Elucidation of intrathrombotic mechanism of autophagic functions in the deep vein thrombosis formation and its application to forensic medicine

2017 Fiscal Year Annual Research Report

• Project/Area Number: 15K08880
• Research Institution: Wakayama Medical University
• Principal Investigator: 野坂 みずほ 和歌山県立医科大学, 医学部, 助教 (00244731)
• Co-Investigator(Kenkyū-buntansha): 近藤 稔和 和歌山県立医科大学, 医学部, 教授 (70251923) ; 木村 章彦 和歌山県立医科大学, 医学部, 博士研究員 (60136611) ; 石田 裕子 和歌山県立医科大学, 医学部, 講師 (10364077)
• Project Period (FY): 2015-04-01 – 2018-03-31
• Keywords: 深部静脈血栓塞栓症 / オートファジー / LC3 / p62

Outline of Annual Research Achievements

下大静脈結紮による深部静脈血栓症（DVT）モデルを用いて，これまでに血栓陳旧度を炎症に関わる因子について，免疫組織化学的に判定する指標を検索してきた．本研究課題ではオートファジーの，血栓形成・溶解過程における動態を明らかにし，さらに血栓陳旧度判定への応用の可能性を検討した．
DVTモデルは，野生型マウスの下大静脈（IVC）を，麻酔下で結紮して作成した．結紮後1～21日目にIVCを採取し，パラフィン包埋組織標本を作成した．オートファジー検出を目的とした免疫染色には抗LC3抗体ならびに抗p62抗体を用いた．この染色像について蛍光顕微鏡観察下にて，LC3陽性細胞数（1000倍，5視野）と，p62陽性細胞の有無により，血栓内におけるオートファジーの動態を評価し，血栓陳旧度との関係を明らかにした（各グループn=5）．
結紮から1日目の血栓内にはLC3陽性細胞は観察されなかったが，3日目以降の全ての血栓について，主に辺縁部に観察され，10日目に最も多く（25.2±0.93），以降は減少傾向を認めた．またp62陽性細胞は3日目以降の全ての血栓について，LC3と同様に主に血栓辺縁部に観察され，特に5～10日では明瞭に観察された．また蛍光二重染色の結果，血栓内のp62の主な産生細胞はマクロファージやCD31であることが分かった．
以上の結果，血栓溶解過程では，血栓内に生じたオートファジーが関与していることが明らかとなり，特に血管新生に寄与している可能性が示唆された．P62陽性細胞が観察される場合は3日以上経過した血栓であり，特にLC3陽性細胞数が10以上で，多数のp62陽性細胞が明瞭に認められる場合は，5～10日経過した血栓と推定できた．以上の結果，法医診断学的には，血栓中のオートファジーの動態が，剖検例においても血栓の陳旧度判定の有用な指標となると考えられた．

Research Products

• [Journal Article] Protective Roles of Interferon-γ in Cardiac Hypertrophy Induced by Sustained Pressure Overload (2018)
  • Author(s): Kimura A, Ishida Y, Furuta M, Nosaka M, Kuninaka Y, Taruya A, Mukaida N, Kondo T
  • Journal Title: J Am Heart Assoc Volume: 7(6) Pages: e008145
  • DOI: 10.1161/JAHA.117.008145
  • Peer Reviewed / Open Access
• [Journal Article] Fatal acute intoxication of accidentally ingested nifedipine in an infant ? A case report (2017)
  • Author(s): Yamamoto H, Takayasu T, Nosaka M, Kimura A, Ishida Y, Kawaguchi T, Fukami M, Okada M, Kondo T
  • Journal Title: Legal Medicine Volume: 24 Pages: 12--18
  • DOI: 10.1016/j.legalmed.2016.11.002
  • Peer Reviewed / Open Access
• [Journal Article] Ventricular androgenic-anabolic steroid-related remodeling: an immunohistochemical study (2017)
  • Author(s): Cecchi R, Muciaccia B, Ciallella C, Di Luca NM, Kimura A, Sestili C, Nosaka M, Kondo T
  • Journal Title: Int J Legal Med Volume: 131(6) Pages: 1589-1595
  • DOI: 10.1007/s00414-017-1589-3
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Detection of intrathrombotic endothelial progenitor cells and its application to thrombus age estimation in a murine deep vein thrombosis model (2017)
  • Author(s): Nosaka M, Ishida Y, Kimura A, Yamamoto, Kato T, Kuninaka Y, Shimada E, Kondo T
  • Journal Title: Int J Legal Med Volume: 131(6) Pages: 1633-1638
  • DOI: 10.1007/s00414-017-1668-5
  • Peer Reviewed / Open Access
• [Journal Article] Severer lupus erythematosus-like skin lesions in MRL/lpr mice with homozygous Kitwsh/wsh mutation (2017)
  • Author(s): Inaba Y, Kanazawa N, Yoshimasu T, Shimokawa T, Nosaka M, Kondo T, Furukawa F
  • Journal Title: Mod Rheumatol Volume: 28(2) Pages: 319-326
  • DOI: 10.1080/14397595.2017.1341591
  • Peer Reviewed / Open Access
• [Journal Article] Postmortem distribution of chlorpyrifos-methyl, fenitrothion, and their metabolites in body fluids and organ tissues of an intoxication case (2017)
  • Author(s): Takayasu T, Yamamoto H, Ishida Y, Nosaka M, Kawaguchi M, Kuninaka Y, Kimura A, Kondo T
  • Journal Title: Legal Medicine Volume: 29 Pages: 44-50
  • DOI: 10.1016/j.legalmed.2017.10.002.
  • Peer Reviewed / Open Access
• [Journal Article] Essential involvement of the CX3CL1-CX3CR1 axis in bleomycininduced pulmonary fibrosis via regulation of fibrocyte and M2 macrophage migration (2017)
  • Author(s): Ishida Y, Kimura A, Nosaka M, Kuninaka Y, Hemmi H, Sasaki I, Kaisho T, Mukaida N, Kondo T
  • Journal Title: Scientific Reports Volume: 7(1) Pages: 16833
  • DOI: 10.1038/s41598-017-17007-8.
  • Peer Reviewed / Open Access
• [Journal Article] Influence of Circadian Rhythm on Thrombus Formation of Murine Deep Vein Thrombosis Model (2017)
  • Author(s): Nosaka M, Ishida Y, Kimura A, Yamamoto H, Kuninaka Y, Shimada E, Okumoto K, Okada M, Hata S, and Kondo T
  • Journal Title: Ann Hematol Oncol Volume: 4(9) Pages: 1171
  • DOI: 10.26420/annhematoloncol.2017.1171
  • Peer Reviewed / Open Access
• [Presentation] アジ化物による中毒死の1剖検例 (2017)
  • Author(s): 山本寛記，工藤恵子，木村章彦，野坂みずほ，石田裕子，川口敬士，近藤稔和
  • Organizer: 第37回日本中毒学会西日本地方会
• [Presentation] 卵巣癌の進展におけるケモカイン受容体CCR5の病態生理学的役割 (2017)
  • Author(s): 近藤稔和，小林彩，佐々木宗一郎，石田裕子，野坂みずほ，木村章彦，井箟一彦，向田直史
  • Organizer: 金沢大学がん進展制御研究所共同利用・共同研究拠点シンポジウム
• [Presentation] Autophagy in skin wounds: a novel marker for vital reactions (2017)
  • Author(s): Kondo T, Kimura A, Ishida Y, Nosaka M, Kuninaka Y, E Shimada, Kawaguchi M, H.Yamamoto
  • Organizer: 26th Spring Meeting of German Society of Legal Medicine in North
• [Presentation] The absence of Spi-B exaggerates acute aortic dissection in mice (2017)
  • Author(s): 樽谷 玲，木村章彦，石田裕子，野坂みずほ，國中由美，島田栄美，山本寛記，川口真理子，近藤稔和
  • Organizer: 第101次日本法医学会学術全国集会
• [Presentation] 深部静脈血栓におけるオートファジーと法医診断学的応用に関する研究 (2017)
  • Author(s): 野坂みずほ，石田裕子，山本寛記，木村章彦，國中由美，川口真理子，近藤稔和
  • Organizer: 第101次日本法医学会学術全国集会
• [Presentation] The influence of circadian rhythm on thrombus formation, infection and intoxication (2017)
  • Author(s): Kuninaka Y, Yamamoto H, Nosaka M, Ishida Y, Shimada E, Kawaguchi M, Kimura A, Kondo T
  • Organizer: 第101次日本法医学会学術全国集会
• [Presentation] ジルチアゼム，プロプラノロールやミルタザピンなどを大量に服用し死亡した１事例 (2017)
  • Author(s): 高安達典，山本寛記，石田裕子，野坂みずほ，川口真理子，國中由美，木村章彦，近藤稔和
  • Organizer: 第101次日本法医学会学術全国集会
• [Presentation] Immunohistochemical detection of intrathrombotic endothelial progenitor cell and its application to thrombus age estimation in murine deep vein thrombosis model (2017)
  • Author(s): Nosaka M, Ishida Y, Kimura A, Kuninaka Y, Yamamoto H, Shimada E, Kondo T
  • Organizer: 26. Frhjahrstauuugung der Deutschen Gesellschaft fr Rechtsmedizin (Region Sd) in Verbindung mit dem 47. Treffen der Oberrheinischen Rechtsmediziner
  • Int'l Joint Research
• [Presentation] Fatal acute intoxication of accidentally ingested nifedipine in an infant (2017)
  • Author(s): Yamamoto H, Takayasu T, Ishida Y, Nosaka M, Kimura A, Shimada E, Kondo T
  • Organizer: 26. Fruhjahrstagung der Deutschen Gesellschaft fur Rechtsmedizin (Region Sud) in Verbindung mit dem 47. Treffen der Oberrheinischen Rechtsmediziner
  • Int'l Joint Research
• [Presentation] Immunohistochemical study on thrombus age determination (2017)
  • Author(s): Nosaka M
  • Organizer: The Seminar for the Post-graduate Course of Legal Medicine
  • Invited
• [Presentation] Spi-Bは圧負荷による心不全の病態形成において炎症と線維化を抑制して保護的に機能する (2017)
  • Author(s): 木村章彦，石田裕子，野坂みずほ，佐々木 泉，山本寛記，國中由美，加藤 喬，福田有里，改正恒康，近藤稔和
  • Organizer: 第38回日本炎症・再生医学会
• [Presentation] The absence of CCL3 exaggerated CaCl2-induced aortic aneurysm (2017)
  • Author(s): 石田裕子，國中由美，野坂みずほ，木村章彦，向田直史，近藤稔和
  • Organizer: 第38回日本炎症・再生医学会
• [Presentation] 深部静脈血栓症モデルにおけるCCR5システムの病態生理学的役割の解明 (2017)
  • Author(s): 野坂みずほ，石田裕子，木村章彦，山本寛記，國中由美，向田直史，近藤稔和
  • Organizer: 第38回日本炎症・再生医学会
• [Presentation] Molecular Pathophysiological Study on Thrombus Age Determination (2017)
  • Author(s): Nosaka M
  • Organizer: The 1st Wakayama Medical University International Symposium of Forensic Molecular Pathology
• [Presentation] いわゆる「ペットボトル症候群」による突然死の１剖検例 (2017)
  • Author(s): 秦 聡，岡田美里，奥本航平，北村謙伍，野坂みずほ，石田裕子，山本寛記，國中由美，島田栄美，木村章彦，近藤稔和
  • Organizer: 第２４回法医病理セミナー
• [Presentation] Forensic diagnosis of compression based on expression of aquaporin-3 in human neck skin (2017)
  • Author(s): Kondo T, Ishida Y, Nosaka M, Kimura A, Kuninaka Y, Yamamoto H, Kawaguchi M, Shimada E, Hata S
  • Organizer: 10th International Symposium Advances in Legal Medicine
  • Int'l Joint Research
• [Presentation] A retrospective study on forensic autopsy ceases of elder persons in Wakayama prefecture (2017)
  • Author(s): Hashizume Y, Hata S, Yamamoto H, Shimada E, Kuninaka Y, Kawaguchi M, .Ishida Y, Nosaka M, Kimura A, Takatsu A, Kondo T
  • Organizer: 10th International Symposium Advances in Legal Medicine
  • Int'l Joint Research
• [Presentation] The influence of circadian rhythm on thrombus formation, infection and intoxication (2017)
  • Author(s): Ishida Y, Kuninaka Y, Hata S, Nosaka M, Kimura A, Yamamoto H, Shimada E, .Kawaguchi M, Murakami K, Sakamoto S, Kondo T
  • Organizer: 10th International Symposium Advances in Legal Medicine
  • Int'l Joint Research
• [Presentation] Acetaminophen-induced fulminant hepatic failure altered the biological clock in mouse heart (2017)
  • Author(s): Kimura A, Yamamoto H, Ozaki M, Hata S, Ishida Y, Nosaka M, Kuninaka Y, Hashizume Y, Kondo T
  • Organizer: 10th International Symposium Advances in Legal Medicine
  • Int'l Joint Research
• [Presentation] Immunohistochemical detection of intrathrombotic fibrocytes and its application to .thrombus age estimation in murine deep vein thrombosis model (2017)
  • Author(s): Nosaka M, Ishida Y, Kuninaka Y, Yamamoto H, Shimada E, Kimura A, Hashizume Y, Kondo T
  • Organizer: 10th International Symposium Advances in Legal Medicine
  • Int'l Joint Research
• [Presentation] The absence of Spi-B exaggerates acute aortic dissection in mice (2017)
  • Author(s): Taruya A, Kimura A, Ishida Y, Nosaka M, Kondo T
  • Organizer: 10th International Symposium Advances in Legal Medicine
  • Int'l Joint Research
• [Presentation] The absence of CCL3 exaggerated CaCl2-induced aortic aneurysm (2017)
  • Author(s): Ishida Y, Kuninaka Y, Nosaka M, Kimura A, Yamamoto H, Shimada E, Kawaguchi M, Kondo T
  • Organizer: 10th International Symposium Advances in Legal Medicine
  • Int'l Joint Research
• [Presentation] Molecular toxicological study on the influence of circadian rhythms on acetaminophen hepatotoxicity (2017)
  • Author(s): Yamamoto H, Ishida Y, Nosaka M, Kuninaka Y, Shimada E, Kawaguchi M, Kimura A, Kondo T
  • Organizer: 10th International Symposium Advances in Legal Medicine
  • Int'l Joint Research
• [Presentation] Molecular Pathophysiological Study on Thrombus Age Determination. (2017)
  • Author(s): Nosaka M
  • Organizer: The 1st Wakayama Medical University International Symposium of Forensic Molecular Pathology
  • Int'l Joint Research / Invited
• [Presentation] Absence of CCR5 axis inhibits thrombus resolution through reduced uPA, tPA and VEGF expression in murine DVT model (2017)
  • Author(s): Nosaka M, Ishida Y, Kimura A, Yamamoto H, Kuninaka Y, Shimada E, Mukaida N, Kondo T
  • Organizer: 5th Annual Meeting of the International Cytokine & Interferon Society
  • Int'l Joint Research
• [Presentation] The absence of CCL3 exaggerated CaCl2-induced aortic aneurysm (2017)
  • Author(s): Ishida Y, Kuninaka Y, Nosaka M, Kimura A, Mukaida N, Kondo T
  • Organizer: 5th Annual Meeting of the International Cytokine & Interferon Society
  • Int'l Joint Research
• [Presentation] Prevention of lipopolysaccharide-induced preterm labor by the lack of CX3CL1-CX3CR1 interaction (2017)
  • Author(s): Mizoguchi M, Ishida Y, Nosaka M, Kimura A, Yahata T, Kuninaka Y, Nanjo S, Minami S, Ino K, Mukaida N, Kondo T
  • Organizer: 5th Annual Meeting of the International Cytokine & Interferon Society
  • Int'l Joint Research
• [Presentation] クエン酸と重曹の混合による若年者の自殺例 (2017)
  • Author(s): 村上圭秀，北村謙伍，岡田美里，奥本航平，石田裕子，野坂みずほ，國中由美，山本寛記，島田栄美，木村章彦，高津光洋，近藤稔和
  • Organizer: 第64回日本法医学会学術近畿地方集会
• [Presentation] 和歌山県の法医解剖例における高齢者事例の実態 (2017)
  • Author(s): 橋爪祐示子，秦 聡，北村謙伍，石田裕子，野坂みずほ，山本寛記，島田栄美，國中由美，川口真理子，村上圭秀，木村章彦，高津光洋，近藤稔和
  • Organizer: 第64回日本法医学会学術近畿地方集会
• [Presentation] CCL2は糖尿病マウスの創傷部に血管内皮前駆細胞浸潤を増加させて創傷治癒を促進する (2017)
  • Author(s): 石田裕子，國中由美，野坂みずほ，木村章彦，近藤稔和
  • Organizer: 第47回日本創傷治癒学会
• [Presentation] Absence of CCR5 axis exaggerates thrombus formation through reduced uPA, tPA and VEGF expression in murine DVT model (2017)
  • Author(s): Nosaka M, Ishida Y, Kimura A, Kuninaka Y, Mukaida N, Kondo T
  • Organizer: 第46回日本免疫学会学術集会
• [Presentation] The absence of CCL3 exaggerates CaCl2-induced aortic aneurysm (2017)
  • Author(s): Kondo T, Ishida Y, Kuninaka Y, Nosaka M, Kimura A, Mukaida N
  • Organizer: 第46回日本免疫学会学術集会
• [Presentation] Decline of lysyl oxidase protein causes acute aortic dissection in Spi-B knockout mice (2017)
  • Author(s): Taruya A, Kimura A, Ishida Y, Nosaka M, Kuninaka Y, Sasaki I, Kaisho T, Kondo T
  • Organizer: 第46回日本免疫学会学術集会