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<p>Skeletal muscle is dependent upon its microvasculature to deliver oxygen and substrates to support the metabolic demands of muscle contraction. Skeletal muscle capillary density is determined by a variety of factors including muscle fiber metabolic phenotype and mitochondrial volume as well as prior exercise training status. Additionally, muscle microvascular density and function can diminish with age, contributing to several age-related muscle dysfunctions. Skeletal muscle fibers regulate their surrounding microvasculature through the release of angiogenic and angiostatic signaling factors. A robust increase in angiogenic signaling from skeletal muscle facilitates increases in muscle capillarization following endurance exercise. Extracellular vesicles (EV) are membrane bound signaling factors secreted by every cell type. Skeletal muscle-derived EVs (SkM-EVs) may help facilitate numerous signaling functions of skeletal muscle including between skeletal muscle and its microvasculature.</p> <p>The primary aim of my dissertation research was to determine the signaling roles that SkM-EVs in regulating endothelial cell homeostasis and angiogenesis in states of aging and health. Chapter 1 provides an overview of the relevant literature. Chapter 2 represents an investigation into how age-related cellular senescence impacts the angiogenic potential of skeletal muscle progenitor cells. We found that stress-induced senescence increases release of small EVs and has pro-senescent and angiostatic effects on culture endothelial cells. In Chapter 3 we compared the release, contents, and angiogenic potential of SkM-EVs collected from primarily oxidative or primarily glycolytic skeletal muscle tissue in mice. We found that oxidative muscle tissue secretes more EVs than glycolytic muscle tissue, and the miR contents of EVs differ greatly between the two phenotypes. Additionally, EVs from oxidative tissue enhanced endothelial cell migration and tube formation compared to glycolytic tissue EVs, in a potentially nitric oxide mediated fashion. In Chapter 4, we tested how PGC-1α overexpression effected myotube EV release and angiogenic potential. We found that PGC-1α overexpression did not impact myotube EV release, but increased the angiogenic signaling potential of SkM-EVs. Chapter 5 is a brief summary of the results and limitations of the projects presented in Chapters 2-4, with a short discussion of potential future research directions.</p>

Almost half of patients recovering from open chest surgery experience atrial fibrillation (AF) that results principally from inflammation in the pericardial space surrounding the heart. Given that post-operative AF is associated with increased mortality, effective measures to prevent AF after open-chest surgery are highly desirable. In this study, we tested the concept that extracellular vesicles (EVs) isolated from human atrial explant-derived cells can prevent post-operative AF. Middle-aged female and male rats were randomized to undergo sham operation or induction of sterile pericarditis followed by trans-epicardial injection of human EVs or vehicle into the atrial tissue. Pericarditis increased the probability of inducing AF while EV treatment abrogated this effect in a sex independent manner. EV treatment reduced infiltration of inflammatory cells and production of pro-inflammatory cytokines. Atrial fibrosis and hypertrophy seen after pericarditis was markedly attenuated by EV pre-treatment; an effect attributable to suppression of fibroblast proliferation by EVs. Our study demonstrates that injection of extracellular vesicles at the time of open-chest surgery shows prominent anti-inflammatory effects and prevents AF due to sterile pericarditis. Translation of this finding to patients might provide an effective new strategy to prevent post-operative AF by reducing atrial inflammation and fibrosis.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13561号 / 論医博第2290号 / 新制||医||1067(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 武藤 学, 教授 今中 雄一, 教授 村川 泰裕 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24798号 / 医博第4990号 / 新制||医||1066(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 萩原 正敏, 教授 妹尾 浩, 教授 竹内 理 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Arsenic is a toxic metalloid that continues to contaminate the water and food sources of millions of people globally. Among the numerous health effects of arsenic exposure are urothelial toxicity and cancer. In recent years, small extracellular vesicles (SEVs) have been shown to be vital in intracellular communication and have been used in clinical studies as biomarkers of disease. The overall goal of this thesis is to understand the mechanisms of cell communication during arsenic exposure and to develop minimally invasive biomarkers for the toxic responses. The specific objectives are to: a) determine if SEVs released from arsenic exposed urothelial cells are responsible for mediating urothelial toxicity; and b) assess the application of urinary SEVs as novel biomarkers of arsenic exposure in an exposed population. The hypothesis leading this research is that the biology and protein packaging profile of urothelial SEVs are altered following arsenic exposure because of the induction of cell stress signaling pathways. I also hypothesize that urinary SEV proteins can be used as biomarkers of arsenic exposure because they are positively correlated with urinary arsenic concentrations in an exposed population. SVHUC1 human urothelial cells were dosed with sodium meta arsenite (1, 2, and 5 uM) for 48 hours. T24 urothelial carcinoma cells were also grown in parallel to compare for carcinogenicity. A label-free quantitative proteomics approach was used to assess the differentially expressed proteins in the cell lysate and the SEVs extracted from the culture media to determine the mechanistic pathways involved and how well the protein profiles in SEVs correlate with those in the cell lysate. SEVs were isolated from the archived urine samples of participants (n=36) enrolled in the Yellow Knife Health Effects Monitoring Program (YKHEMP) and two potential biomarkers, transforming growth factor beta receptor 1 (TGFBR1) and ribonuclease inhibitor 1 (RNH1), were measured by an enzyme linked immunosorbent assay (ELISA). SEVs in all samples were successfully characterized based on their size (50-200 nm) and positive antibody array for eight protein markers indicating their endosomal biogenesis. The total number of SEVs was not shown to increase following arsenic exposure in the in vitro study. However, the cancerous T24 cells had nearly four times higher numbers of SEVS compared to the non-cancerous SVHUC1 cells. The changes in the protein profiles in SEVs released following arsenic dosage indicated activation of pathways important for cell survival, viability, and migration and inactivation of pathways related to cell death and necrosis which were also observed in the paired cell lysate samples. Comparison between paired SEV and cell lysate samples, however, indicated selective SEV packaging of proteins which may be for the purpose of intracellular communication. Comparative assessment of SEVs from T24 and arsenic exposed SVHUC1 cells showed similar activation of cancer related pathways including those responsible for malignant tumors and increased proliferation rates. From the in vitro study results, we identified 8 potential SEV biomarkers. Of which, TGFBR1 showed the most promising association, having been positively associated with both inorganic arsenic and cadmium concentrations in urine samples. This thesis showed that SEVs are important mediators of arsenic exposure in urothelial cells and highlighted the comparability of SEV and cell lysate analysis. Furthermore, TGFBR1 was identified as a promising biomarker of arsenic exposure for its positive association with increased arsenic both in vitro and in human biomonitoring analysis.

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.

Indiana University-Purdue University Indianapolis (IUPUI) / The proliferation and differentiation of osteoblasts are influenced by mechanical and geometrical growth environments. A specific aim of my thesis was the elucidation of signaling pathways involved in mechanical stimulation and geometric alterations of the extracellular matrix (ECM). A pair of questions addressed herein was (a) Does mechanical stimulation modulate translational regulation through the phosphorylation of eukaryotic initiation factor 2 (eIF2)? (b) Do geometric alterations affect the phosphorylation patterns of mitogen-activated protein kinase (MAPK) signaling? My hypothesis was mechanical stress enhances the proliferation and survival of osteoblasts through the reduction in phosphorylation of eIF2, while 3-dimensional (3D) ECM stimulates differentiation of osteoblasts through the elevation of phosphorylation of p38 MAPK. First, mechanical stimulation reduced the phosphorylation of eIF2. Furthermore, flow pre-treatment reduced thapsigargin-induced cell mortality through suppression of phosphorylation of protein kinase RNA-like ER kinase (Perk). However, H2O2-driven cell mortality, which is not mediated by Perk, was not suppressed by mechanical stimulation. Second, in the ECM geometry study, the expression of the active (phosphorylated) form of p130Cas, focal adhesion kinase (FAK) and extracellular signal-regulated protein kinase (ERK) was reduced in cells grown in the 3D matrix. Conversely, phosphorylation of p38 MAPK was elevated in the 3D matrix and its up-regulation was linked to an increase in mRNA levels of dentin matrix protein 1 and bone sialoprotein. In summary, our observations suggest the pro-survival role of mechanical stimulation and the modulation of osteoblastic fates by ECM geometry.