Analysis of cartilage protection by mechanical stress -miRNA and extracellular vesicles functions in osteoarthritis-

• Project/Area Number: 16K10905
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Orthopaedic surgery
• Research Institution: Okayama University
• Principal Investigator: Ohtsuki Takashi 岡山大学, 保健学研究科, 非常勤研究員 (10534802)
• Co-Investigator(Kenkyū-buntansha): 古松 毅之 岡山大学, 大学病院, 講師 (20432651) ; 西田 圭一郎 岡山大学, 医歯薬学総合研究科, 准教授 (80284058) ; 廣畑 聡 岡山大学, 保健学研究科, 教授 (90332791) ; 品岡 玲 岡山大学, 医歯薬学総合研究科, 助教 (90724500)
• Project Period (FY): 2016-04-01 – 2019-03-31
• Project Status: Completed (Fiscal Year 2018)
• Budget Amount: ¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000); Fiscal Year 2018: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2017: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2016: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
• Keywords: エクソソーム / miRNA / マトリックス / ADAMTS / アグリカン / ヒアルロン酸 / コラーゲン / 変形性関節症 / micro RNA / メカニカルストレス

Outline of Final Research Achievements

We established the method how to purify Extracellular vesicles (EVs) from synovial fluid. Furthermore, we analyzed the effects of adding EVs in culture medium in mRNA expressions (matrix components and matrix degradative enzymes) by RT-PCR method. Using fluorescence labelled EVs and some inhibitors, we revealed EVs were up-taken by clathrin mediated endocytosis in chondrocyte.
We isolated several miRNAs that reduced inflammatory cytokine induced matrix degradative enzyme protein expression.

Academic Significance and Societal Importance of the Research Achievements

関節液からエクソソーム（EVs）の精製する方法を確立し、軟骨細胞へのEVs添加試験系を用いて滑液中EVsが周辺組織に作用するかを解析可能にした。また軟骨細胞のEVs取り込み機構を解明したことでEVs が変形性関節症（OA）にどのように関与するか、EVsの内包するどの成分（mRNA、miRNA、タンパク）が作用するのかという解析を行うことができるようになった。また、本研究でマトリックス分解酵素発現を抑制する可能性のあるmiRNAを同定し、これらmiRNAのタンパクレベルでのマトリックス分解因子産生抑制を確認した。OA治療薬としてのmiRNAの可能性が示された。

Research Products

• [Journal Article] A High-Fat and High-Cholesterol Diet Induces Cardiac Fibrosis, Vascular Endothelial, and Left Ventricular Diastolic Dysfunction in SHRSP5/Dmcr Rats (2018)
  • Author(s): Watanabe S, Kumazaki S, Kusunoki K, Inoue T, Maeda Y, Usui S, Shinohata R, Ohtsuki T, Hirohata S, Kusachi S, Kitamori K, Mori M, Yamori Y, Oka H.
  • Journal Title: Journal of Atherosclerosis and Thrombosis Volume: 25 Issue: 5 Pages: 439-453
  • DOI: 10.5551/jat.40956
  • NAID: 130006729837
  • ISSN: 1340-3478, 1880-3873
  • Year and Date: 2018-05-01
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Host-produced ADAMTS4 inhibited early stage of tumor growth. (2018)
  • Author(s): Asano K, Edamatsu M, Hatipoglu O F, Cilek M Z, Inagaki J, Ohtsuki T, Oohashi T, Hirohata S.
  • Journal Title: Acta Med Okayama. Volume: 2018 Pages: 257-266
  • NAID: 120006482820
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] High molecular weight hyaluronan protects cartilage from degradation by inhibiting aggrecanase expression. (2018)
  • Author(s): Ohtsuki T, Asano K, Inagaki J, Shinaoka A, Kumagishi-Shinaoka K, Cilek MZ, Hatipoglu OF, Oohashi T, Nishida K, Komatsubara I, Hirohata S.
  • Journal Title: J Orthop Res. Volume: 36 Pages: 3247-3255
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Diverse Functions of a Disintegrin and Metalloproteinase with Thrombospondin Motif-1 (2017)
  • Author(s): 廣畑 聡、稲垣 純子、大月 孝志
  • Journal Title: YAKUGAKU ZASSHI Volume: 137 Issue: 7 Pages: 811-814
  • DOI: 10.1248/yakushi.16-00236-4
  • NAID: 130007287836
  • ISSN: 0031-6903, 1347-5231
  • Year and Date: 2017-07-01
  • Peer Reviewed / Open Access
• [Journal Article] Stromal Versican Regulates Tumor Growth by Promoting Angiogenesis (2017)
  • Author(s): Asano Keiichi、Nelson Courtney M.、Nandadasa Sumeda、Aramaki-Hattori Noriko、Lindner Daniel J.、Alban Tyler、Inagaki Junko、Ohtsuki Takashi、Oohashi Toshitaka、Apte Suneel S.、Hirohata Satoshi
  • Journal Title: Scientific Reports Volume: 7 Issue: 1 Pages: 1-11
  • DOI: 10.1038/s41598-017-17613-6
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] Isolation and characterization of extracellular microvesicles in osteoarthritis (2019)
  • Author(s): Liu Guole, Takashi Ohtsuki, Akira Shinaoka, Kana Komatsu, Yurika Notsu, Omer Faruk Hatipoglu, Takuto Nishimur, Miho Tatii, Junko Inagaki, Hiroshi Yamada, Keiichiro Nishida, Sun Liankun, Satoshi Hirohata
  • Organizer: The 32th Annual meeting of the Japanese Society of Cartilage metabolism
• [Presentation] Isolation and characterization of Osteoarthritis concerning microRNA (2018)
  • Author(s): Takashi Ohtsuki, Omer Faruk Hatipoglu, Akira Shinaoka, Mehmet Zeynel Cilek, Takuto Nishimura, Keiichi Asano, Junko Inagaki, Toshitaka Oohashi, Keiichiro Nishida, Yasunori Okada, Satoshi Hirohata
  • Organizer: Joint Conference of 50th Annual Meeting of the Japanese Society of Connective Tissue Research (JSCTR)
• [Presentation] Silencing of Important Molecules in Pathogenesis of Idiopathic Pulmonary Fibrosis via RNA interference (2018)
  • Author(s): OF Hatipoglu, E Uctepe, E. Gunduz, M. Gunduz, M. Aca, T Ohtsuki, J Inagaki, S Hirohata
  • Organizer: Joint Conference of 50th Annual Meeting of the Japanese Society of Connective Tissue Research (JSCTR)
• [Presentation] Eosinophyl derived cationic protein purification and cardiomyoblast protection activity (2018)
  • Author(s): Takuto Nishimura, Miho Tachiki, Junko Inagaki, Omer faruk Hatipogl, Takashi Ohtsuki, Kentaro Ikemura, Liu Guole, Satoshi Hirohata
  • Organizer: The 13th JAPANESE ASSOCIATION OF MEDICAL TECHONOLOGY EDUCATION
• [Presentation] Isolation and characterization of matrix degradative microRNA (2018)
  • Author(s): Takashi Ohtsuki, Omer Faruk Hatipoglu, Akira Shinaoka, Mehmet Zeynel Cilek, Takuto Nishimura, Keiichi Asano, Junko Inagaki, Toshitaka Oohashi, Keiichiro Nishida, Yasunori Okada, Satoshi Hirohata
  • Organizer: 10th Annual Meeting of the Japan CCN family study group
• [Presentation] Analysis of cartilage matrix degradation and regeneration mechanism (2018)
  • Author(s): Takashi Ohtsuki, Liu Guole, Omer Faruk Hatipoglu, Akira Shinaoka, Junko Inagaki, Ken Takahashi, Hiroshi Yamada, Toshitaka Ohashi, Keiichiro Nishida, Yasunori Okada, Suneel S, Apte, Satoshi Hirohata
  • Organizer: Brainstorming 2018
• [Presentation] Purification and characterization of exosome from chondrocyte conditioned medium and analysis of exosome up-taking mechanism in chondrocyte (2018)
  • Author(s): Liu Guole, Takashi Ohtsuki, Akira Shinaoka, Kana Komatsu, Yurika Notsu, Omer Faruk Hatipoglu, Takuto Nishimura1, Miho Tatiki, Junko Inagaki, Hiroshi Yamada, Keiichiro Nishida, Sun Liankun, Satoshi Hirohata
  • Organizer: Brainstorming 2018
• [Presentation] Mechanical stress attenuated inflammatory cytokines induced matrix degradative enzymes expression in chondrocyte by inhibition of NF-kB translocation (2018)
  • Author(s): Takashi Ohtsuki, Omer Faruk Hatipoglu, Guole Liu, Akira Shinaoka, Keiichi Asano, Takuto Nishimura, Junko Inagaki, Keiichiro Nishida, Toshitaka Oohashi, Satoshi Hirohata
  • Organizer: American Society for Matrix Biology Biennial Meeting 2018
  • Int'l Joint Research
• [Presentation] Isolation and characterization of extracellular microvesicles in osteoarthritis (2018)
  • Author(s): Liu Guole, Takashi Ohtsuki, Akira Shinaoka, Kana Komatsu, Yurika Notsu, Omer Faruk Hatipoglu, Takuto Nishimur, Miho Tatii, Junko Inagaki, Hiroshi Yamada, Keiichiro Nishida, Sun Liankun, Satoshi Hirohata
  • Organizer: The 41st Annual Meeting of the molecular Biology Society of Japan
• [Presentation] HYBID (HYaluronan-Binding protein Involved in hyaluronan Depolymerization) 遺伝子の炎症性サイトカインによる発現機構解析 (2017)
  • Author(s): 大月孝志、吉田浩之、品岡玲、熊岸-品岡加苗、メフメット・ゼイネル・チレッキ、オメル・ファルク・ハティポール、稲垣純子、西田圭一郎、岡田保典 、廣畑聡 "
  • Organizer: 第30回日本軟骨代謝学会
  • Place of Presentation: 京都
  • Year and Date: 2017-03-03
• [Presentation] INFLAMMATORY CYTOKINE INDUCED HYBID (HYALURONAN-BINDING PROTEIN INVOLVED IN HYALURONAN DEPOLYMERAZATION) -EXPRESSION MECHANISM- (2017)
  • Author(s): Takashi Ohtsuki, Lyu Chen, Hiroyuki Yoshida, Akira Shinaoka, Kanae Kumagishi-Shinaoka, Mehmet Zeynel Cilek, Omer Faruk Hatipoglu, Junko Inagaki, Keiichiro Nishida, Yasunori Okada, Satoshi Hirohata
  • Organizer: HYALRONAN 2017
  • Int'l Joint Research
• [Presentation] メカニカルストレスの軟骨細胞に及ぼす多彩な作用-マトリックス合成促進と炎症性サイトカイン誘導性マトリックス分解活性抑制 (2017)
  • Author(s): 大月孝志、品岡玲、熊岸-品岡加苗、メフメット・ゼイネル・チレッ、オメル・ファルク・ハティポール、稲垣純子、西田圭一郎、廣畑聡
  • Organizer: 第49回日本結合組織学会学術大会
• [Presentation] がん微小環境におけるバーシカン分解酵素ADAMTSの局在と血管新生におけるバーシカンの意義 (2017)
  • Author(s): 浅野恵一、廣畑 聡、大月孝志、オメル・ファルク・ハティポール、稲垣純子、大橋俊孝
  • Organizer: 第49回日本結合組織学会学術大会
• [Presentation] 炎症性サイトカイン誘導性マトリックス分解酵素発現のイオンチャネルを介した抑制機構解析 (2017)
  • Author(s): 大月孝志、品岡玲、熊岸-品岡加苗、オメル・ファルク・ハティポール、山之口奈保、岡田真澄、西村拓人、稲垣純子、西田圭一郎、廣畑聡
  • Organizer: 第40回日本分子生物学会年会 第90回日本生化学会大会 合同大会
• [Presentation] Expression and proteolysis of the proteoglycan versican in the tumor microenvironment (2016)
  • Author(s): Keiichi Asano, Tyler Alban, Courtney Nelson, Junko Inagaki, Takashi Ohtsuki, Shozo Kusachi, Yoshifumi Ninomiya, Toshitaka Oohashi, Suneel Apte, Satoshi Hirohata
  • Organizer: American Society for Matrix Biology Biennial Meeting 2016
  • Place of Presentation: Florida, USA
  • Year and Date: 2016-11-13
  • Int'l Joint Research
• [Presentation] 変形性膝関節症の治療効果に関する組織学的解析-ラットモデルを用いての検討- (2016)
  • Author(s): 熊岸-品岡 加苗、大月孝志、廣畑聡、河村顕治、大塚愛二 、坂田賢一郎、坂田俊輔
  • Organizer: 第29回日本臨床整形外科学会学術集会
  • Place of Presentation: 札幌
  • Year and Date: 2016-08-31
• [Presentation] 変形性関節症へのヒアルロン酸投与、いつ投与すべきか？～動物モデルからの知見～ (2016)
  • Author(s): 大月孝志、品岡玲、熊岸加苗、河野真優美、篠原真歩、浅野恵一、稲垣純子、大橋俊孝、西田圭一郎、廣畑聡
  • Organizer: 第48回日本結合組織学会学術大会
  • Place of Presentation: 長崎
  • Year and Date: 2016-06-24