京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21003号 / 医博第4349号 / 新制||医||1028(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 木村 剛, 教授 小西 靖彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21009号 / 医博第4355号 / 新制||医||1028(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 羽賀 博典, 教授 前川 平, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Chapter 1: Molecular Mechanism of Mitochondrial Fragmentation and Glucose Metabolism under Disturbed Flow in Endothelial Cells: Focus on the Role of Dynamin-Related Protein 1. The luminal surface of the endothelium is continually exposed to dynamic blood flow patterns that is known to alter immunometabolic phenotypes of the endothelial cells (ECs). Recent literature reported that inhibition of the metabolic reprogramming to glycolysis or enhancement of oxidative phosphorylation (OXPHOS) is considered as an effective strategy to prevent EC proinflammatory activation and eventually the progression of vascular diseases. Endothelial mitochondria are highly dynamic organelles playing versatile roles in maintaining endothelial cell homeostasis working as bioenergetic, biosynthetic, and signaling organelles. The balance between fusion and fission processes modulates mitochondrial network, which is essential for maintaining mitochondrial homeostasis. Disruption of the orchestrated balance, especially toward excessive fission resulting in fragmented and dysfunctional mitochondria, has been shown to be associated with atheroprone phenotypes of ECs. However, there is a key knowledge gap with respect to morphology of EC mitochondria under different flow conditions and its role on EC immunometabolic phenotypes.In chapter 1, the purpose of this study was to investigate the effect of different flow patterns on mitochondrial morphology in ECs and its implication in immunometabolic endothelial phenotype. The overarching hypothesis of the Chapter 1 was that disturbed flow (DF) will increase mitochondrial fragmentation, which will facilitate glycolysis and inflammatory activation in ECs. In the study, mitochondrial morphology was analyzed in ECs at multiple segments of the aorta and arteries in EC-specific photo-activatable mitochondria (EC-PhAM) mice. Increased mitochondrial fragmentation was observed at atheroprone regions (e.g., lesser curvature of the aortic arch, LC) with increased dynamin-related protein 1 (Drp1) activity, compared with the atheroprotective regions (e.g., thoracic aorta, TA). The atheroprone regions also showed a higher level of endothelial activation and glycolysis. Carotid artery partial ligation surgery, as a surgical model of DF, significantly induced mitochondrial fragmentation with elevated Drp1 activity and increased EC activation. in vitro experiments recapitulated in vivo observations. Inhibition of Drp1 activity by mdivi-1 attenuated the DF-induced atheroprone EC phenotypes, showing the close relationship between mitochondrial morphology and atheroprone phenotypes of ECs. As for the molecular mechanism, hypoxia-inducible factor 1 α (HIF-1α) stabilization and its nuclear translocation was significantly increased under DF, which was attenuated by mdivi-1 treatment. Mitochondrial reactive oxygen species (mtROS) and succinate, which are known to reduce prolyl hydroxylase domain 2 (PHD2) activity thereby increasing HIF-1α stabilization, were significantly elevated under DF, but those were attenuated by mdivi-1 treatment. Finally, a 7-week voluntary wheel-running exercise training significantly decreased mitochondrial fragmentation with a down-regulation of VCAM-1 expression at the LC. In conclusion, our data suggest that DF induces mitochondrial fragmentation with increased Drp1 activity, which is associated with an atheroprone EC phenotype. In addition, regular practice of aerobic exercise reduces mitochondrial fragmentation and prevents ECs from an atheroprone endothelial phenotype at the atheroprone regions. Chapter 2: Molecular Mechanisms for Unidirectional Flow (UF)/Exercise-Induced improvement of Mitochondrial Integrity: Focus on phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) /PARKIN-Dependent Mitochondrial Autophagy (Mitophagy) Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) is an essential molecule in the mitophagy process and known to act as a cytoprotective protein involved in several cellular mechanisms in mammalian cells. It has been documented that the loss of PINK1 expression in mice and various cell types enhance susceptibility to stress-induced cell damage, while the overexpression of PINK1 significantly attenuates stress-induced mitochondrial and cellular dysfunction.In chapter 2, the purpose of this study was to investigate PINK1 expression and its subcellular localization under an exercise-mimic laminar shear stress (LSS) condition in human primary endothelial cells and in exercizing mice, and its implications on endothelial homeostasis and cardiovascular disease (CVD) prevention. The overarching hypothesis of the Chapter 2 was that unidirectional flow (UF) will increase cytosolic PINK1 expression through which UF-preconditioned ECs will be more protective against an accumulation of dysfunctional mitochondria via a greater mitophagy induction. In this study, we measured the full-length PINK1 (FL-PINK1) mRNA and protein expression levels in ECs under unidirectional laminar shear stress (LSS). LSS significantly elevated both FL-PINK1 mRNA and protein expressions in ECs. Mitochondrial fractionation assays showed a decrease in FL-PINK1 accumulation in the mitochondria with an increase in the cytosolic FL-PINK1 level under LSS. Confocal microscopic analysis confirmed these subcellular localization patterns suggesting downregulation of mitophagy induction. Indeed, mitophagy flux was decreased under LSS, determined by a mtKeima probe. Mitochondrial morphometric analysis and mitochondrial membrane potential determined by tetraethylbenzimidazolylcarbocyanine iodide (JC-1) showed mitochondrial elongation and increased mitochondrial membrane potential under LSS respectively, suggesting that an elevation of cytosolic PINK1 is not related to an immediate induction of mitophagy. However, increased cytosolic PINK1 elevated mitophagic sensitivity toward dysfunctional mitochondria in pathological conditions. Preconditioned ECs with LSS showed lower mtDNA lesions under angiotensin II stimulation. Moreover, LSS-preconditioned ECs showed rapid Parkin recruitment and mitophagy induction in response to mitochondrial toxin (i.e., carbonyl cyanide chlorophenylhydrazone, CCCP) treatment compared to the control. We measured PINK1 expression at ECs of the thoracic aorta in exercised mice, a physiological LSS-enhanced model, which was significantly elevated compared to sedentary animals. In addition, exercise-preconditioned mice were more protective to angiotensin II-induced mtDNA lesion formation in the mouse abdominal aorta than sedentary mice, suggesting a potential protective mechanism of exercise in a PINK1-dependent manner. In conclusion, LSS increases a cytosolic pool of FL-PINK1, which may elevate the mitophagic sensitivity toward dysfunctional mitochondria or activate other cytoprotective mechanisms in ECs. Our data suggest that exercise may support mitochondrial homeostasis in vascular ECs by enhancing PINK1-dependent cell protection mechanisms. / Kinesiology

>Magister Scientiae - MSc / Cancer is the most common leading cause of death worldwide. Glioblastoma and breast cancer are the most aggressive solid tumour. The survival rate of these tumours depends on their ability to progress and spread. These cancers use their high proliferative capabilities for survival, increasing their malignancies. Glioblastoma is considered the most aggressive tumour initiated in the brain, whereas breast cancer is the most common metastatic cancer in the brain, both types of cancer are known as high infiltrated cancer and their invasiveness due to their capability to release factors that can alter the neighbouring cells to facilitate their progression.

The vasculature is a highly intricate system of "highways" that shuttles blood from the heart to every tissue and organ in the human body. These vessels are responsible for carrying oxygen, trafficking hormones, delivering nutrients, and removing waste products from the body. The formation of a functioning vascular system depends on the close coordination of many cell types and, on the capillary level, specifically endothelial cells and pericytes as well as the surrounding protein microenvironment, known as the extracellular matrix (ECM). Impaired coordination amongst the cellular and protein constituents results in the improper functioning of the vascular network and can eventually contribute to the failure of organ systems. This dissertation research focuses on how improper ECM deposition affects vascular assembly. We utilized several approaches to affect ECM composition, specifically: 1) hypoxia exposure and 2) reducing ECM pharmacologically and utilizing lentiviral-mediated silencing of Type IV Collagen (Col-IV, gene Col4a1) expression. In these experimental settings, we observed downstream changes in the coordination between endothelial cells and pericytes while forming vascular networks. In short, this dissertation work suggests that excess ECM deposition, and particularly that of Col-IV, has unique deleterious effects on the developing vasculature as compared to reduced ECM deposition. The findings from this work suggest mechanisms underlying how the vasculature may be destabilized in hypoxia-associated pathologies, such as preeclampsia. / Doctor of Philosophy / Every tissue and organ in the human body receives blood from the heart via the extremely complex network of "highways" known as the vasculature. These vessels oversee moving nutrients, oxygen, hormones, and waste materials out of the body. At the capillary level, endothelial cells and pericytes, as well as the surrounding protein milieu known as the extracellular matrix (ECM), are required for the development of a functional vascular system. If the vascular network fails to develop and operate properly because of poor protein and cellular coordination, it can eventually lead to the failure of organ systems. The study for this dissertation focuses on how vascular development is impacted by insufficient ECM deposition. We used several strategies to modify the composition of the ECM, including 1) hypoxia exposure, 2) pharmaceutical ECM reduction, and 3) lentiviral-mediated delivery of shRNA to silence Type IV Collagen (Col-IV, gene Col4a1) production. We noticed alterations in the coordination between endothelial cells and pericytes as vascular networks were being formed in these experimental environments. In summary, this dissertation work contends that, in contrast to reduced ECM deposition, excess ECM deposition, and specifically that of Col-IV, has distinct detrimental consequences on the developing vasculature. The results of this study offer methods by which diseases associated with hypoxia, such preeclampsia, may cause the vasculature to become unstable.

A newly discovered activity in human serum protects porcine aortic endothelial cells in culture from injury by very low density lipoproteins (VLDL). This factor, toxicity-preventing activity (TxPA), was measured in 29 relatively young men (43 ± 8 years) who had undergone coronary angiography. The level of TxPA was found to be significantly reduced (P < 0.001) in men who demonstrated more than 15% narrowing of their coronary arteries. Men (n = 18) who had 15% or less narrowing were found to have 104 ± 48 units of TxPA while men (n = 11) with coronary artery disease had 48 ± 24 units of TxPA. A value derived from the product of TxPA and the high density lipoprotein cholesterol (HDL-C) level divided by the non-HDL-C (total cholesterol-HDL-C) accurately separated 97% of the men into 2 groups. TxPA thus appears to be a new protective factor in coronary artery disease, which, when combined with total cholesterol and high density lipoprotein cholesterol values, provides an accurate classification of established coronary artery disease in these subjects.

Heat therapy may be an alternative or adjunct intervention to exercise training for improving cardiovascular function and health. However, its prescription must be refined in order to overcome the feasibility and tolerability issues associated with current whole-body heating modes. There is substantial evidence to support the beneficial effects of high doses (e.g., frequency, duration, and intensity) of heating typically achieved using whole-body modes, but there is limited knowledge on whether lower doses of heating administered through local hot water immersion of the limbs can still have an impact on vascular function. All studies were conducted in heathy young men and women. In the first study, we found that regardless of whether local heating was applied to the lower limbs up to the ankles or knees, upper limb endothelial function and lower limb arterial stiffness improved acutely. In the second study, we proceeded to prescribe ankle-level heating in a chronic intervention and compared its effects to that of moderate-intensity cycling exercise training. We observed no changes in endothelial function, but decreases in central arterial stiffness and increases in cardiorespiratory fitness in those who performed heat therapy and exercise training combined with heat therapy. In the third study, we evaluated the ability of acute vascular function responses to predict chronic vascular function responses with heating and exercise interventions, and found significant positive associations between the acute and chronic responses for absolute and relative brachial artery flow-mediated dilation and femoral-foot pulse wave velocity. These findings suggest that, in healthy young men and women, local heating through ankle-level hot water immersion can improve indices of cardiovascular function both acutely and chronically, alone or combined with exercise training. Further, acute responses may be used to determine an individual’s chronic responsiveness to a heat therapy and/or exercise training intervention. More research in larger, more diverse samples and with a longer duration of therapy and/or training should be conducted to determine if the results are replicable. / Dissertation / Doctor of Philosophy (PhD) / Regular participation in whole-body heat therapy can extend health and life span, but it is used infrequently because of a lack of feasibility from a cost, accessibility, and tolerability standpoint. This thesis explored whether local heat therapy in young healthy men and women would be effective for improving blood vessel health defined as endothelial function and arterial stiffness, both of which are linked to the risk of developing many chronic diseases. Furthermore, the effects of local heat therapy were compared to that of exercise training. We found that there were beneficial short- and long-term effects of lower limb hot water immersion that manifested in different areas of the body. Local heat therapy improved upper limb endothelial function and lower limb arterial stiffness immediately after a session, whereas with repeated exposure, it may have improved central arterial stiffness and cardiorespiratory fitness. Exercise training only had beneficial effects on the blood vessels when combined with heat therapy. Finally, short-term vascular responses can predict long-term vascular responses to both heat therapy and/or exercise training. Overall, our findings suggest that there may be some utility for local heat therapy to promote healthy blood vessels, but more work must be done to replicate our findings and explore its effects on other populations.

Damage to the endothelium is an important contributor to the initiation and progression of atherosclerosis. GRP78 is an endoplasmic reticulum (ER)-resident molecular chaperone in normal healthy endothelium that functions to assist in the correct folding of newly synthesized proteins and to prevent the aggregation of folding intermediates. In addition, GRP78 is present as a transmembrane protein on the surface of lesion-resident endothelial cells. Surface GRP78 is known to act as a surface signaling receptor in cancer cells and is activated by anti-GRP78 autoantibodies (GRP78a-Abs) isolated from the serum of cancer patients. However, the role of cell surface GRP78 on endothelial cells and the influence of GRP78a-Abs in atherosclerosis is unknown. The objectives of this study were to investigate the effects of GRP78a-Abs on lesion development, examine whether engagement of cell surface GRP78 by GRP78a-Abs modulates endothelial cell function, and determine whether GRP78a-Abs were associated with cardiovascular disease (CVD) in humans. This research showed that ApoE-/- mice with advanced atherosclerotic lesions have elevated serum levels of GRP78a-Abs and ApoE-/- mice immunized against recombinant GRP78 demonstrated a significant increase in GRP78a-Abs titers as well as accelerated lesion growth. Furthermore, this work demonstrated that activation of surface GRP78 on endothelial cells by GRP78a-Abs significantly increases gene expression of adhesion molecules ICAM-1 and VCAM-1 as well as leukocyte adhesion through the NFκB pathway. Additionally, middle-aged to elderly adults at risk for CVD showed a tendency toward elevated circulating GRP78a-Ab levels. Our results suggest that signaling through cell surface GRP78 can activate intracellular pathways that contribute to endothelial cell activation and augment atherosclerotic lesion development. These findings demonstrate a novel role for GRP78a-Abs and surface GRP78 receptor activity in endothelial cell function and the early stages of lesion development, as well as establish an initial framework for future work involving circulating GRP78a-Abs and atherosclerotic disease in humans. Furthermore, this work indicates inhibiting the interaction of GRP78a-Abs with cell surface GRP78 could present a novel therapeutic strategy to modulate lesion growth, thereby reducing the risk for atherosclerosis and cardiovascular disease. / Thesis / Doctor of Philosophy (PhD)