Fluvastatin upregulates the expression of tissue factor pathwav inhibitor in human umbilical vein endothelial cells

13301甲第4205号博士（保健学）金沢大学博士論文本文Full 以下に掲載：Journal of Atherosclerosis and Thrombosis 22(7) pp.660-668 2015. Japan Atherosclerosis Society. 共著者：Akiko Sekiya, Eriko Morishita, Keiko Maruyama, Hiroki Torishima, Shigeki Ohtak

Stabilization of Microtubule-Unbound Tau via Tau Phosphorylation at Ser262/356 by Par-1/MARK Contributes to Augmentation of AD-Related Phosphorylation and Aβ42-Induced Tau Toxicity.

Abnormal accumulation of the microtubule-interacting protein tau is associated with neurodegenerative diseases including Alzheimer\u27s disease (AD). β-amyloid (Aβ) lies upstream of abnormal tau behavior, including detachment from microtubules, phosphorylation at several disease-specific sites, and self-aggregation into toxic tau species in AD brains. To prevent the cascade of events leading to neurodegeneration in AD, it is essential to elucidate the mechanisms underlying the initial events of tau mismetabolism. Currently, however, these mechanisms remain unclear. In this study, using transgenic Drosophila co-expressing human tau and Aβ, we found that tau phosphorylation at AD-related Ser262/356 stabilized microtubule-unbound tau in the early phase of tau mismetabolism, leading to neurodegeneration. Aβ increased the level of tau detached from microtubules, independent of the phosphorylation status at GSK3-targeted SP/TP sites. Such mislocalized tau proteins, especially the less phosphorylated species, were stabilized by phosphorylation at Ser262/356 via PAR-1/MARK. Levels of Ser262 phosphorylation were increased by Aβ42, and blocking this stabilization of tau suppressed Aβ42-mediated augmentation of tau toxicity and an increase in the levels of tau phosphorylation at the SP/TP site Thr231, suggesting that this process may be involved in AD pathogenesis. In contrast to PAR-1/MARK, blocking tau phosphorylation at SP/TP sites by knockdown of Sgg/GSK3 did not reduce tau levels, suppress tau mislocalization to the cytosol, or diminish Aβ-mediated augmentation of tau toxicity. These results suggest that stabilization of microtubule-unbound tau by phosphorylation at Ser262/356 via the PAR-1/MARK may act in the initial steps of tau mismetabolism in AD pathogenesis, and that such tau species may represent a potential therapeutic target for AD

Loss of axonal mitochondria promotes tau-mediated neurodegeneration and Alzheimer\u27s disease-related tau phosphorylation via PAR-1.

Abnormal phosphorylation and toxicity of a microtubule-associated protein tau are involved in the pathogenesis of Alzheimer\u27s disease (AD); however, what pathological conditions trigger tau abnormality in AD is not fully understood. A reduction in the number of mitochondria in the axon has been implicated in AD. In this study, we investigated whether and how loss of axonal mitochondria promotes tau phosphorylation and toxicity in vivo. Using transgenic Drosophila expressing human tau, we found that RNAi-mediated knockdown of milton or Miro, an adaptor protein essential for axonal transport of mitochondria, enhanced human tau-induced neurodegeneration. Tau phosphorylation at an AD-related site Ser262 increased with knockdown of milton or Miro; and partitioning defective-1 (PAR-1), the Drosophila homolog of mammalian microtubule affinity-regulating kinase, mediated this increase of tau phosphorylation. Tau phosphorylation at Ser262 has been reported to promote tau detachment from microtubules, and we found that the levels of microtubule-unbound free tau increased by milton knockdown. Blocking tau phosphorylation at Ser262 site by PAR-1 knockdown or by mutating the Ser262 site to unphosphorylatable alanine suppressed the enhancement of tau-induced neurodegeneration caused by milton knockdown. Furthermore, knockdown of milton or Miro increased the levels of active PAR-1. These results suggest that an increase in tau phosphorylation at Ser262 through PAR-1 contributes to tau-mediated neurodegeneration under a pathological condition in which axonal mitochondria is depleted. Intriguingly, we found that knockdown of milton or Miro alone caused late-onset neurodegeneration in the fly brain, and this neurodegeneration could be suppressed by knockdown of Drosophila tau or PAR-1. Our results suggest that loss of axonal mitochondria may play an important role in tau phosphorylation and toxicity in the pathogenesis of AD

Carbon monoxide (CO)-releasing molecule-derived CO regulates tissue factor and plasminogen activator inhibitor type 1 in human endothelial cells

Introduction: Heme oxygenase-1 (HO-1) is the rate limiting enzyme that catalyzes the conversion of heme into biliverdin, free iron, and carbon monoxide (CO). The first human case of HO-1 deficiency showed abnormalities in blood coagulation and the fibrinolytic system. Thus, HO-1 or HO-1 products, such as CO, might regulate coagulation and the fibrinolytic system. This study examined whether tricarbonyldichlororuthenium (II) dimer (CORM-2), which liberates CO, modulates the expression of tissue factor (TF) and plasminogen activator inhibitor type 1 (PAI-1) in human umbilical vein endothelial cells (HUVECs), and TF expression in peripheral blood mononuclear cells (PBMCs). Additionally, we examined the mechanism by which CO exerts its effects. Materials and Methods: HUVECs were pretreated with 50 μM CORM-2 for 3 hours, and stimulated with tumor necrosis factor-α (TNF-α, 10 ng/ml) for an additional 0-5 hours. PBMCs were pretreated with 50-100 μM CORM-2 for 1hour followed by stimulating with lipopolysaccharid (LPS, 10 ng/ml) for additional 0-9 hours. The mRNA and protein levels were determined by RT-PCR and western blotting, respectively. Results: Pretreatment with CORM-2 significantly inhibited TNF-α-induced TF and PAI-1 up-regulation in HUVECs, and LPS-induced TF expression in PBMCs. CORM-2 inhibited TNF-α-induced activation of p38 MAPK, ERK1/2, JNK, and NF-κB signaling pathways in HUVECs. Conclusions: CORM-2 suppresses TNF-α-induced TF and PAI-1 up-regulation, and MAPKs and NF-κB signaling pathways activation by TNF-α in HUVECs. CORM-2 suppresses LPS-induced TF up-regulation in PBMCs. Therefore, we envision that the antithrombotic activity of CORM-2 might be used as a pharmaceutical agent for the treatment of various inflammatory conditions. © 2012 Elsevier Ltd.Thesis of Keiko Maruyama / 丸山 慶子 博士論文 金沢大学医薬保健学総合研究科（保健学専攻

初年次導入教育における「多職種連携学習（IPE）」の評価：PBL/ ポスターツアーの実践から

　現代医療の高度化と複雑化の中で，多職種協働の教育は必須となっている。本研究は，保健学類5 専攻の初年次生を対象とした，多職種連携学習（Interprofessional Education:IPE）を評価するものである。教材に＜社会問題を焦点化した６ 課題＞を準備し，PBL（Problem/Project-Based Learning）およびポスターツアー（ジグソー法）を行ったが，この方法が目的達成に適したものであったかを，学生の反応から評価する。　分析対象は，授業後に提出された187 名のレポート，成果物（ポスター），事後アンケートである。レポートは内容分析の計量テキスト分析（KH Coder (ver.3.) 使用）を行い，アンケートは記述統計を行った。　レポートの内容分析では，総文数は4,508，総段落数は2,913，抽出語数67,510，異なる語数3,302 であった。抽出された高い出現単語は，「専攻：1,104」「医療：813」「自分：478」「意見：407」「考える：405」等だった。さらに，共起ネットワーク（サブグラフ検出modularity）では，＜自分－他専攻－違う－視点＞がネットとなり，また＜異なる－学生－保健－意義－考える＞と＜職種－理解－連携―思う＞，＜様々－意見－聞く＞がつながっていた。KWIC コンコーダンスでの文脈分析からは，IPE の「交わる」や「つながる」，互いの考えを「取り入れる」，「広がる」などで，その連携や知の交流の意味は多面的にとらえられていた。一方で，PBL/ ポスターツアーに関連する，「討論する」「難しい」「面白い」などの，問いを立て，討議を通して深めていく知的探求の語の出現数は少なかった。　グループワークの成果物であるポスターの評価は，30 グループで，優：10，良：11，可：9 であり，課題の明確化，論旨の一貫性および自分たちの主張の面で不足があり，知識の寄せ集め的ポスターとなっていると評価したグループが3 割あった。　事後アンケートでは，＜課題の発見＞＜レジュメ作成＞＜レジュメの説明＞で，身についたが7 割強に留まり，＜ポスターの発表力＞は６ 割だった。　全体として，IPE の理念の理解や態度形成は達成できた。一方PBL/ ポスターツアーの社会的課題を明確にしてプロジェクトとして課題解決をはかっていく，は課題が残り，授業設計の改善が示唆された

ポスターツアー，ワールドカフェによる高大接続プログラム「将来の医療と医療人について考える」：定量テキスト分析による教育実践の効果検証

The purpose of this study was to summarize a program that connects high schools and universities (involving a “Poster Tour” and “World Café”), which was carried out through the cooperation of five health science departments in the School of Health Sciences and to discuss whether it was a success with regard to achieving its objectives, based on responses from participating university and high school students. 　About ２00 first-year students in five departments and ３１ high school students participated in the program. The first-year students prepared posters in advance that summarized their opinions related to social problems in the medical field based on the methodology of problem/project-based learning. They gave their presentations in the “Poster Tour” and attended the “World Café” with the high school students. We analyzed the contents of the statements, which were written on 60 pieces of round paper put on round cardboard desks during the World Café session, as well as the results of ex-post questionnaires from the participants. We performed quantitative text analysis of the contents of description using KH Coder ver. ３ and totalized the results of the ex-post questionnaires. 　The quantitative text analysis involved a total of ２３,２７8 extracted words and １,98７ types of words. Some of the extracted high-frequency words were artificial intelligence (AI) (４１２ times), medicine (iryo, ２４１ times) and technology (gijutsu, 89 times). Some extracted words such as hungry (onakasuita, ２9 times) suggest that the participants were able to discuss frankly and freely with others. 80%, 50%, and ７0% of the high school students rated the atmosphere of the sessions as “excellent”, and stated that they were able to give their opinions “very well”, and that they were able to learn “very much”, respectively. There was a tendency for the participants to discuss a lot about “medical care provided by AI”, “AI changes job requirements”, and “induced pluripotent stem (iPS) cells and robots”, topics that have been prominent in the media. 　Based on these results, the program connecting high schools and universities seemed to be effective as an opportunity for conversations among students in five departments and between university and high school students. However, it did not lead them to deepen their conversations by analyzing future medical care multilaterally and thinking of the issues as concerns of their own

The effect of direct oral anticoagulants on blood protein C activity

[Aim] In this study, the effect of direct oral anticoagulants (DOACs) on protein C (PC) activity was examined using several measuring reagents. [Materials and Methods] In total, 90 patients (60 male and ３0 female) with nonvalvular atrial fibrillation or venous thromboembolism (VTE) who were on anticoagulation therapy with DOACs (rivaroxaban, apixaban, or edoxaban) were studied. The plasma levels of PC activity were measured by means of a clotting assay and chromogenic substrate assay, using three reagents for each type of assay. [Result] Prothrombin time (PT) and activated partial thromboplastin time (APTT) were significantly prolonged in a dose-dependent manner in patients who were taking rivaroxaban or edoxaban. PC activity, as measured by all three reagents using the clotting assay, was influenced only by rivaroxaban, indicating an increase in PC activity in a dose-dependent manner. Apixaban did not have any influence on the measurements made using all three reagents in the clotting assay. On the other hand, none of the three FXa inhibitors had any influence on PC activity when it was measured using the three reagents in the chromogenic substrate assay. Plasma samples were collected before, as well as two and four to eight weeks after rivaroxaban administration in seven patients with AF or VTE sequentially. In all three regents using the clotting assay, plasma levels of PC activity had increased after the administration of rivaroxaban. On the other hand, all three regents using the chromogenic assay had very little influence on PC activity after the administration of rivaroxaban. [Conclusion] The inhibitory effects of the different types of DOACs on clotting activity interfere with clotting test measurement systems in patients receiving DOAC therapy. When measuring PC activity while the patient is taking rivaroxaban or edoxaban, it is necessary to use the chromogenic substrate assay to avoid false highs. Moreover, collecting specimens when blood levels of drugs are low, e.g. during the trough phase, whenever possible, would be one way to minimize interference