動脈壁と周囲脂肪組織における慢性炎症の可視化と病態解明

2013 Fiscal Year Annual Research Report

• Project/Area Number: 25293184
• Research Institution: The University of Tokushima
• Principal Investigator: 佐田 政隆 徳島大学, ヘルスバイオサイエンス研究部, 教授 (80345214)
• Co-Investigator(Kenkyū-buntansha): 堀川 一樹 徳島大学, ヘルスバイオサイエンス研究部, 教授 (70420247) ; 福田 大受 徳島大学, ヘルスバイオサイエンス研究部, 特任講師 (40637568) ; 東邦 康智 東京大学, 医学部附属病院, 助教 (10586481)
• Project Period (FY): 2013-04-01 – 2016-03-31
• Keywords: 慢性炎症 / 脂肪 / 血管 / 可視化 / コンストラクト / FRET / 動脈硬化 / カスパーゼ

Outline of Annual Research Achievements

この血管壁と血管周囲脂肪組織における慢性炎症の起点となるインフラマソームの活性化を可視化する手段の開発を開始した。インフラマソームが活性化すると、Caspase1 (ICE, Interleukin-1β変換酵素) が活性化する。このCaspase1活性をFRET(fluorescence energy transfer)現象を用いて可視化することを図った。青色蛍光タンパクCFPと黄色蛍光タンパクYFP をICE認識配列であるY E V D G W というアミノ酸配列を含んだリンカーによって結合したFRETコンストラクトを作成し、その感度、特異度を培養細胞を用いて確認した。

Caspase1によって認識されるYEVDGW をCFP（青色蛍光タンパク）とYFP（黄色蛍光タンパク）間に挟んだFRETタンパクを発現するコンストラクトを pcDNA3発現ベクターに組み込んだ。蛍光タンパクとして、CFPΔC11とcp173Venusの組み合わせがFRET効果が最大であった。YEVDGW配列を一個ならびに、二個、三個含む組み換え型タンパク質をCasepase1 で一時間処理すると、YEVDGW配列を二個ならびに三個含むコンストラクトにおいて、発光スペクトラムが変化し、FRET効果を反映して、蛍光が黄色から青色に変化することを確認した。次に、YEVDGW配列を三個含むタンパクを発現するプラスミドSCAT1.43をRAW264.7細胞にNucleofection を用いてトランスフェクションした。そののち、stable line を６株樹立した。1 ng/ml のLPS２時間刺激後に、1mMのATPを負荷した。425nmのレーザー光で励起した時の放出光を解析したところ、わずか３０分でCFP/YFPの上昇が認められ、Caspase1 の活性化を可視化することに成功した。

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

順調に計画は進行して、DNAコンストラクトの作製に成功し、その特異性を確認することができた。

Strategy for Future Research Activity

細胞レベルにおいて、提案した手法が有効であることを確認できた。今後は、インフラマソーム活性を効率的に可視化するトランスジェニックマウスのラインを樹立、増殖して、疾患モデルにおける慢性炎症の病態解明に応用する。

Research Products

• [Journal Article] Effects of telmisartan on inflammatory cytokines and the coronary plaque component as assessed on integrated backscatter intravascular ultrasound in hypertensive patients. (2014)
  • Author(s): Yamaguchi K, Wakatsuki T, Soeki T, Niki T, Taketani Y, Oeduka H, Kusunose K, Ise T, Iwase T, Yamada H, Sata M.
  • Journal Title: Circ J. Volume: 78 Pages: 240-247
  • DOI: 10.1253/circj.CJ-13-0741
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Beneficial effect of a synthetic prostacyclin agonist, ONO-1301, in rat autoimmune myocarditis model. (2013)
  • Author(s): Hirata Y, Kurobe H, Uematsu E, Yagi S, Soeki T, Yamada H, Fukuda D, Shimabukuro M, Nakayama M, Matsumoto K, Sakai Y, Kitagawa T, Sata M.
  • Journal Title: Eur J Pharmacol. Volume: 699 Pages: 81-87
  • DOI: 10.1016/j.ejphar.2012.11.045
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Exendin-4, a glucagon-like peptide-1 receptor agonist, attenuates neointimal hyperplasia after vascular injury. (2013)
  • Author(s): Hirata Y, Kurobe H, Nishio C, Tanaka K, Fukuda D, Uematsu E, Nishimoto S, Soeki T, Harada N, Sakaue H, Kitagawa T, Shimabukuro M, Nakaya N, Sata M.
  • Journal Title: Eur J Pharmacol. Volume: 699 Pages: 51-53
  • DOI: 10.1016/j.ejphar.2012.11.057
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Ulcer-like projection caused by a penetrating branch to the bronchial artery. (2013)
  • Author(s): Ise T, Fujioka K, Iwase T, Hotchi J, Akaike M, Sata M.
  • Journal Title: J Am Coll Cardiol. Volume: 61 Pages: e147
  • DOI: 10.1016/j.jacc.2012.10.047
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Interval from the onset of transmitral flow to annular velocity is a marker of LV filling pressure. (2013)
  • Author(s): Kusunose K, Yamada H, Nishio S, Tomita N, Niki T, Yamaguchi K, Taketani Y, Iwase T, Soeki T, Wakatsuki T, Sata M.
  • Journal Title: JACC Cardiovasc Imaging. Volume: 6 Pages: 528-530
  • DOI: 10.1016/j.jcmg.2012.10.025
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Epicardial adipose tissue volume and adipocytokine imbalance are strongly linked to human coronary atherosclerosis. (2013)
  • Author(s): Shimabukuro M, Hirata Y, Tabata M, Dagvasumberel M, Sato H, Kurobe H, Fukuda D, Soeki T, Kitagawa T, Takanashi S, Sata M.
  • Journal Title: Arterioscler Thromb Vasc Biol. Volume: 33 Pages: 1077-1084
  • DOI: 10.1161/ATVBAHA.112.300829
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Androgen receptor promotes sex-independent angiogenesis in response to ischemia and is required for activation of vascular endothelial growth factor receptor signaling. (2013)
  • Author(s): Yoshida S, Aihara KI, Ikeda Y, Sumitomo-Ueda Y, Uemoto R, Ishikawa K, Ise T, Yagi S, Iwase T, Mouri Y, Sakari M, Matsumoto T, Takeyama KI, Akaike M, Matsumoto M, Sata M, Walsh K, Kato S, Matsumoto T.
  • Journal Title: Circulation Volume: 128 Pages: 60-71
  • DOI: 10.1161/CIRCULATIONAHA
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] HMGB1 plays a critical role in vascular inflammation and lesion formation via toll-like receptor 9. (2013)
  • Author(s): Hirata Y, Kurobe H, Higashida M, Fukuda D, Shimabukuro M, Higashikuni Y, Kitagawa T, Sata M.
  • Journal Title: Atherosclerosis. Volume: 231 Pages: 227-233
  • DOI: 10.1016/j.atherosclerosis.2013.09.010
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Protective effects of selective mineralocorticoid receptor antagonist against aortic aneurysm progression in a novel murine model. (2013)
  • Author(s): Kurobe H, Hirata Y, Matsuoka Y, Sugasawa N, Higashida M, Nakayama T, Maxfield MW, Shimabukuro M, Kitagawa T, Sata M
  • Journal Title: J Surg Res. Volume: 185 Pages: 455-462
  • DOI: 10.1016/j.jss.2013.05.002
  • Peer Reviewed
• [Journal Article] Quercetin glucosides promote ischemia-induced angiogenesis, but do not promote tumor growth. (2013)
  • Author(s): Sumi M, Tateishi N, Shibata H, Ohki T, Sata M.
  • Journal Title: Life Sciences. Volume: 93 Pages: 814-819
  • DOI: 10.1016/j.lfs.2013.09.005
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Renin-angiotensin-aldosterone system has a pivotal role in cognitive impairment. (2013)
  • Author(s): Yagi S, Akaike M, Ise T, Ueda Y, Iwase T, Sata M.
  • Journal Title: Hypertens Res. Volume: 36 Pages: 753-758
  • DOI: 10.1038/hr.2013.51
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Ghrelin protects the heart against ischemia-induced arrhythmias by preserving connexin-43 protein. (2013)
  • Author(s): Soeki T, Niki T, Uematsu E, Bando S, Matsuura T, Kusunose K, Ise T, Ueda Y, Tomita N, Yamaguchi K, Koshiba K, Yagi S, Fukuda D, Taketani Y, Iwase T, Yamada H, Wakatsuki T, Akaike M, Shimabukuro M, Kishimoto I, Kangawa K, Sata M.
  • Journal Title: Heart Vessels. Volume: 28 Pages: 795-801
  • DOI: 10.1007/s00380-013-0333-2
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Toll-like receptor-2 mediates adaptive cardiac hypertrophy in response to pressure overload through interleukin-1β upregulation via nuclear factor κB activation. (2013)
  • Author(s): Higashikuni Y, Tanaka K, Kato M, Nureki O, Hirata Y, Nagai R, Komuro I, Sata M.
  • Journal Title: J Am Heart Assoc. Volume: 2 Pages: e000267
  • DOI: 10.1161/JAHA.113.000267
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Presentation] Post-Transcriptional Regulation of Adiponectin by Micro-RNA 378 in Human Visceral and Subcutaneous Adipose Tissue. (2013)
  • Author(s): Ishida M, Shimabukuro M, Yagi S, Fukuda D, Soeki T, Sato H, Uematsu E, Izaki H, Kanayama H, Sata M.
  • Organizer: AHA Scientific Sessions 2013
  • Place of Presentation: ﾀﾞﾗｽｺﾝﾍﾞﾝｼｮﾝｾﾝﾀｰ（Dallas, America）
  • Year and Date: 2013-11-16 – 2013-11-20
• [Presentation] Macrophage Toll-like Receptor 9 Signaling Contributes to the Development of Insulin Resistance through the Promotion of Inflammation in Adipose Tissue. (2013)
  • Author(s): Nishimoto S, Fukuda D, Shimabukuro M, Yagi S, Soeki T, Sakaue H, Nakaya Y, Sata M.
  • Organizer: AHA Scientific Sessions 2013
  • Place of Presentation: ﾀﾞﾗｽｺﾝﾍﾞﾝｼｮﾝｾﾝﾀｰ（Dallas, America）
  • Year and Date: 2013-11-16 – 2013-11-20
• [Presentation] Time-dependent Changes Of Local Vascular Response And Angiographic Peri-stent Contrast Staining Up To Five Years After Zotarolimus-eluting, Sirolimus-eluting, And Bare Metal Stent Implantation. (2013)
  • Author(s): Yamaguchi K, Wakatsuki T, Ise T, Tobiume T, Yagi S, Iwase T, Yamada H, Soeki T, Sata M.
  • Organizer: AHA Scientific Sessions 2013
  • Place of Presentation: ﾀﾞﾗｽｺﾝﾍﾞﾝｼｮﾝｾﾝﾀｰ（Dallas, America）
  • Year and Date: 2013-11-16 – 2013-11-20
• [Presentation] Decreased Local Hypercoagulation after Implantation of Everolimus-Eluting and Zotarolimus-Eluting Stents Compared with Early-Generation Drug-Eluting Stents in patients with stable angina. (2013)
  • Author(s): Yamaguchi K, Wakatsuki T, Ise T, Tobiume T, Yagi S, Iwase T, Yamada H, Soeki T, Sata M.
  • Organizer: AHA Scientific Sessions 2013
  • Place of Presentation: ﾀﾞﾗｽｺﾝﾍﾞﾝｼｮﾝｾﾝﾀｰ（Dallas, America）
  • Year and Date: 2013-11-16 – 2013-11-20
• [Presentation] Rivaroxaban, A Novel Oral Anticoagulant, Attenuates Plaque Progression in ApoE-Deficient Mice Through the Inhibition of Pro-Inflammatory Activation of Macrophages. (2013)
  • Author(s): Hara T, Fukuda D, Tanaka K, Higashikuni Y, Hirata Y, Ise T, Tobiume T, Yamaguchi K, Yagi S, Iwase T, Yamada H, Soeki T, Wakatsuki T, Shimabukuro M, Sata M.
  • Organizer: AHA Scientific Sessions 2013
  • Place of Presentation: ﾀﾞﾗｽｺﾝﾍﾞﾝｼｮﾝｾﾝﾀｰ（Dallas, America）
  • Year and Date: 2013-11-16 – 2013-11-20