Faculty Profiles - TAKEFUJI Mikito

Assessment of In-Stent Restenosis Using High-Definition Computed Tomography With a New Gemstone Detector

Tsuda Takuma, Ishii Hideki, Ichimiya Satoshi, Kanashiro Masaaki, Watanabe Jyunji, Takefuji Mikito, Aoyama Toru, Suzuki Susumu, Tanaka Akihito, Matsubara Tatsuaki, Murohara Toyoaki

Circulation Journal Vol. 79 ( 7 ) page： 1542 - 8 2015

Language：English Publishing type：Research paper (scientific journal) Publisher：The Japanese Circulation Society

Background:Until now, there have been few reports on the accuracy of in-stent restenosis (ISR) detection using high-definition computed tomography (HDCT). The purpose of this study was to assess ISR using HDCT with a new gemstone detector and to examine the diagnostic accuracy compared with invasive coronary angiography.Methods and Results:We evaluated 162 consecutive patients with 316 stents and the image quality (IQ) scores used to assess ISR, and analyzed whether stent strut thickness and diameter affected IQ score and assessability. In the 316 stents, 278 were diagnosed as assessable with HDCT (88.0%). IQ score for stent diameter ≥3 mm was significantly higher than that for stent diameter <3 mm, for stents with both thick struts ≥140 μm in thickness (mean IQ: 2.04±0.97 vs. 2.83±1.06, P<0.001) and thin struts <140 μm (mean IQ: 1.92±0.87 vs. 2.64±0.96, P=0.01). Assessability for stent diameter ≥3 mm was significantly higher than that for stent diameter <3 mm only for stents with thick struts (92.8% vs. 76.1%, P<0.001). Stent strut thickness, however, was not statistically significantly associated with either IQ score or assessability.Conclusions:In-stent lumens have high HDCT assessability, and HDCT is useful to evaluate thick-strut stents with diameter <3 mm. (Circ J 2015; 79: 1542–1548)

DOI： 10.1253/circj.CJ-14-1344

Akt-dependent Girdin phosphorylation regulates repair processes after acute myocardial infarction. Reviewed

Hayano S, Takefuji M, Maeda K, Noda T, Ichimiya H, Kobayashi K, Enomoto A, Asai N, Takahashi M, Murohara T

J Mol Cell Cardiol. Vol. 88 page： 55-63 2015

RhoGEF-mediated vasoconstriction in hypertension.

Takefuji M, Yura Y, Kaibuchi K, Murohara T

Hypertension research : official journal of the Japanese Society of Hypertension Vol. 36 ( 11 ) page： 930 - 1 2013.11

RhoGEF12 controls cardiac remodeling by integrating G protein- and integrin-dependent signaling cascades.

Takefuji M, Krüger M, Sivaraj KK, Kaibuchi K, Offermanns S, Wettschureck N

The Journal of experimental medicine Vol. 210 ( 4 ) page： 665 - 73 2013.4

Language：English Publisher：Journal of Experimental Medicine

Structural cardiac remodeling, including hypertrophy and fibrosis, plays a crucial role in the pathogenesis of heart failure. In vitro studies suggested a role of the small GTPase RhoA in hypertrophic cardiomyocyte growth, but neither the molecular mechanisms leading to RhoA activation nor their relevance in vivo are known. We use here a mass spectrometric approach to identify Rho guanine nucleotide exchange factors (RhoGEFs) activated during cardiac pressure overload in vivo and show that RhoGEF12 is a central player during cardiac remodeling. We show that RhoGEF12 is required for stretch-induced RhoA activation and hypertrophic gene transcription in vitro and that its activation depends on integrin β1 and heterotrimeric G proteins of the G12/13 family. In vivo, cardiomyocyte-specific deletion of RhoGEF12 protects mice from overload-induced hypertrophy, fibrosis, and development of heart failure. Importantly, in mice with preexisting hypertrophy, induction of RhoGEF12 deficiency protects from cardiac decompensation, resulting in significantly increased longterm survival. Collectively, RhoGEF12 acts as an integrator of stretch-induced signaling cascades in cardiomyocytes and is an interesting new target for therapeutic intervention in patients with pressure overload-induced heart failure. © 2013 Takefuji et al.

DOI： 10.1084/jem.20122126

Mutation of ARHGAP9 in patients with coronary spastic angina.

RhoGEF-mediated vasoconstriction in hypertension Reviewed

Takefuji M, Yura Y, Kaibuchi K, Murohara T

Hypertens Res. Vol. 36 ( 11 ) page： 930-931 2013

G13 controls angiogenesis through regulation of VEGFR-2 expression Reviewed International coauthorship

Sivaraj KK, Takefuji M, Schmidt I, Adams RH, Offermanns S, Wettchureck N

Dev Cell Vol. 25 ( 4 ) page： 427-434 2013

RhoGEF12 controls cardiac remodeling by intergrating G protein- and integrin-dependent signaling cascades. Reviewed

Takefuji M, Kruger M, Sivaraj KK, Kaibuchi K, Offermanns S, Wettshureck N

J Exp Med. Vol. 210 ( 4 ) page： 665-673 2013

G(13)-mediated signaling pathway is required for pressure overload-induced cardiac remodeling and heart failure.

Takefuji M, Wirth A, Lukasova M, Takefuji S, Boettger T, Braun T, Althoff T, Offermanns S, Wettschureck N

Circulation Vol. 126 ( 16 ) page： 1972 - 82 2012.10

Procontractile G protein-mediated signaling pathways antagonistically regulate smooth muscle differentiation in vascular remodeling. Reviewed International coauthorship

Althoff TF, Juarez JA, Troidl K, Wirth A, Takefuji M, Wettschureck N,

J Exp Med. Vol. 209 ( 12 ) page： 2277-2290 2012

Takefuji M, Asano H, Mori K, Amano M, Kato K, Watanabe T, Morita Y, Katsumi A, Itoh T, Takenawa T, Hirashiki A, Izawa H, Nagata K, Hirayama H, Takatsu F, Naoe T, Yokota M, Kaibuchi K

Journal of human genetics Vol. 55 ( 1 ) page： 42 - 9 2010.1

Language：English Publisher：Journal of Human Genetics

Coronary artery spasm has an important function in the etiology of variant angina and other acute coronary syndromes. Abnormal activation of Rho-family GTPases has been observed in cardiovascular disorders, but the function of genetic variability in Rho-family GTPases remains to be evaluated in cardiovascular disorders. We examined the genetic variability of Rho-family GTPases and their regulators in coronary artery spasm. We performed a comprehensive candidate gene analysis of 67 single nucleotide polymorphisms with amino-acid substitution in Rho-family GTPases and their regulators in 103 unrelated Japanese patients with acetylcholine-induced coronary artery spasm and 102 control Japanese subjects without acetylcholine-induced coronary artery spasm. We noted an association of the single nucleotide polymorphism of ARHGAP9 (rs11544238, Ala370Ser) with coronary artery spasm (odds ratio=2.67). We found that ARHGAP9 inactivated Rac as RacGAP and that the mRNA level of ARHGAP9 was strongly detected in hematopoietic cells. ARHGAP9 negatively regulated cell migration. The Ala370Ser polymorphism counteracted ARHGAP9-reduced cell migration, spreading and adhesion. The Ala370Ser polymorphism in the ARHGAP9 gene is associated with coronary artery spasm. These data suggest that the polymorphism of ARHGAP9 has a critical function in the infiltration of hematopoietic cells into the endothelium and inflammation leading to endothelial dysfunction. © 2010 The Japan Society of Human Genetics. All rights reserved.

DOI： 10.1038/jhg.2009.120

Rho-kinase Contributes to Sustained RhoA Activation through Phosphorylation of p190A RhoGAP Reviewed

Kazutaka Mori, Mutsuki Amano, Mikito Takefuji, Katsuhiro Kato, Yasuhiro Morita, Tomoki Nishioka, Yoshiharu Matsuura, Toyoaki Murohara, Kozo Kaibuchi

JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 284 ( 8 ) page： 5067 - 76 2009.2

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

RhoA is transiently activated by specific extracellular signals such as endothelin-1 (ET-1) in vascular smooth muscle cells. RhoGAP negatively regulates RhoA activity: thus, RhoA becomes the GDP-bound inactive form afterward. Sustained activation of RhoA is induced with high doses of the extracellular signals and is implicated in certain diseases such as vasospasms. However, it remains largely unknown how prolonged activation of RhoA is induced. Here we show that Rho-kinase, an effector of RhoA, phosphorylated p190A RhoGAP at Ser(1150) and attenuated p190A RhoGAP activity in COS7 cells. Binding of Rnd to p190A RhoGAP is thought to enhance its activation. Phosphorylation of p190A RhoGAP by Rho-kinase impaired Rnd binding. Stimulation of vascular smooth muscle cells with a high dose of ET-1 provoked sustained RhoA activation and p190A RhoGAP phosphorylation, both of which were prohibited by a Rho-kinase inhibitor. The phosphomimic mutation of p190A RhoGAP weakened Rnd binding and RhoGAP activities. Taken together, these results suggest that ET-1 induces Rho-kinase activation and subsequent phosphorylation of p190A RhoGAP, leading to prolonged RhoA activation.

DOI： 10.1074/jbc.M806853200

NOVEL MECHANISM OF SUSTAINED RHOA ACTIVATION IN VASCULAR SMOOTH MUSCLE; ITS POSSIBLE ROLE IN VASOSPASM

Katsuhiro Kato, Kazutaka Mori, Mutsuki Amano, Mikito Takefuji, Toyoaki Murohara, Kozo Kaibuchi

JOURNAL OF VASCULAR RESEARCH Vol. 46 page： 14 - 14 2009

39) 企業におけるAEDを用いた救命蘇生体験と救命救急体制づくり(第131回日本循環器学会東海地方会)

下方敬子, 近藤隆久, 大杉茂樹, 竹藤幹人, 梶口雅弘, 大林利博, 鈴木克昌

Circulation journal : official journal of the Japanese Circulation Society Vol. 72 ( 0 ) page： 1079 - 1079 2008.10

Rho-kinase modulates the function of STEF, a Rac GEF, through its phosphorylation. International journal

Mikito Takefuji, Kazutaka Mori, Yasuhiro Morita, Nariko Arimura, Takashi Nishimura, Masanori Nakayama, Mikio Hoshino, Akihiro Iwamatsu, Toyoaki Murohara, Kozo Kaibuchi, Mutsuki Amano

Biochemical and biophysical research communications Vol. 355 ( 3 ) page： 788 - 94 2007.4

Language：English Publishing type：Research paper (scientific journal)

Rho family GTPases are key regulators of various physiological processes. Several recent studies indicated that the antagonistic relationship between Rho and Rac is essential for cell polarity and that the Rac activity is negatively regulated by Rho. In this study, we found that Rho-kinase, an effector of Rho, counteracted the Rac GEF STEF-induced Rac1 activation in COS7 cells. Rho-kinase phosphorylated STEF at Thr1662 in vitro, and Y-27632, a Rho-kinase inhibitor, suppressed lysophosphatidic acid-induced phosphorylation of STEF in PC12D cells. STEF interacted with specific molecules such as microtubule-associated protein 1B, and the phosphorylation of STEF by Rho-kinase diminished its interaction with these molecules. STEF promoted nerve growth factor-induced neurite outgrowth in PC12D cells, while the phosphomimic mutant of STEF had a weakened ability to enhance neurite outgrowth. Taken together, these results suggest that the phosphorylation of STEF by Rho-kinase exerts the inhibitory effect on the function of STEF.

DOI： 10.1016/j.bbrc.2007.02.028

Rho mediates endocytosis of epidermal growth factor receptor through phosphorylation of endophilin A1 by Rho-kinase. International journal

Takako Kaneko, Akio Maeda, Mikito Takefuji, Hiroki Aoyama, Masanori Nakayama, Saeko Kawabata, Yoji Kawano, Akihiro Iwamatsu, Mutsuki Amano, Kozo Kaibuchi

Genes to cells : devoted to molecular & cellular mechanisms Vol. 10 ( 10 ) page： 973 - 87 2005.10

Language：English Publishing type：Research paper (scientific journal)

After binding of epidermal growth factor (EGF), the EGF receptor is activated, internalized by endocytosis, and subsequently degraded in the lysosomal pathway. Endocytotic trafficking of the activated EGF receptor is essential for controlling EGF signaling. Upon ligand-induced activation of EGF receptors, Cbl (ubiquitin ligase) binds to the activated receptor and leads to translocation of the CIN85 (Cbl-interacting protein of 85 kDa)/endophilin complex in the vicinity of the activated EGF receptors. Endophilin is known as a key regulator of clathrin-mediated endocytosis, and the translocation of endophilin in the vicinity of active EGF receptor is thought to promote receptor internalization. The constitutively active mutant of small GTPase Rho inhibits EGF receptor endocytosis. In this study, we found that this inhibitory effect was canceled by the dominant negative form of Rho-associated kinase (Rho-kinase), which is an effector of Rho. To clarify the molecular mechanisms of endocytosis downstream of Rho/Rho-kinase signal, we searched for and identified endophilin A1 as a novel substrate of Rho-kinase. We identified the phosphorylation site of endophilin A1 at Thr-14 and made endophilin T14D (substitution of Thr-14 by Asp), which is expected to mimic the phosphorylation state of endophilin A1. Endophilin T14D inhibited EGF receptor internalization. Furthermore, phosphorylation of endophilin by Rho-kinase inhibited the binding to CIN85. Taken together, these results suggest that Rho-kinase phosphorylates endophilin downstream of Rho and regulates EGF receptor endocytosis through the inhibition of binding between endophilin and CIN85.

DOI： 10.1111/j.1365-2443.2005.00895.x

Potential role of Rap1 GTPase for cadherin turnover in MDCK cells

Jun Noritake, Kazumasa Sato, Masato Nakagawa, Nanae Izumi, Yuki Taya, Takashi Watanabe, Shuijie Wang, Mikito Takefuji, Masaki Fukata, Kozo Kaibuchi

CELL STRUCTURE AND FUNCTION Vol. 30 page： 35 - 35 2005.6

Rho-kinase and myosin II activities are required for cell type and environment specific migration. International journal

Masanori Nakayama, Mutsuki Amano, Akira Katsumi, Takako Kaneko, Saeko Kawabata, Mikito Takefuji, Kozo Kaibuchi

Genes to cells : devoted to molecular & cellular mechanisms Vol. 10 ( 2 ) page： 107 - 17 2005.2

Language：English Publishing type：Research paper (scientific journal)

Cell migration is important in the development of atherosclerotic lesions. Macrophages and smooth muscle cells migrate into the subendothelial space of arteries, leading to plaque formation. Long-term inhibition of the activity of Rho-kinase induces a regression of atherosclerotic coronary lesions, probably by preventing migration of macrophages and smooth muscle cells. Previous reports concerning the effect of Rho-kinase inhibitors on cell migration are contradictory, however. We examined here the cell type specificity of Rho-kinase inhibitors and found that migration of endothelial cells, macrophages, and smooth muscle cells was inhibited by treatment with Rho-kinase inhibitors in a dose-dependent fashion in a three-dimensional migration assay, whereas that of fibroblasts and epithelial cells was not inhibited. Myosin II inhibitor prevented cell migration in a manner similar to Rho-kinase inhibitors. In contrast, in a two-dimensional migration assay, cell migration was not inhibited by Rho-kinase or myosin II inhibitors for any of the cell types examined. Taken together, these results indicate that Rho-kinase inhibitors suppress migration of specific cell types under specific conditions through the regulation of myosin II activity. Our findings suggest that Rho-kinase is the therapeutic target of atherosclerosis accompanied with invasion by leukocytes and smooth muscle cells.

DOI： 10.1111/j.1365-2443.2005.00823.x

Interaction with IQGAP1 links APC to Rac1, Cdc42, and actin filaments during cell polarization and migration.

Watanabe T, Wang S, Noritake J, Sato K, Fukata M, Takefuji M, Nakagawa M, Izumi N, Akiyama T, Kaibuchi K

Developmental cell Vol. 7 ( 6 ) page： 871 - 83 2004.12

Language：English Publisher：Developmental Cell

Rho family GTPases, particularly Rac1 and Cdc42, are key regulators of cell polarization and directional migration. Adenomatous polyposis coli (APC) is also thought to play a pivotal role in polarized cell migration. We have found that IQGAP1, an effector of Rac1 and Cdc42, interacts directly with APC. IQGAP1 and APC localize interdependently to the leading edge in migrating Vero cells, and activated Rac1/Cdc42 form a ternary complex with IQGAP1 and APC. Depletion of either IQGAP1 or APC inhibits actin meshwork formation and polarized migration. Depletion of IQGAP1 or APC also disrupts localization of CLIP-170, a microtubule-stabilizing protein that interacts with IQGAP1. Taken together, these results suggest a model in which activation of Rac1 and Cdc42 in response to migration signals leads to recruitment of IQGAP1 and APC which, together with CLIP-170, form a complex that links the actin cytoskeleton and microtubule dynamics during cell polarization and directional migration. Copyright © 2004 Cell Press.

DOI： 10.1016/j.devcel.2004.10.017