fasting in WT mice and was associated with steatosis, whereas both of these changes were absent in KO mice. Compared with Langendorff-perfused hearts isolated from fasted WT mice, the isolated KO hearts also displayed higher contractile function and a blunted response to FA. Although cardiac glucose uptake in KO mice was comparable with WT mice under all conditions tested, cardiac VLDL uptake and lipoprotein lipase (LPL) activity were twofold higher in KO mice during fasting. The KO hearts showed undetectable activity of neutral cholesteryl esterase and 40% lower non-LPL triglyceride lipase activity compared with WT hearts in refed conditions accompanied by overt steatosis, normal cardiac function, and increased mRNA expression of adipose differentiation-related protein. Thus, the dissociation between cardiac steatosis and functional sequelae observed in HSL-KO mice suggests that excess FA influx, rather than steatosis per se, appears to play an important role in the pathogenesis of cardiac lipotoxicity.
  • Abe H, Takahashi M, Yaegashi H, Eda S, Kitahara H, Tsunemoto H, Kamikozawa M, Koyama J, Yamazaki K, Ikeda U
    Circulation journal : official journal of the Japanese Circulation Society 73 (11) 2148 - 2153 1346-9843 2009/11 [Refereed][Not invited]
  • Masafumi Takahashi
    ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY 29 (10) 1407 - 1408 1079-5642 2009/10 [Refereed][Not invited]
  • Masafumi Takahashi, Atsushi Izawa, Yoshiaki Ishigatsubo, Kazuteru Fujimoto, Masaaki Miyamoto, Takashi Horie, Yoshifusa Aizawa, Jun Amano, Seiji Minota, Toyoaki Murohara, Hiroaki Matsubara, Uichi Ikeda
    CURRENT PHARMACEUTICAL DESIGN 15 (24) 2778 - 2783 1381-6128 2009/08 [Refereed][Not invited]
    Vasculopathy in patients with connective tissue diseases (CTDs), including systemic sclerosis (SSc) and systemic lupus erythematosus (SLE), is a serious complication that mainly affects small arteries and capillaries, reduces the blood flow and causes progressive tissue ischemia. Recently, CTD patients have been reported to have abnormalities in circulating endothelial progenitor cells (EPCs); these abnormalities are believed to contribute to the pathophysiology of vasculopathy and to the premature and accelerated development of atherosclerosis in CTD patients. Furthermore, we are currently conducting a clinical pilot study to determine the efficacy of implanting autologous mononuclear cells obtained from the bone marrow and peripheral blood into the ischemic digits or limbs of CTD patients. In this review, we discuss the role of EPCs in the process of neovascularization and in the pathophysiology of CTDs, and we describe a clinical pilot study on the use of autologous cell therapy for treating ischemic digits in patients with CTDs.
  • Yukitomo Arao, Yoji Hakamata, Yuka Igarashi, Yuki Sato, Fujio Kayama, Masafumi Takahashi, Eiji Kobayashi, Takashi Murakami
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 382 (1) 46 - 50 0006-291X 2009/04 [Refereed][Not invited]
    We previously created the Alb-DsRed2 transgenic (Tg) rat that specifically expresses the red fluorescent protein, DsRed2, in the liver. Herein, we demonstrate that the DsRed2 expression is sexually dimorphic and exhibits a male-specific pattern. The profiling of sexual dimorphism in DsRed2 expression during pre-pubertal development was investigated using an in vivo fluorescent imaging analysis. The DsRed2 expression decreased gradually in both sexes until 28 days after birth. While DsRed2 expression was not persistent in the female liver, the male hepatic expression increased again at 35 days. Sexual dimorphic DsRed2 expression did not change in gonadectomized male and female Tg-rats. However, female hepatic DsRed2 was induced 72 h after the hypophysectomy. Hepatocytes isolated from the female Tg-rats also revealed DsRed2 induction by 96 h in culture. These results suggest that the pituitary hormone suppresses the female hepatic DsRed2 expression causing the sexual dimorphism of DsRed2 expression. (C) 2009 Elsevier Inc. All rights reserved.
  • Hiroshi Doi, Tatsuya Iso, Yuji Shiba, Hiroko Sato, Miki Yamazaki, Yoshiaki Oyama, Hideo Akiyama, Toru Tanaka, Tomoyuki Tomita, Masashi Arai, Masafumi Takahashi, Uichi Ikeda, Masahiko Kurabayashi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 381 (4) 654 - 659 0006-291X 2009/04 [Refereed][Not invited]
    Bone marrow- (BM-) derived cells can differentiate into smooth muscle-like cells (SMLC), resulting in vascular pathogenesis. However, the molecular mechanism of the differentiation remains unknown. We have recently reported that Notch signaling promotes while a Notch target HERP1 inhibit the differentiation of mesenchymal cells to SMC. During the differentiation of BM-derived mononuclear cells into smooth muscle alpha-actin (SMA)-positive cells, expression of Jagged1 and SMC-specific Notch3 was increased. Blocking Notch with gamma-secretase inhibitor prevented the induction of SMA. Wire-mediated vascular injury was produced in femoral arteries in mice transplanted with green fluorescent protein (GFP)-positive cells. Many double-positive cells for GFP/Jagged1 or GFP/Notch3 were detected in the thickened neointima. In contrast, only a few SMA-positive cells were positive for GFP in neointima where HERP1, a suppressor for Notch, were abundantly expressed. In conclusion, Notch-HERP1 pathway plays an important role in differentiation of BM-derived mononuclear cells into SMLC. (C) 2009 Elsevier Inc. All rights reserved
  • Ryosuke Misawa, Junpei Soeda, Hirohiko Ise, Masafumi Takahashi, Koji Kubota, Atsuyoshi Mita, Takenari Nakata, Shinichi Miyagawa
    TRANSPLANTATION 87 (8) 1147 - 1154 0041-1337 2009/04 [Refereed][Not invited]
    Background. Bone marrow cells (BMCs) are believed to have the ability to generate functional hepatocytes and have some merits as a therapeutic modality for metabolic liver diseases. However, the appearance of BMC-derived hepatocytes (BMDHs) is low. We hypothesized that early BMC injection would be feasible for creating BMDHs for two main reasons: (1) the liver is a hematopoietic organ in neonatal rats and (2) it may allow sufficient time to generate more BMDHs before severe liver injury occurs. Methods. We used Long Evans Cinnamon (LEC) rats as recipients, a model of (1) Wilson disease and (2) liver carcinogenesis. Green fluorescent protein-expressing BMCs were injected into newborn LEC rats through the spleen. The oxidative activity of ceruloplasmin, which is low in LEC rats, was measured to evaluate the treatment. In addition, we performed fluorescence in situ hybridization to clarify the origin of the BMDHs and immunohistochemical analysis to confirm whether these BMDHs had malignant potential. Results. At 18 months after injection, we found some green fluorescent protein-expressing areas macroscopically in the liver of treated LEC rats. The oxidative activity of ceruloplasmin increased in treated LEC rats (n=7) and were much higher than that in untreated LEC rats (P=0.015). Moreover, we confirmed that the BMDHs were generated by cell fusion and was not detected in any of the neoplastic lesions or cholngiofibrotic regions. Conclusion. Our results suggest that this novel strategy for creating BMDHs is effective and safe.
  • Toru Hiraga, Tadashi Ninomiya, Akihiro Hosoya, Masafumi Takahashi, Hiroaki Nakamura
    JOURNAL OF BONE AND MINERAL METABOLISM 27 (2) 149 - 157 0914-8779 2009/03 [Refereed][Not invited]
    Periodontal ligament (PDL) is a unique connective tissue that not only connects cementum and alveolar bone to support teeth, but also plays an important role in reconstructing periodontal tissues. Previous studies have suggested that PDL cells have osteogenic potential; however, they lack precise histological examinations. Here, we studied bone-like matrix formation by PDL cells in rats using morphological techniques. Rat and human PDL cells exhibited substantial alkaline phosphatase activity and induced mineralization in vitro. RT-PCR analyses showed that PDL cells expressed the osteoblast markers, Runx2, osterix, and osteocalcin. These results suggest that PDL cells share similar phenotypes with osteoblasts. To examine the bone-like matrix formation in vivo, PDL cells isolated from green fluorescent protein (GFP)-transgenic rats were inoculated with hydroxyapatite (HA) disks into wild-type rats. Five weeks after the implantation, the pores in HA disks were occupied by GFP-positive cells. Mineralized matrix formation was also found on the surface of HA pores. At 12 weeks, some of the pores were filled with bone-like mineralized matrices (BLMM), which were positive for the bone matrix proteins, osteopontin, bone sialoprotein, and osteocalcin. Immunohistochemical examination revealed that most of the osteoblast- and osteocyte-like cells on or in the BLMM were GFP-positive, suggesting that the BLMM were directly formed by the inoculated PDL cells. On the pore surfaces, Sharpey's fiber-like structures embedded in cementum-like mineralized layers were also observed. These results collectively suggest that PDL cells have the ability to form periodontal tissues and could be a useful source for regenerative therapies of periodontal diseases.
  • Megumi Koshikawa, Kazunori Aizawa, Hiroki Kasai, Atsushi Izawa, Takeshi Tomita, Setsuo Kumazaki, Hiroshi Tsutsui, Jun Koyama, Shigetaka Shimodaira, Masafumi Takahashi, Uichi Ikeda
    ANGIOLOGY 60 (1) 42 - 45 0003-3197 2009/02 [Refereed][Not invited]
    This study was carried out to compare concentrations of osteopontin (OPN) and osteoprotegerin (OPG) in peripheral arterial disease (PAD). The study population consisted of 200 consecutive subjects in whom both OPN/OPG and ankle-brachial index were measured. It was found that OPN levels, but not OPG levels, were significantly more increased in patients with PAD than those without PAD. Serum OPN levels were significantly lower in subjects with angiotensin converting enzyme inhibitors or angiotensin II receptor blockers than those without these agents. In this study, it has been demonstrated for the first time that serum OPN levels are related to PAD. Inhibition of reninangiotensin system could decrease OPN levels and prevent the progression of PAD.
  • Satoshi Kobayashi, Hirohiko Ise, Masafumi Takahashi, Mitsuaki Goto, Toshihiro Akaike, Uichi Ikeda
    BIOMATERIALS 30 (4) 574 - 582 0142-9612 2009/02 [Refereed][Not invited]
    Bone marrow implantation (BMII) has been performed clinically for the treatment of ischemic cardiovascular diseases. To achieve BMI effectively, accumulation of many bone marrow cells (BMCs) in an infarcted area of the myocardium is important. Previously, we reported that cardiomyocytes show strong interaction with N-acetylglucosamine (GIcNAc) and they can take up GIcNAc-conjugated liposomes. Thus, we examined whether GlcNAc-coated BMCs exhibit Strong interaction with cardiomyocytes. The Cell Surface of BMCs was coated with GIcNAc without causing cell injury by GIcNAc-lipophilic polymers. it was found that the GIcNAc-coated BMCs exhibited strong interaction with cardiomyocytes. At 7 days of coculturing the GlcNAc-coated BMCs with cardiomyocytes, BMC-derived cardiomyocytes were generated. The number of BMC-derived cardiomyocytes was higher following coculture with GlcNAc-coated BMCs than following coculture with uncoated and maltose (MA)-coated BMCs. In this study, we demonstrated that the surface coating of BMCs with GIcNAc can be performed easily by using GIcNAc-lipophilic polymers and that GIcNAc-coated BMCs exhibited strong interaction with cardiomyocytes. Therefore, we think that cell Surface Coating with GIcNAc would help promote accumulation of BMCs in the infarcted area of the myocardium and that this accumulation Would be helpful in the treatment of ischemic cardiovascular diseases with BMI. (C) 2008 Elsevier Ltd. All rights reserved.
  • Shin-ichi Aso, Yoshikazu Yazaki, Hiroki Kasai, Masafumi Takahashi, Taku Yoshio, Keiji Yamamoto, Uichi Ikeda
    INTERNATIONAL JOURNAL OF CARDIOLOGY 131 (2) 240 - 245 0167-5273 2009/01 [Refereed][Not invited]
    Background: The autoantibodies stimulate the beta1-adrenoreceptors on cardiac myocytes similar to norepinephrine, and are associated with reduced cardiac function. Iodine-123 metaiodobenzylguanidine ((123)I-MIBG) is metabolized similarly to norepinephrine. This study was undertaken to investigate the relationship between cardiac stimulation by anti-beta1-adrenoreceptor autoantibodies and myocardial sympathetic nervous activity in patients with chronic heart failure. Methods: We screened for the anti-beta1-adrenoreceptor autoantibodies in 52 patients with chronic heart failure by conducting an enzyme-linked immunosorbent assay, and underwent (123)I-MIBG scintigraphy in 27 of the patients. Anterior planar images of (123)I-MIBG were obtained 15 min and 3 h after the injection. We determined the heart to mediastinum radioactivity ratio (H/M), and calculated the rate of washout of (123)I-MIBG from the heart. Results: Patients with New York Heart Association functional class III or IV had higher levels of anti-beta1-adrenoreceptor autoantibodies than those with class I or II (p<0.01). The autoantibody level was significantly correlated with delayed H/M (r =-0.65, p<0.001) and washout rate (r = 0.65, p<0.001). Sixteen patients with a cardiac event showed higher levels of the autoantibodies (p<0.05). Cardiac event-free survival was poorer in patients with the autoantibody levels >10 U/ml than that <10 U/ml (log-rank= 12.1, p<0.001). Conclusion: The anti-beta1-adrenoreceptor autoantibodies are closely associated with cardiac sympathetic nervous activity assessed by (123)I-MIBG and cardiac event in patients with chronic heart failure. (C) 2008 Elsevier Ireland Ltd. All rights reserved.
  • Yuji Shiba, Masafumi Takahashi, Takeki Hata, Hideki Murayama, Hajime Morimoto, Hirohiko Ise, Takashi Nagasawa, Uichi Ikeda
    CARDIOVASCULAR RESEARCH 81 (1) 169 - 177 0008-6363 2009/01 [Refereed][Not invited]
    The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor (CXCR4, CXC chemokine receptor 4) play a critical role in the process of post-natal neovascularization. Here, we investigated the role of CXCR4(+) bone marrow cells (BMCs) in neovascularization in a murine hindlimb ischaemia model. We found that the expression of CXCR4 in BMCs was specifically upregulated by cultivation; therefore, we used freshly isolated BMCs and cultivated BMCs, designated as BMC(Fr) and BMC(Cul), respectively. The increased CXCR4 expression corresponded to the migratory capacity in response to SDF-1 alpha. Real-time reverse transcription-polymerase chain reaction and immunohistochemical analyses revealed that SDF-1 alpha expression was significantly increased in the ischaemic limbs of mice. Blood flow perfusion and capillary density were significantly accelerated in mice implanted with BMC(Cul) as compared with those in mice implanted with BMC(Fr). The stimulatory effect of BMC(Cul) on neovascularization was significantly impaired when BMC(Cul) derived from CXCR4(+/-) mice were implanted. The implanted BMC(Cul) showed high retention in the ischaemic limbs. Further, the implantation of BMC(Cul) significantly increased the expression of interleukin (IL)-1 beta and vascular endothelial growth factor-A in the ischaemic limbs. The upregulation of CXCR4 expression by cultivation may serve as a useful source of BMCs for accelerating therapeutic angiogenesis in ischaemic cardiovascular diseases.
  • Takahashi M
    Current pharmaceutical design 15 (24) 2759  1381-6128 2009 [Refereed][Not invited]
  • Hideki Murayama, Masafumi Takahashi, Masaya Takamoto, Yuji Shiba, Hirohiko Ise, Jun Koyama, Yoh-ichi Tagawa, Yoichiro Iwakura, Uichi Ikeda
    CARDIOVASCULAR RESEARCH 80 (2) 175 - 180 0008-6363 2008/11 [Refereed][Not invited]
    Aims Neointimal formation after percutaneous coronary intervention (PCI), termed restenosis, limits therapeutic revascularization. Since it is now known that vascular injury involves an inflammatory response, we examined the role of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) in the neointimal formation after injury. Methods and results Control (BALB/c), TNF-alpha-deficient (Tnf(-/-)), IFN-gamma-deficient Ifng(-/-)), or double-deficient (Tnf(-/-)Ifng(-/-)) mice were subjected to wire-mediated vascular injury of the right femoral artery. Neointimal formation after injury was significantly reduced after the injury in the Tnf(-/-)Ifng(-/-)) mice, compared to that in the control, Tnf(-/-), and Ifng(-/-) mice. Immunohistochemical analysis showed that TNF-alpha and IFN-gamma were expressed in neointimal lesions in the control mice, but not in mice with deficiency of the corresponding cytokine. No significant difference in re-endothelialization was observed among these groups. The number of proliferating cell nuclear antigen in the neointimal lesions was significantly decreased in the Tnf(-/-)Ifng(-/-) mice. Bone marrow transplantation experiments revealed that deficiency of TNF-alpha and IFN-gamma specifically in bone marrow cells significantly inhibited neointimal formation after vascular injury. Conclusion The absence of TNF-alpha and IFN-gamma in bone marrow cells synergistically inhibits neointimal formation following vascular injury, and thus, may provide new insights into the mechanisms underlying restenosis after PCI.
  • Masafumi Takahashi
    CARDIOVASCULAR RESEARCH 80 (1) 5 - 6 0008-6363 2008/10 [Refereed][Not invited]
  • Hajime Morimoto, Masamichi Hirose, Masafumi Takahashi, Masanori Kawaguchi, Hirohiko Ise, Pappachan E. Kolattukudy, Mitsuhiko Yamada, Uichi Ikeda
    CARDIOVASCULAR RESEARCH 78 (3) 554 - 562 0008-6363 2008/06 [Refereed][Not invited]
    Aims Monocyte chemoattractant protein-1 (MCP-1: CCL2) has been demonstrated to be involved in the pathophysiology of ischaemic heart disease; however, the precise rote of MCP-1 in ischaemia/reperfusion (I/R) injury is controversial. Here, we investigated the role of cardiac MCP-1 expression on left ventricular (I]V) dysfunction after global I/R in Langendorff-perfused hearts isolated from transgenic mice expressing the mouse JE-MCP-1 gene under the control of the alpha-cardiac myosin heavy chain promoter (MHC/MCP-1 mice). Methods and results In vitro experiments showed that MCP-1 prevented the apoptosis of murine neonatal cardiomyocytes after hypoxia/reoxygenation. I/R significantly increased the mRNA expression of MCP-1 in the Langendorff-perfused hearts of wild-type mice. Cardiac MCP-1 overexpression in the MHC/MCP-1 mice improved LV dysfunction after I/R without affecting coronary flow; in particular, it ameliorated LV diastolic pressure after reperfusion. This improvement was independent of both sarcolemmal. and mitochondrial K-ATP channels. Cardiac MCP-1 overexpression prevented superoxide generation in the I/R hearts, and these hearts showed decreased expression of the NADPH oxidase family proteins Nox1, gp91phox, and Nox3 compared with the hearts of wild-type mice. Further, superoxide dismutase activity in the hearts of MHC/MCP-1 mice was significantly increased compared with that in the hearts of wild-type mice. Conclusion These findings suggest that cardiac MCP-1 prevented LV dysfunction after global I/R through a reactive oxygen species-dependent but K-ATP channel-independent pathway; this provides new insight into the beneficial role of MCP-1 in the pathophysiology of ischaemic heart diseases.
  • Noriyuki Yajima, Masafumi Takahashi, Hajime Morimoto, Yuji Shiba, Yasuko Takahashi, Junya Masumoto, Hirohiko Ise, Junji Sagara, Jun Nakayama, Shun'ichiro Taniguchi, Uichi Ikeda
    CIRCULATION 117 (24) 3079 - 3087 0009-7322 2008/06 [Refereed][Not invited]
    Background-Inflammatory cytokines such as interleukin (IL)-1 beta and IL-18 play an important role in the development of atherosclerosis and restenosis. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an adaptor protein that regulates caspase-1-dependent IL-1 beta and IL-18 generation; however, the role of ASC in vascular injury remains undefined. Here, we investigated the contribution of ASC to neointimal formation after vascular injury in ASC-deficient (ASC(-/-)) mice. Methods and Results-Wire-mediated vascular injury was produced in the femoral artery of ASC(-/-) and wild-type mice. Immunohistochemical analysis revealed that ASC was markedly expressed at the site of vascular injury. Neointimal formation was significantly attenuated in ASC(-/-) mice after injury. IL-1 beta and IL-18 were expressed in the neointimal lesion in wild-type mice but showed decreased expression in the lesion of ASC(-/-) mice. To investigate the contribution of bone marrow-derived cells, we developed bone marrow-transplanted mice and found that neointimal formation was significantly decreased in wild-type mice in which bone marrow was replaced with ASC(-/-) bone marrow cells. Furthermore, in vitro experiments showed that the proliferation activity of ASC(-/-) vascular smooth muscle cells was not impaired. Conclusions-These findings suggest that bone marrow-derived ASC is critical for neointimal formation after vascular injury and identify ASC as a novel therapeutic target for atherosclerosis and restenosis.
  • Hongo M, Tsutsui H, Mawatari E, Hidaka H, Kumazaki S, Yazaki Y, Takahashi M, Kinoshita O, Ikeda U
    Circulation journal : official journal of the Japanese Circulation Society 72 (5) 722 - 728 1346-9843 2008/05 [Refereed][Not invited]
  • Yasuo Zenimaru, Sadao Takahashi, Masafumi Takahashi, Kazuya Yamada, Tadao Iwasaki, Hiroaki Hattori, Michiko Imagawa, Masami Ueno, Jinya Suzuki, Isamu Miyamori
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 368 (3) 716 - 722 0006-291X 2008/04 [Refereed][Not invited]
    Glucose and fatty acids are major energy sources in skeletal muscle. Very low-density lipoprotein receptor (VLDL-R), which is highly expressed in heart, skeletal muscle and adipose tissue, plays a crucial role in metabolism of triglyceride (TG)-rich lipoproteins. To explore energy switching between glucose and fatty acids, we studied expression of VLDL-R and lipoprotein uptake in rat L6 myoblasts. L-Glucose or D-glucose deprivation in the medium noticeably induced the AMPK (AMP-activated protein kinase) activation and VLDL-R expression. Dose-dependent induction of VLDL-R expression was observed when D-glucose was less than 4.2 mM. The same phenomenon was also observed in rat primary skeletal myoblasts and cultured vascular smooth muscle cells. The uptake of P-VLDL but not LDL was accompanied by induction of VLDL-R expression. Our study suggests that the VLDL-R-mediated uptake of TG-rich lipoproteins might compensate for glucose shortfall through AMPK activation in skeletal muscle. (c) 2008 Elsevier Inc. All rights reserved.
  • Akihiro Hosoya, Tadashi Ninomiya, Toru Hiraga, Chen Zhao, Kunihiko Yoshiba, Nagako Yoshiba, Masafumi Takahashi, Takahiro Okabe, Shigeyuki Wakitani, Hirohito Yamada, Etsuo Kasahara, Hidehiro Ozawa, Hiroaki Nakamura
    BONE 42 (2) 350 - 357 8756-3282 2008/02 [Refereed][Not invited]
    Regeneration of alveolar bone is essential for periodontal treatment. Recently, cell replacement therapy has been focused on periodontal disease, but the source of the cells that regenerate alveolar bone is still uncertain. Therefore, to clarify the source of these bone-regenerating cells, we transplanted GFP-transgenic rat molars into the subcutaneous tissues of wild-type rats. Five days after transplantation, the tooth was surrounded by connective tissue containing many blood vessels. At 10 days, bone-like tissue was formed in the connective tissue between the branches of the bifurcated root. This hard tissue expanded to almost all of this bifurcation area without osseous ankylosis after 20 days. All ostcoblast-like cells in the newly formed matrix were immunopositive for GFP. In addition, these cells and the peripheral cells of the matrix showed intense immunoreactivity for BMP4, Runx2, BSP, and OPN. These results demonstrate that periodontal ligament tissue contains osteoprogenitor cells that have the ability to regenerate alveolar bone. Our model suggests that these regeneration processes might be similar to normal alveolar bone formation. (c) 2007 Elsevier Inc. All rights reserved.
  • Koji Kubota, Junpei Soeda, Ryousuke Misawa, Motohiro Mihara, Shiro Miwa, Hirohiko Ise, Masafumi Takahashi, Shinichi Miyagawa
    CARCINOGENESIS 29 (2) 448 - 454 0143-3334 2008/02 [Refereed][Not invited]
    Bone marrow cells (BMCs) have been reported to behave as tissue-specific stem cells in some organs and to participate in tumorigenesis. However, the roles of BMCs in hepatic regeneration and carcinogenesis are still unknown. A choline-deficient, ethionine-supplemented (CDE) diet leads to the appearance of oval cells, a type of hepatic progenitor cell, and activates their replication. Furthermore, this type of diet induces preneoplastic nodules and hepatocellular carcinomas (HCCs) derived from oval cell progenitors. The aims of this study were to determine whether oval cells are derived from BMCs and whether preneoplastic nodules or HCCs originate from BMCs in the CDE diet rat model. To clarify the origin of constituent cells in the liver, we transplanted BMCs from green fluorescent protein (GFP) transgenic female rats into male Lewis rats, which were then exposed to a CDE diet to induce hepatocarcinogenesis. Some oval cells showed both donor-derived GFP expression and the recipient-specific Y chromosome, indicating that donor BMCs fused with recipient oval cells. Several preneoplastic nodules (precancerous lesions) identified by their glutathione S-transferase placental (GSTp) positivity were induced by CDE treatment. However, these preneoplastic GSTp-positive nodules were not GFP positive. In conclusion, this study has produced two major findings. First, BMCs fuse with some oval cells. Second, BMC-fused oval cells and BMCs might not have malignant potential in the CDE-treated rat model.
  • Yuji Shiba, Masafumi Takahashi, Uichi Ikeda
    CURRENT PHARMACEUTICAL DESIGN 14 (4) 371 - 377 1381-6128 2008/02 [Refereed][Not invited]
    Cardiovascular disease remains a principal cause of mortality in Western countries. Novel strategies for enhancing angiogenesis (such as gene or cell therapy) provide alternative choices for patients without any current treatment options. This progress has contributed towards understanding the mechanisms underlying vascular formation. The establishment of new experimental models could lead to the development of new treatments.
  • Atsushi Izawa, Takuya Ueno, Mollie Jurewicz, Toshiro Ito, Katsunori Tanaka, Masafumi Takahashi, Uichi Ikeda, Olga Sobolev, Paolo Fiorina, Rex Neal Smith, Richard O. Hynes, Reza Abdi
    JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 18 (11) 2929 - 2936 1046-6673 2007/11 [Refereed][Not invited]
    The selectins expressed on activated endothelial cells (E- and P-selectin), leukocytes (L-selectin), and platelets (P-selectin) play crucial roles in the rolling and tethering of leukocytes. We explored the importance of donor and recipient selectins in acute and chronic cardiac allograft rejection using mice deficient in all three selectins (ELP-/-). In BALB/c recipients, survival of fully allomismatched hearts from ELP-/- C57BL/6 donors was almost double that of wild-type grafts. In ELP-/- cardiac allografts, mononuclear cell infiltration and vasculitis of intramyocardial coronary arteries were significantly reduced. Interestingly, ELP-/- grafts were rejected similarly in both the presence and the absence of recipient selectins, and both wild-type and ELP-/- recipients promptly rejected wild-type hearts. Alternative adhesive molecules such as alpha 4 beta 7 integrin may compensate for the lack of selectins and may mediate rejection in ELP-/- recipients. Chronic rejection was evaluated in a major histocompatibility complex (MHC) class II mismatch model using C57BL/6.C-H2(bm12) mice. While lack of selectins in recipients did not offer protection against chronic rejection, luminal stenosis of coronary arteries in ELP-/- grafts was markedly diminished. In conclusion, donor-derived selectins contribute to the development of both acute and chronic cardiac allograft rejection, and targeting donor selectins may open novel therapeutic approaches in clinical transplantation.
  • Chen Zhao, Akihiro Hosoya, Hiroshi Kurita, Tao Hu, Toru Hiraga, Tadashi Ninomiya, Kunihiko Yoshiba, Nagako Yoshiba, Masafumi Takahashi, Kenji Kurashina, Hidehiro Ozawa, Hiroaki Nakamura
    ARCHIVES OF ORAL BIOLOGY 52 (10) 945 - 953 0003-9969 2007/10 [Refereed][Not invited]
    While mineralized tissue is formed in the pulp cavity after tooth replantation or transplantation, little is known of this hard tissue formation. Therefore, we conducted histological and immunohistochemical evaluations of hard tissue formed in the pulp of rat maxillary molars after tooth replantation. At S days after replantation, degenerated odontoblasts were lining the pulp cavity. At 14 days, dentin- or bone-like tissue was present in the pulp cavity. Immunore activity for osteopontin (OPN) and bone sialoprotein (BSP) was strong in the bone-like tissue, but weak in the dentin-like tissue. Conversely, dentin sialoprotein (DSP) was localized in the dentin-like tissue, but not in the bone-like tissue. Cells positive for BMP4, Smad4, Runx2, and Osterix were found around the blood vessels of the root apex at 5 days. At 14 days, these cells were also localized around the bone-like tissue. Cells expressing alpha-smooth muscle actin (SMA) were seen around the newly formed bone-like tissue, whereas no such cells were found around the newly formed dentin-like tissue. In an experiment involving the transplantation of a green fluorescent protein (GFP)-transgenic rat tooth into a wild-type rat tooth socket, GFP-positive cells were detected on the surface of the bone-like tissue and over all dentin-like tissue. These results indicate that the original pulp cells had the ability to differentiate into osteoblast-like cells as well as into odontoblast-like cells. (C) 2007 Elsevier Ltd. All rights reserved.
  • Takayuki Ito, Takashi Okada, Jun Mimuro, Hiroshi Miyashita, Ryosuke Uchibori, Masashi Urabe, Hiroaki Mizukami, Akihiro Kume, Masafumi Takahashi, Uichi Ikeda, Yoichi Sakata, Kazuyuki Shimada, Keiya Ozawa
    HYPERTENSION 50 (3) 531 - 536 0194-911X 2007/09 [Refereed][Not invited]
    Prostacyclin synthase (PGIS) is the final committed enzyme in the metabolic pathway of prostacyclin production. The therapeutic option of intravenous prostacyclin infusion in patients with pulmonary arterial hypertension is limited by the short half-life of the drug and life-threatening catheter-related complications. To develop a better delivery system for prostacyclin, we examined the feasibility of intramuscular injection of an adenoassociated virus (AAV) vector expressing PGIS for preventing monocrotaline-induced pulmonary arterial hypertension in rats. We developed an AAV serotype 1-based vector carrying a human PGIS gene (AAV-PGIS). AAV-PGIS or the control AAV vector expressing enhanced green fluorescent protein was injected into the anterior tibial muscles of 3-week-old male Wistar rats; this was followed by the monocrotaline administration at 7 weeks. Eight weeks after injecting the vector, the plasma levels of 6-keto-prostaglandin F-1 alpha increased in a vector dose-dependent manner. At this time point, the PGIS transduction (1x10(10) genome copies per body) significantly decreased mean pulmonary arterial pressure (33.9 +/- 2.4 versus 46.1 +/- 3.0 mm Hg; P < 0.05), pulmonary vascular resistance (0.26 +/- 0.03 versus 0.41 +/- 0.03 mm Hg . mL(-1) . min(-1) . kg(-1); P < 0.05), and medial thickness of the peripheral pulmonary artery (14.6 +/- 1.5% versus 23.5 +/- 0.5%; P < 0.01) as compared with the controls. Furthermore, the PGIS-transduced rats demonstrated significantly improved survival rates as compared with the controls (100% versus 50%; P < 0.05) at 8 weeks postmonocrotaline administration. An intramuscular injection of AAV-PGIS prevents monocrotaline-pulmonary arterial hypertension in rats and provides a new therapeutic alternative for preventing pulmonary arterial hypertension in humans.
  • Hajime Morimoto, Masafumi Takahashi, Yuji Shiba, Atsushi Izawa, Hirohiko Ise, Minoru Hongo, Kiyohiko Hatake, Kazuo Motoyoshi, Uichi Ikeda
    AMERICAN JOURNAL OF PATHOLOGY 171 (3) 755 - 766 0002-9440 2007/09 [Refereed][Not invited]
    The monocyte/macrophage lineage might affect the healing process after myocardial infarction (MI). Because macrophage colony-stimulating factor (M-CSF) stimulates differentiation and proliferation of this lineage, we examined the effect of M-CSF treatment on infarct size and left ventricular (LV) remodeling after MI. MI was induced in C57BL/6J mice by ligation of the left coronary artery. Either recombinant human M-CSF or saline was administered for 5 consecutive days after MI induction. M-CSF treatment significantly reduced the infarct size (P < 0.05) and scar formation (P < 0.05) and improved the LV dysfunction (percent fractional shortening, P < 0.001) after the MI. Immunohistochemistry revealed that M-CSF increased macrophage infiltration (F4/80) and neovascularization (CD31) of the infarct myocardium but did not increase myofibroblast accumulation (a-smooth muscle actin). M-CSF mobilized CXCR4(+) cells into peripheral circulation, and the mobilized CXCR4(+) cells were then recruited into the infarct area in which SDF-1 showed marked expression. The CXCR4 antagonist AMD3100 deteriorated the infarction and LV function after the MI in the M-CSF-treated mice. In conclusion, M-CSF reduced infarct area and improved LV remodeling after MI through the recruitment of CXCR4(+) cells into the infarct myocardium by the SDF-1-CXCR4 axis activation; this suggests that the SDF-1-CXCR4 axis is as a potential target for the treatment of MI.
  • Ryo Ogawa, Masafumi Takahash, Sho Chi Hirose, Hajime Morimoto, Me Mori, Hirohiko Ise, Takashi Murakami, Tokutaro Yasue, Kazuhiko Kuriyama, Minoru Hongo, Eiji Kobayashi, Uichi Ikeda
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 361 (3) 621 - 628 0006-291X 2007/09 [Refereed][Not invited]
    Sphingosine-1-phosphate (S1P) is an active sphingolipid metabolite that exerts important biological effects. Recently, we demonstrated that KRP-203 is a novel S1P receptor agonist that can alter lymphocyte homing and act as an immunomodulating agent. We investigated the efficacy of KRP-203 in the treatment of rat experimental autoimmune myocarditis. KRP-203 significantly attenuated the inflammation area, heart weight/body weight ratio, and left ventricular function. Immunohistochemical analysis and RT-PCR revealed that KRP-203 significantly decreased the infiltration of macrophages and CD4 T cells in the myocardium and the expression of inflammatory cytokines. Flow cytometric analysis revealed that treatment with KRP-203 effectively reduced the number of peripheral CD4 and CD8 T cells but not that of B cells and granulocytes. Further, late KRP-203 treatment was effective even against established EAM. These results demonstrate the therapeutic potential of KRP-203 for the treatment of human myocarditis and provide new insights into the pathogenesis of this disease. (c) 2007 Elsevier Inc. All rights reserved.
  • Takayuki Ito, Takashi Okada, Hiroshi Miyashita, Tatsuya Nomoto, Mutsuko Nonaka-Sarukawa, Ryosuke Uchibori, Yoshikazu Maeda, Masashi Urabe, Hiroaki Mizukami, Akihiro Kume, Masafumi Takahashi, Uichi Ikeda, Kazuyuki Shimada, Keiya Ozawa
    CIRCULATION RESEARCH 101 (7) 734 - 741 0009-7330 2007/09 [Refereed][Not invited]
    Pulmonary arterial hypertension (PAH) is a fatal disease associated with inflammation and pathological remodeling of the pulmonary artery (PA). Interleukin (IL)-10 is a pleiotropic antiinflammatory cytokine with vasculoprotective properties. Here, we report the preventive effects of IL-10 on monocrotaline-induced PAH. Three-week-old Wistar rats were intramuscularly injected with an adeno-associated virus serotype 1 vector expressing IL-10, followed by monocrotaline injection at 7 weeks old. IL-10 transduction significantly improved survival rates of the PAH rats 8 weeks after monocrotaline administration compared with control gene transduction (75% versus 0%, P < 0.01). IL-10 also significantly reduced mean PA pressure (22.8 +/- 1.5 versus 29.7 +/- 2.8 mm Hg, P < 0.05), a weight ratio of right ventricle to left ventricle plus septum (0.35 +/- 0.04 versus 0.42 +/- 0.05, P < 0.05), and percent medial thickness of the PA (12.9 +/- 0.3% versus 21.4 +/- 0.4%, P < 0.01) compared with controls. IL-10 significantly reduced macrophage infiltration and vascular cell proliferation in the remodeled PA in vivo. It also significantly decreased the lung levels of transforming growth factor-beta(1) and IL-6, which are indicative of PA remodeling. In addition, IL-10 increased the lung level of heme oxygenase-1, which strongly prevents PA remodeling. In vitro analysis revealed that IL-10 significantly inhibited excessive proliferation of cultured human PA smooth muscle cells treated with transforming growth factor-beta(1) or the heme oxygenase inhibitor tin protoporphyrin IX. Thus, IL-10 prevented the development of monocrotaline-induced PAH, and these results provide new insights into the molecular mechanisms of human PAH.
  • Shin-ichi Aso, Hirohiko Ise, Masafumi Takahashi, Satoshi Kobayashi, Hajime Morimoto, Atsushi Izawa, Mitsuaki Goto, Uichi Ikeda
    JOURNAL OF CONTROLLED RELEASE 122 (2) 189 - 198 0168-3659 2007/09 [Refereed][Not invited]
    A drug delivery system (DDS) that targets the injured myocardium would serve as a novel therapeutic tool for cardiac diseases. To develop such a DDS, we investigated the interaction of 2 types of glycoside-conjugated liposomes containing a fluorescence substrate with cardiomyocytes. Flow cytometry revealed that cardiomyocytes adequately interact with N-acetylglucosamine-conjugated liposomes (GlcNAc-Ls). Furthermore, to confirm whether the agents encapsulated in GlcNAc-Ls affect the intracellular environment of cardiomyocytes, we prepared GlcNAc-Ls-containing pravastatin and examined the effect of pravastatin on cardiomyocytes. Pravastatin is a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor (statin) and is hydrophilic. It is reported that lipophilic statins enhance nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression by interieukin-1 beta (IL-1 beta)-stimulated cardiomyocytes. The hydrophilic nature of pravastatin prevents its entry into cardiomyocytes; therefore, it cannot enhance both these processes. Treatment with GlcNAc-Ls-containing pravastatin specifically enhanced NO production and iNOS expression by IL-1 beta-stimulated cardiomyocytes. Based on these results, we found that cardiomyocytes exhibit a high degree of interaction with GlcNAc-Ls, and GlcNAc-Ls-encapsulated agents can be effectively taken up by cardiomyocytes. We suggest that GlcNAc-Ls can be utilized therapeutically as a DDS for the injured myocardium. (C) 2007 Elsevier B.V. All rights reserved.
  • Eiichiro Mawatari, Minoru Hongo, Akio Sakai, Fumiko Terasawa, Masafumi Takahashi, Yoshikazu Yazaki, Osamu Kinoshita, Uichi Ikeda
    CLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY 34 (7) 594 - 600 0305-1870 2007/07 [Refereed][Not invited]
    1. The present study was designed to examine the role of amlodipine in preventing and reversing monocrotaline (MCT)induced pulmonary arterial hypertension (PAH) in rats. 2. Rats were injected with MCT (40 mg/kg, s.c.) and randomly given either 6 mg/kg per day of amlodipine in drinking water or placebo for 3 weeks. Any animals treated with MCT that survived for 3 weeks were given either amlodipine or placebo for the next 3 weeks. 3. Blood pressure was not different between the groups. Amlodipine immediately following MCT markedly inhibited PAH with severe pulmonary vascular remodelling. The survival rate at 3 weeks after treatment was increased significantly in the amlodipine group compared with the placebo group (77% vs 43%; P < 0.01). The placebo group showed markedly diminished expression of endothelial nitric oxide synthase (eNOS) protein and mRNA levels, increased numbers of proliferating cell nuclear antigen-positive cells, enhanced mRNA expression of matrix metalloproteinase-2 and pro-inflammatory cytokines in the lung tissue and upregulation of P-selectin on the endothelium of the pulmonary arteries, whereas these effects were suppressed in the amlodipine-treated group. Furthermore, late treatment with amlodipine did not palliate PAH or improve survival. 4. Amlodipine inhibited the development of PAH and improved survival in rats independent of its effect on lowering blood pressure. These effects were associated with marked inhibition of the downregulation of eNOS and improvement of pulmonary vascular endothetial activation, as well as anti-inflammatory, antiproliferative and antifibrotic effects in the lung tissue. However, amlodipine failed to reverse established PAH. This study may provide an insight into therapeutic strategy of amlodipine in PAH.
  • Yuji Shiba, Masafumi Takahashi, Toru Yoshioka, Noriyuki Yajima, Hajime Morimoto, Atsushi Izawa, Hirohiko Ise, Kiyohiko Hatake, Kazuo Motoyoshi, Uichi Ikeda
    ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY 27 (2) 283 - 289 1079-5642 2007/02 [Refereed][Not invited]
    Objective-Since the macrophage colony-stimulating factor ( M-CSF) has been shown to stimulate differentiation and proliferation of monocyte/macrophage lineage and to be involved in the process of neointimal formation after vascular injury, we tested the effects of M-CSF on the recruitment of bone marrow-derived progenitor cells in neointimal formation after vascular injury in mice. Methods and Results-Wire-mediated vascular injury was produced in the femoral artery of C57BL/6 mice. Recombinant human M-CSF [ 500 mu g/(] or saline ( control) was administered for 10 consecutive days, starting 4 days before the injury. Treatment with M-CSF accelerated neointimal formation in the early phase after injury, and this neointimal lesion mainly consisted of bone marrow - derived cells. M-CSF treatment had no effect on the mobilization of endothelial progenitor cells ( EPCs: CD34(+)/Flk-1(+)) and reendothelialization after injury. The stromal cell-derived factor-1 ( SDF-1) was markedly expressed in the neointima and media after injury, whereas CXCR4(+) cells were observed in the neointima. Further, a novel CXCR4 antagonist, AMD3100, significantly attenuated the M-CSF - induced neointimal formation. Conclusions-These findings suggest that M-CSF accelerated neointimal formation after vascular injury via the SDF-1-CXCR4 system, and the inhibition of this system has therapeutic potential for the treatment of cardiovascular diseases.
  • Sho-ichi Hirose, Masafumi Takahashi, Ryo Ogawa, Hajime Morimoto, Atsushi Izawa, Hajime Sato, Hirohiko Ise, Minoru Hongo, Uichi Ikeda
    CARDIOVASCULAR DRUGS AND THERAPY 21 (1) 17 - 27 0920-3206 2007/02 [Refereed][Not invited]
    Objective Erythropoietin (EPO) has been shown to not only have cardioprotective effects but also attenuate autoimmune diseases. In the present study, we investigated the effect of EPO on cardiac inflammation and function, inflammatory cell infiltration, and cytokine expression in a rat model of experimental autoimmune myocarditis (EAM). Methods and results Male Lewis rats (6-8 weeks old) were immunized on day 0 with porcine cardiac myosin to establish EAM. The rats were subcutaneously administered either vehicle (saline) or human recombinant EPO (6,000 U/kg, 3 days/week) from day 0 to 20, and they were evaluated on day 21. In the EPO group, the inflammation area and heart weight/body weight ratio were significantly attenuated as compared with those in the vehicle group. Blood pressure and cardiac function were also improved in the EPO group. Immunohistochemistry revealed that EPO decreased the infiltration of macrophages and CD4 T cells, and degranulated mast cells in the myocardium. Real-time RT-PCR analysis demonstrated that inflammatory cytokine expression in the myocardium and lymphocytes was suppressed in the EPO group. However, in vitro experiments showed that EPO had no effect on antigen-induced proliferation and cytokine expression in lymphocytes. Conclusion EPO attenuates inflammatory cell infiltration and cytokine expression, and it improves cardiac function and reduces cardiac inflammation in EAM. This beneficial effect of EPO is unlikely to arise from a direct anti-inflammatory action on lymphocytes. These findings suggest the therapeutic potential of EPO for the treatment of myocarditis.
  • Chihiro Suzuki, Masafumi Takahashi, Hajime Morimoto, Atsushi Izawa, Hirohiko Ise, Minoru Hongo, Yasushi Hoshikawa, Takayuki Ito, Hiroshi Miyashita, Eiji Kobayashi, Kazuyuki Shimada, Uichi Ikeda
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 349 (2) 781 - 788 0006-291X 2006/10 [Refereed][Not invited]
    Pulmonary arterial hypertension (PAH) is characterized by abnormal proliferation of smooth muscle cells (SMCs), leading to occlusion of pulmonary arterioles, right ventricular (RV) hypertrophy, and death. We investigated whether mycophenolate mofetil (MMF), a potent inummosuppresssant, prevents the development of monocrotaline (MCT)-induced PAH in rats. MMF effectively decreased RV systolic pressure and RV hypertrophy, and reduced the medial thickness of pulmonary arteries. MMF significantly inhibited the number of proliferating cell nuclear antigen (PCNA)-positive cells, infiltration of macrophages, and expression of P-selectin and interleukin-6 on the endothelium of pulmonary arteries. The infiltration of T cells and mast cells was not affected by MMF. In vitro experiments revealed that mycophenolic acid (MPA), an active metabolite of MMF, dose-dependently inhibited proliferation of human pulmonary arterial SMCs. MMF attenuated the development of PAH through its anti-inflammatory and anti-proliferative properties. These findings provide new insight into the potential role of immunosuppressants in the treatment of PAR (c) 2006 Elsevier Inc. All rights reserved.
  • Hajime Morimoto, Masafumi Takahashi, Atsushi Izawa, Hirohiko Ise, Minoru Hongo, Pappachan E. Kolattukudy, Uichi Ikeda
    CIRCULATION RESEARCH 99 (8) 891 - 899 0009-7330 2006/10 [Refereed][Not invited]
    Myocardial infarction (MI) is accompanied by inflammatory responses that lead to the recruitment of leukocytes and subsequent myocardial damage, healing, and scar formation. Because monocyte chemoattractant protein-1 (MCP-1) ( also known as CCL2) regulates monocytic inflammatory responses, we investigated the effect of cardiac MCP-1 overexpression on left ventricular (LV) dysfunction and remodeling in a murine MI model. Transgenic mice expressing the mouse JE-MCP-1 gene under the control of the alpha-cardiac myosin heavy chain promoter (MHC/MCP-1 mice) were used for this purpose. MHC/MCP-1 mice had reduced infarct area and scar formation and improved LV dysfunction after MI. These mice also showed induction of macrophage infiltration and neovascularization; however, few bone marrow-derived endothelial cells were detected in MHC/MCP-1 mice whose bone marrow was replaced with that of Tie2/LacZ transgenic mice. Flow cytometry analysis showed no increase in endothelial progenitor cells (CD34(+)/Flk-1(+) cells) in MHC/MCP-1 mice. Marked myocardial interleukin (IL)-6 secretion, STAT3 activation, and LV hypertrophy were observed after MI in MHC/MCP-1 mice. Furthermore, cardiac myofibroblasts accumulated after MI inMHC/MCP-1 mice. In vitro experiments revealed that a combination of IL-6 with MCP-1 synergistically stimulated and sustained STAT3 activation in cardiomyocytes. MCP-1, IL-6, and hypoxia directly promoted the differentiation of cardiac fibroblasts into myofibroblasts. Our results suggest that cardiac overexpression of MCP-1 induced macrophage infiltration, neovascularization, myocardial IL- 6 secretion, and accumulation of cardiac myofibroblasts, thereby resulting in the prevention of LV dysfunction and remodeling after MI. They also provide a new insight into the role of cardiac MCP-1 in the pathophysiology of MI.
  • Jun Fujishiro, Shinji Kudou, Satomi Iwai, Masafumi Takahashi, Yoji Hakamata, Miki Kinoshita, Satoru Iwanami, Shigeru Izawa, Tokutaro Yasue, Kohei Hashizume, Takashi Murakami, Eiji Kobayashi
    TRANSPLANTATION 82 (6) 804 - 812 0041-1337 2006/09 [Refereed][Not invited]
    Background. We demonstrate the long-term effectiveness of KRP-203 treatment in combination with a subtherapeutic dose of cyclosporine A (CsA) on rat renal allografts. Methods. We tested the effect of KRP-203 in combination with CsA using a rat skin allograft model. The Pharmacokinetic interaction between CsA and KRP-203 was evaluated. The selectivity of KRP-203 for sphingosine-I-phosphate (S1P)(1) and S1P(3) receptors were investigated in vitro. Heart rate alteration following bolus injection of phosphorylated KRP-203 (KRP-203-P) or FTY720 (FTY720-P) was also monitored in rats. Finally, the long-term effectiveness of KRP-203 in conjunction with a low dose of CsA was investigated in a rat renal transplantation model. Results. Administration of KRP-203 with CsA prolonged skin allograft survival. KRP-203 and CsA had no effect on the pharmacokinetics of the other. While FTY720-P activated both S1P(1), and S1P(3) receptors, KRP-203-P selectively activated S1P(1), but not the S1P(3) receptor (EC(50): > 1000 nM). Compared to FTY720-P, a tenfold higher dose of KRP-203-P was necessary to induce transient bradycardia. With a low dose of CsA (1 mg/kg/day), KRP-203 (0.3 mg/kg/day) significantly prolonged renal allograft survival (P < 0.05, survival time: 9.8 days (CsA) vs. > 27.4 days (CsA+KRP)). Although a higher dose of CsA (3, mg/kg/day) alone kept recipients alive, this caused severe renal graft dysfunction. Use of KRP-203 (3 mg/kg/day) in conjunction with CsA markedly improved graft function (P < 0.05, creatinine clearance: 0.41 +/- 0.25 ml/min [CsA] vs. 1.15 +/- 0.16 ml/min [CsA+KRP]). Conclusions. The selectivity of KRP-203 for S1P(1) reduces the risk of bradycardia, and the combination therapy of KRP-203 with CsA represents a safe and effective strategy for use in renal transplantation.
  • Masafumi Takahashi
    CARDIOVASCULAR RESEARCH 71 (1) 4 - 5 0008-6363 2006/07 [Refereed][Not invited]
  • Jun Fujishiro, Chihiro Suzuki, Shinji Kudou, Tokutaro Yasue, Yoji Hakamata, Masafumi Takahashi, Takashi Murakami, Kohei Hashizume, Eiji Kobayashi
    JOURNAL OF HEART AND LUNG TRANSPLANTATION 25 (7) 825 - 833 1053-2498 2006/07 [Refereed][Not invited]
    Background: Replacement of calcineurin inhibitor (CI) with anti-metabolic agents in transplant patients with CI-induced nephrotoxiciry is performed clinically and improves renal function, but increases the risk of rejection. We investigated whether the change from cyclosporine (CsA) to a limited dose of mycophenolic acid (MPA) together with a new sphihgosine-1-phosphate (S1P) receptor agonist, KRP-203, is sufficient to prevent both transplant vasculopathy and CsA-induced nephrotoxicity. Methods: Orthotopic aortic transplantation was conducted in a high-responder rat combination of Dark Agouti (DA; major histocompatibiliry complex [MHC] haplotype RT-1(a)) to Lewis (RT-1(l)). After CsA administration (15 mg/kg/day) for 2 weeks, the recipients were divided into the following treatment groups for 6 weeks: MPA (10 mg/kg); KRP-203 (KRP; 1 mg/kg); and MPA + KRP. Serum creatinine (Cr), arteriolar hyalinosis and expression of transforming growth factor (TGF)-beta 1 in the recipient kidney were examined as parameters indicating nephrotoxicity. Intimal hyperplasia was assessed by vascular occlusion, and graft-infiltrated cells were semi-quantitatively evaluated histologically and then characterized immunohistochemically. Results: Continuous CsA treatment attenuated intimal hyperplasia and cell infiltration (2.9 +/- 0.3% and 0.4 +/- 0.1; p < 0.01 vs vehicle), but increased Cr and hyalinosis (0.43 +/- 0.03 mg/dl and 57.2 +/- 0.4%; p < 9.01) with upregulated TGF-beta 1. Replacement of CsA by MPA or KRP treatment alone improved nephrotoxicity, but worsened intimal hyperplasia and cell infiltration. Conversion to MPA + KRP treatment prevented nephrotoxicity (Cr, 0.32 +/- 0.02 mg/dl; hyalinosis, 5.6 +/- 1.3%; p < 0.01 vs CsA) and markedly suppressed intimal hyperplasia and cell infiltration (3.6 +/- 1.2% and 1.0 +/- 0.3;p = not significant vs CsA), with reduced T-cell infiltrates in the graft. Conclusions: Changing from CsA to a combined therapy of MMF with S1P agonist is a promising strategy in clinical transplantation to overcome Cl-induced nephrotoxicity and chronic rejection.
  • T Kaneko, T Murakami, H Kawana, M Takahashi, T Yasue, E Kobayashi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 345 (1) 85 - 92 0006-291X 2006/06 [Refereed][Not invited]
    T cell-mediated immune responses play a critical role in a variety of liver injuries including autoimmune hepatitis. Injection of concanavalin A (Con A) into mice mimics the histological and pathological phenotype of T cell-mediated hepatitis. Recent advances in host immune control of organ transplantation include the development of sphingosine-1-phosphate (SIP) receptor agonists such as FTY720, which alter lymphocyte homing but do not suppress host general immunity. Herein we examined the effect of the new SIP receptor agonist KRP-203 on the Con A-induced liver damage model. In normal liver lymphocytes of BALB/c mice, both FTY720 and KRP203 promoted lymphocyte sequestering from the liver to secondary lymph nodes and significantly reduced the number of liver lymphocytes (p < 0.05). Based on this observation, KRP203 was employed in the Con A-induced hepatitis model. KRP203 markedly reduced the number of CD4(+) lymphocytes that infiltrate Con A-treated liver (p < 0.05) and successfully reduced serum transaminase elevation (p = 0.017), therefore protecting mice from Con A-induced liver injury. Interestingly this homing modulation less occurs in natural hepatic T cell homing through the chemokine receptor, CXCR4. Therefore, S1P receptor agonists preferentially target CXCR4(+)CD4(+) peripheral blood T lymphocytes and suppress the occurrence of Con A-induced hepatitis, suggesting their therapeutic usefulness against T cell-mediated hepatic injury. (c) 2006 Elsevier Inc. All rights reserved.
  • T Okada, R Uchibori, M Iwata-Okada, M Takahashi, T Nomoto, M Nonaka-Sarukawa, T Ito, Y Liu, H Mizukami, A Kume, E Kobayashi, K Ozawa
    MOLECULAR THERAPY 13 (4) 738 - 746 1525-0016 2006/04 [Refereed][Not invited]
    The transduction of cancer cells using recombinant adeno-associated virus (rAAV) occurs with low efficiency, which limits its utility in cancer gene therapy. We have previously sought to enhance rAAV-mediated transduction of cancer cells by applying DNA-damaging stresses. In this study, we examined the effects of the histone deacetylase inhibitor FR901228 on tumor transduction mediated by rAAV types 2 and 5. FR901228 treatment significantly improved the expression of the transgene in four cancer cell lines. The cell surface levels of alpha v integrin, FGF-R1, and PDGF-R were modestly enhanced by the presence of FR901228. These results suggest that the superior transduction induced by the HDAC inhibitor was due to an enhancement of transgene expression rather than increased viral entry. Furthermore, we characterized the association of the acetylated histone H3 in the episomal AAV vector genome by using the chromatin immunoprecipitation assay. The results suggest that the superior transduction may be related to the proposed histone-associated chromatin form of the rAAV concatemer in transduced cells. In the analysis with subcutaneous tumor models, strong enhancement of the transgene expression as well as therapeutic effect was confirmed in vivo. The use of this HDAC inhibitor may enhance the utility of rAAV-mediated transduction strategies for cancer gene therapy.
  • T Yoshioka, M Takahashi, Y Shiba, C Suzuki, H Morimoto, A Izawa, H Ise, U Ikeda
    CARDIOVASCULAR RESEARCH 70 (1) 61 - 69 0008-6363 2006/04 [Refereed][Not invited]
    Objective: Neointimal formation following percutaneous coronary intervention (PCI), termed restenosis, limits therapeutic revascularization. Since reendothelialization is one of the determinant factors for the development of neointimal formation, we examined the effects of granulocyte colony-stimulating factor (G-CSF) on reendothelialization and neointimal formation after vascular injury in mice. Methods and results: Wire-mediated vascular injury was produced in the femoral artery of C57BL/6 mice. G-CSF pretreatment significantly accelerated reendothelialization and decreased neointimal formation following vascular injury; however, this inhibitory effect of G-CSF was diminished when G-CSF was started following the injury. Flow cytometry analysis revealed that G-CSF treatment increased the number of endothelial progenitor cells (EPCs: CD34(+)/Flk-1(+)) in the peripheral circulation. Vascular injury was also produced in 2 types of mice whose bone marrow was replaced with that of enhanced green fluorescent protein- and Tie2/LacZ-transgenic mice. In the reendothelialized artery of these mice, few bone marrow-derived EPCs were detected. Furthermore, G-CSF treatment reduced the serum level of interleukin (IL)-6. Conclusion: G-CSF treatment accelerated reendothelialization and decreased neointimal formation following vascular injury, although there was little contribution of bone marrow-derived EPCs to the reendothelialization of the artery. These results suggest that G-CSF pretreatment has a therapeutic potential for prevention of restenosis following PCI. (c) 2005 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
  • R Misawa, H Ise, M Takahashi, H Morimoto, E Kobayashi, SI Miyagawa, U Ikeda
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 342 (2) 434 - 440 0006-291X 2006/04 [Refereed][Not invited]
    Several recent studies have reported that bone marrow cells (BMCs) have the ability to generate functional hepatocytes. However, the efficiency at which BMC transplantation generates functional hepatocytes is rather low. We assumed that if BMCs accumulated directly in liver, the functional BMC-derived hepatocytes should increase efficiently. We tried to increase the accumulation of BMCs directly in liver through the interaction between hepatic asialoglycoprotein receptor and desialylated BMCs. Desialylated BMCs were produced with treatment of neuraminidase. Desialylated BMCs that expressed green fluorescent protein (GFP) were injected into Long Evans Cinnamon (LEC) rats, a human Wilson's disease model, intravenously. At 3 and 5 months after transplantation, GFP-expressing hepatocyte nodules appeared in the liver of these BMC-transplanted LEC rats. These findings suggest that the functional BMC-derived hepatocytes can be generated by the direct accumulation of BMCs and that this strategy is new BMC therapy for liver regeneration. (c) 2006 Elsevier Inc. All rights reserved.
  • H Sato, M Takahashi, H Ise, A Yamada, S Hirose, Y Tagawa, H Morimoto, A Izawa, U Ikeda
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 342 (1) 107 - 112 0006-291X 2006/03 [Refereed][Not invited]
    Ascorbic acid has been reported to promote the differentiation of embryonic stem (ES) cells into cardiomyocytes; however, the specific functions of ascorbic acid have not been defined. A stable form of ascorbic acid, namely, L-ascorbic acid 2-phosphate (A2-P), significantly enhanced cardiac differentiation; this was assessed by spontaneous beating of cardiomyocytes and expression of cardiac-specific markers obtained from mouse ES cells. This effect of ascorbic acid was observed only when A2-P was present during the early phase of differentiation. Treatment with two types of collagen synthesis inhibitors, L-2-azetidine carboxylic acid and cis-4-hydroxy-D-proline, significantly inhibited the A2-P-enhanced cardiac differentiation, whereas treatment with the antioxidant N-acetyl cysteine showed no effect. These findings demonstrated that ascorbic acid enhances differentiation of ES cells into cardiomyocytes through collagen synthesis and suggest its potential in the modification of cardiac differentiation of ES cells. (c) 2006 Elsevier Inc. All rights reserved.
  • C Suzuki, M Takahashi, H Morimoto, A Izawa, H Ise, J Fujishiro, T Murakami, J Ishiyama, A Nakada, J Nakayama, K Shimada, U Ikeda, E Kobayashi
    JOURNAL OF HEART AND LUNG TRANSPLANTATION 25 (3) 302 - 309 1053-2498 2006/03 [Refereed][Not invited]
    Background: To explore a more effective and less toxic immunosuppressive strategy in organ transplantation, we recently developed the novel sphingosine-1-phosphate receptor agonist KRP-203. This study examined the efficacy of KRP-203 combined with mycophenolic acid (MPA), an active metabolite of mycophenolate mofetil, in rat heart allografts. Methods: Heterotopic heart transplantation was performed in a rat combination of DA (MHC haplotype: RT1(a)) to Lewis (RT1(1)). The recipients were divided into 12 groups (n = 5-7): Syngeneic (Lewis to Lewis), Vehicle, KRP-203 (0.3 and 1 mg/kg), MPA (10 and 20 mg/kg), 10 mg/kg MPA with KRP-203 (0.03, 0.3, 1, and 3 mg/kg), and 20 mg/kg MPA with KRP-203 (0.3 and 1 mg/kg). MPA, KRP-203, and vehicle were given orally. Results: The mean days of survival were 5.8 (vehicle), 7 and 7.9 (0.3 and 1 mg/kg KRP-203, respectively), 12.7 and > 54.4 (10 and 20 mg/kg MPA), > 39.6 and > 30.5 (10 mg/kg MPA with 1 and 3 mg/kg KRP-203), > 100 and > 87.8 (20 mg/kg MPA with 0.3 and 1 mg/kg KRP-203). Histologic and immunohistochemical analysis revealed that diffuse mononuclear cell infiltration (macrophages and T cells), hemorrhage, myocardial necrosis and fibrosis, and expression of endothelin-1, transforming growth factor-beta 1, monocyte chemoattractant protein-1, interleukin-8, and E-selectin were markedly diminished in the allografts treated with MPA combined with KRP-203. Pharmacokinetic experiments indicated no interaction between MPA and KRP-203, and both combination regimens were well tolerated. Conclusions: Combination therapy of MPA with KRP-203 has a therapeutic potential as a novel immunosuppressant strategy in clinical transplantation.
  • LJ Jia, M Takahashi, H Morimoto, S Takahashi, A Izawa, H Ise, T Iwasaki, H Hattori, KJ Wu, U Ikeda
    CARDIOVASCULAR RESEARCH 69 (2) 545 - 555 0008-6363 2006/02 [Refereed][Not invited]
    Objective: Sepsis accompanies myocardial dysfunction and dynamic alterations of cardiac metabolism. We have recently demonstrated that the very low-density lipoprotein receptor (VLDL-R), which is abundantly expressed in the heart, plays a key role in energy metabolism of the fasting heart. However, little is known about the function and regulation of the VLDL-R during sepsis. In the present study, we explored lipid accumulation and VLDL-R expression in the lipopolysaccharide (LPS)-stimulated heart in vivo and regulation of VLDL-R expression in vitro. Methods and results: Electron microscopy and immunohistochemistry demonstrated that LPS significantly decreased both lipid accumulation and VLDL-R expression in the hearts of fasting mice. Treatment with LPS also downregulated VLDL-R in rat neonatal cardiac myocytes, and this downregulation was completely reversed by interleukin (IL)-1 beta receptor antagonist. IL-1 beta downregulated the expression of VLDL-R in a time- and dose-dependent manner and markedly reduced the uptake of DiI-labeled beta-VLDL but not DiI-labeled low-density lipoprotein (LDL). Use of specific pharmacologic inhibitors and short interference RNA revealed that Hsp90 was required for IL-1 beta to downregulate VLDL-R expression. Conclusions: These findings suggest that IL-1 beta is a principle mediator of changes in cardiac lipid and energy metabolism during sepsis through the downregulation of myocardial VLDL-R expression. (C) 2005 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
  • A Izawa, K Sano, M Takehara, M Inobe, J Suzuki, H Imamura, M Takahashi, U Ikeda, M Isobe, T Uede
    CARDIOVASCULAR RESEARCH 69 (1) 289 - 297 0008-6363 2006/01 [Refereed][Not invited]
    Objective: Transfer of the CTLA4IgG gene induces long-term and high levels of CTLA41gG expression, which can result in generalized immunosuppression. In this study, we utilized Cre/loxP-mediated on-off switch recombination to eliminate transgene expression of CTLA4IgG following acceptance of murine cardiac allografts. Methods: Fully MHC-mismatched hearts from BALB/c donor mice were transplanted into C3H/He recipient mice. Adenovirus-containing CTLA41gG flanked between two loxP sites was administered via a recipient tail vein immediately after transplantation. Cre-recombinase gene was subsequently transferred at day 30 posttransplantation. Results: Long-term allograft survival was observed in recipients that received the CTLA41gG gene. Cre-mediated recombination reduced CTLA4IgG gene expression without any adverse effect on the graft survival. Secondary skin grafts of donor type and of third party were promptly rejected in the recipients that accepted cardiac allografts. In addition, the B cell response against ovalbumin was suppressed during high levels of serum CTLA41gG, but recovered after Cre-mediated inactivation of CTLA4IgG gene. Conclusion: CTLA4IgG gene transfer promoted long-term survival of murine cardiac allografts; however, this was not sufficient to induce tolerance. Cre/loxP-mediated on-off switch recombination was useful to inactivate the CTLA41gG gene so that recipients' immune responses against neoantigens were restored without an influence on the allograft survival. This system may open novel strategies to orchestrate clinically relevant immunosuppression. (c) 2005 European Society of Cardiology. Published by Elsevier B.V. All fights reserved.
  • Lijing Jia, Masafumi Takahashi, Toru Yoshioka, Hajime Morimoto, Hirohiko Ise, Uichi Ikeda
    Current Vascular Pharmacology 4 (1) 59 - 65 1570-1611 2006 [Refereed][Not invited]
    In the past decade, researchers have defined committed stem or progenitor cells from various tissues, including bone marrow, peripheral blood, brain, liver and reproductive organs, in both adult animals and humans. Recently, endothelial progenitor cells (EPCs) were isolated from peripheral blood mononuclear cells and were shown to be incorporated into foci of neovascularization. This finding that circulating EPCs may home into sites of neovascularization and differentiate into mature endothelial cells in situ is consistent with the concept of 'vasculogenesis' and suggests that vasculogenesis and angiogenesis might constitute complementary mechanisms for postnatal neovascularization. Furthermore, experimental and clinical studies on ischemic cardiovascular diseases suggest a therapeutic potential for EPC transplantation. In this review, we summarize the biological features of EPCs and discuss their therapeutic potential for the treatment of cardiovascular diseases. © 2006 Bentham Science Publishers Ltd.
  • Y Kamiyoshi, M Takahashi, O Yokoseki, Y Yazaki, S Hirose, H Morimoto, N Watanabe, O Kinoshita, M Hongo, U Ikeda
    JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY 39 (3) 467 - 477 0022-2828 2005/09 [Refereed][Not invited]
    Experimental autoimmune myocarditis (EAM) is characterized by the appearance of multinucleated giant cells. EAM leads to severe myocardial damage and is a useful model of human giant cell myocarditis. We investigated whether mycophenolate mofetil (MMF), which is a potent immunosuppressant, prevents the development of myocarditis in a rat EAM model, and focused on the role of osteopontin (OPN) in the pathogenesis of this disorder. Adult Lewis rats were immunized with porcine cardiac myosin to establish EAM. The early MMF treatment completely prevented the development of EAM, and the late MMF treatment was also effective even against established EAM. Echocardiogram demonstrated that left ventricular function was also improved by the treatment with MMR Real-time RT-PCR analysis showed that both early and late MMF treatments significantly inhibited myocarditis-induced OPN mRNA expression in the heart. Immunohistochemistry revealed that OPN expression was prominent in the myocardium on day 14, whereas expression was observed in the infiltrated macrophages on day 21. Mycophenolic acid (MPA) did inhibit agonist-induced OPN expression in cultured cardionnyocytes. These results show the therapeutic potential of MMF for autoimmune myocarditis and provide new insights into the pathogenesis of this disease. (c) 2005 Elsevier Ltd. All rights reserved.
  • Fujishiro J, Takeda S, Takeno Y, Takeuchi K, Ogata Y, Takahashi M, Hakamata Y, Kaneko T, Murakami T, Okada T, Ozawa K, Hashizume K, Kobayashi E
    Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association 20 (7) 1385 - 1391 0931-0509 2005/07 [Refereed][Not invited]
  • Suzuki F, Hashikura Y, Ise H, Ishida A, Nakayama J, Takahashi M, Miyagawa S, Ikeda U
    Transplant international : official journal of the European Society for Organ Transplantation 18 (7) 844 - 853 0934-0874 2005/07 [Refereed][Not invited]
  • K Tahara, T Murakami, J Fujishiro, M Takahashi, S Inoue, K Hashizume, K Matsuno, E Kobayashi
    ANNALS OF SURGERY 242 (1) 124 - 132 0003-4932 2005/07 [Refereed][Not invited]
    Objective: Based on development of stem cell technology, newborn tissue, even undergoing cryopreservation, possesses promising potential as a donor source in, the field of organ transplantation. However, the precise regeneration processes remains unclear. This study was designed to investigate the regenerative potential of newborn intestine with or without cryopreservation in the transplantation. Methods: Newborn rat intestines with or without cryopreservation were transplanted subcutaneously into the syngeneic host, and specimens were evaluated by histology, multiple immunostaining, and comprehensive gene expression analysis. Results: We determined that newborn rat intestine possessed regenerative potential in the syngeneic host even after cryopreservation, where angiogenesis was induced early in the submucosa with subsequent maturation in the crypts. Furthermore, newborn intestinal graft could facilitate the survival of maturation-incompetent 10-day-old graft that lacked regenerating activity (P < 0.01, n = 13). Tissue aggregates from the maturation-incompetent graft underwent reconstitution of their histologic configuration in the presence of newborn intestinal aggregates. Comprehensive gene expression analysis showed that 37 genes were preferentially up-regulated, while 19 genes were downregulated in the regenerating 10-day-old graft (supported by the newborn graft). Conclusions: Regeneration of newborn intestine is implicated in neo-angiogenesis in the host, and the newborn intestinal graft is capable of mediating the survival of the maturation-incompetent 10-day-old graft. Notwithstanding ethical and legal limitations in the clinic, these results may provide new insights into the regenerative role of newborn grafts.
  • H Inoue, Ohsawa, I, T Murakami, A Kimura, Y Hakamata, Y Sato, T Kaneko, M Takahashi, T Okada, K Ozawa, J Francis, P Leone, E Kobayashi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 329 (1) 288 - 295 0006-291X 2005/04 [Refereed][Not invited]
    The ideal goal of regeneration medicine is to restore form and function to damaged tissues. While stem cell transplantation is considered a promising therapeutic approach, knowing the fate of transplanted cells using appropriate markers is essential. We developed new inbred transgenic rat strains with lacZ and GFP based on the transgenic (Tg) animal technique in rats. These Tg animals expressed most of their marker genes ubiquitously, compared to previous Tg rats. Immunological antigenicity against marker proteins was evaluated using conventional skin grafting, and results suggested lacZ-Tg-derived skin was much less immunogenic than that of GFP-Tg. However, GFP-positive cells from parental transgenic rats were still potential candidates for the study of cellular fate in immune privilege sites, such as the brain. Taking advantage of less immunogenic lacZ, we also examined the role of bone marrow-derived cells (BMDCs) in skin wound healing using an in vivo biological imaging system. Although transplantation of BMDCs enhanced wound healing at the injection site, BMDCs were detected only for a short time, suggesting a transient contribution of autologous BMDC-transplantation in wound healing. Our Tg-rat system may provide great benefits for the elucidation of the cellular process of regenerative medicine, including cell and tissue transplantation. (C) 2005 Elsevier Inc. All rights reserved.
  • M Takahashi, S Takahashi, C Suzuki, L Jia, H Morimoto, H Ise, T Iwasaki, H Hattori, J Suzuki, Miyamori, I, E Kobayashi, U Ikeda
    JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY 38 (4) 637 - 646 0022-2828 2005/04 [Refereed][Not invited]
    The very low-density lipoprotein (VLDL) receptor is a member of the low-density lipoprotein (LDL) receptor gene family with distinct tissue distribution and function. VLDL receptors are also expressed in vascular smooth muscle cells (VSMCs) and have been shown to be upregulated in atherosclerotic lesions. In the present study, we examined the effects of interleukin-1 beta (IL-1 beta) on the uptake of beta VLDL and its receptor expression in rat VSMCs. IL-1 beta downregulated expression of the VLDL receptor in a time and dose-dependent manner as shown by Western blotting, Northern blotting, and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Treatment with IL-1 beta significantly reduced the uptake of P-VLDL but not LDL in VSMCs. Use of specific pharmacologic inhibitors indicated that the tyrosine kinase inhibitors, herbimycin A and geldanamycin, completely reversed IL-1 beta-induced downregulation of the VLDL receptor expression. Another tyrosine kinase inhibitor, genistein, the protein kinase C inhibitors, GF109203X and H7, the mitogen-activated protein (MAP) kinase inhibitors (MEK inhibitor PD098059 for [MEK] and SB203580 for p38-MAP kinase), and the protein kinase A inhibitor, KT5270 all had no effect on receptor expression. In addition, the c-Src specific inhibitor PP2 or adenoviral-mediated gene transfer of kinase inactive (KI)-c-Src failed to reverse IL-1 beta-induced downregulation of VLDL receptor expression. These results indicate that IL-1 beta attenuates uptake of VLDL through downregulation of its receptor in VSMCs, and that this downregulation is mediated through a benzoquinone ansamycin-dependent but c-Src-independent pathway. (c) 2005 Elsevier Ltd. All rights reserved.
  • H Shimizu, M Takahashi, T Kaneko, T Murakami, Y Hakamata, S Kudou, T Kishi, K Fukuchi, S Iwanami, K Kuriyama, T Yasue, S Enosawa, K Matsumoto, Takeyoshi, I, Y Morishita, E Kobayashi
    CIRCULATION 111 (2) 222 - 229 0009-7322 2005/01 [Refereed][Not invited]
    Background - A novel immunomodulator, KRP-203, the molecular structure of which has some similarity to FTY720, has been developed for use in organ transplantation. The present study was designed to investigate the potency and safety of KRP-203 on allograft survival against both acute and chronic rejection in rat skin and heart transplantation. Methods and Results - KRP-203 significantly prolonged skin or heart allograft survival of a minor histocompatibility complex (mHC) - disparate (LEW to F344) rat combination. Histopathological and immunohistochemical analysis at 100 days after mHC-disparate rat heart transplantation revealed that KRP-203 treatment significantly inhibited infiltration of inflammatory cells, including macrophages and T cells; expression of endothelin-1 and transforming growth factor-beta(1); and IgG deposition and eventually attenuated neointimal formation and myocardial fibrosis. KRP-203 also prolonged heart allograft survival in a major histocompatibility complex (MHC)-incompatible (DA to LEW) rat combination, but the efficacy was not as significant. However, KRP-203 combined with a subtherapeutic dose of cyclosporin A synergistically prolonged the heart allograft survival. Flow cytometric analysis demonstrated that KRP-203 reduced the number of peripheral blood mononuclear cells ( lymphocytes and monocytes) but not granulocytes and enhanced lymphocyte homing into peripheral lymph nodes. The influence of KRP-203 on heart rate changes in Hartley guinea pigs was examined. KRP-203 had less of a tendency to cause bradycardia than FTY720. Conclusions - These findings demonstrated that KRP-203 prolonged skin and heart allograft survival and significantly attenuated chronic rejection and bradycardia as an adverse effect. Therefore, KRP-203 offers considerable potential as a novel therapeutic immunosuppressant in patients with organ transplantation.

Research Grants & Projects

  • Regenerative Therapy for Cardiovasuclar Diseases
    Grant-in-Aid for Scientific Research
    Date (from‐to) : 2001 -2006
  • 心血管疾患における炎症反応の役割
  • 心血管疾患における再生医療
  • Study on Mechanism of Atherogenesis

Copyright © MEDIA FUSION Co.,Ltd. All rights reserved.