Cortical injury, such as that following stroke, is one of the leading causes of long-term disabilities world-wide. While some neuroprotective agents given within hours of stroke can reduce damage, there are currently no neurorestorative therapeutics that can enhance long-term recovery. To address this, we tested Mesenchymal Stem Cell (MSC) derived Extracellular Vesicles (EVs) as a treatment for cortical injury in rhesus monkeys (Macaca mulatta). Monkeys treated with EVs 24 hours after injury and again at 14 days after injury recovered more completely and more rapidly than monkeys given a vehicle control. However, the cellular changes associated with enhanced recovery remained unknown. In this dissertation, it was hypothesized that EVs modulated cells within the brain to enhance recovery after cortical injury. To explore this hypothesis, three specific aims were tested. Aim 1: To determine the effects of EVs on microglial reactivity. Since EVs in this study were derived from MSCs, it was hypothesized that they would have an immunomodulatory effect. Using immunohistochemistry, image analyses, and 3-D reconstruction, we showed that microglia shifted from reactive, damaging phenotypes towards homeostatic, surveilling functions in EV-treated monkeys. These effects correlated with reduced time to recovery, suggesting that reduced microglial reactivity enhanced recovery. Aim 2: To assess the effects of EVs on myelination. Because MSCs have regenerative effects, it was hypothesized that these MSC-derived EVs would improve neurorestoration. Using immunohistochemistry, qRT-PCR, Spectral Confocal Reflectance microscopy, and ELISA, we assessed myelination after cortical injury with and without EV treatment. EVs limited oligodendrocyte damage and increased densities of mature oligodendrocytes to enhance myelin maintenance. These effects correlated with improved recovery, suggesting the importance of myelination in recovery after cortical injury. Aim 3: To assess the neuroprotective role of EVs on infarct volumes. While it was hypothesized that EVs would reduce the densities of inflammatory cells (astrocytes, macrophages/microglia, T-cells), hemosiderin accumulation, and infarct volume, we found that EVs did not alter these endpoints. Collectively, our results suggest that EVs modulated microglia and oligodendrocytes to promote neurorestoration. Overall, these findings demonstrate the therapeutic potential of EVs for neurorestoration after cortical injury.

No description available.

No description available.

Alzheimer’s disease is characterized by the formation of two major pathological hallmarks: amyloid plaques and neurofibrillary tangles. Although there have been many studies to understand the role of microglia in Alzheimer’s disease, it is not yet known how microglia can promote disease progression while actively phagocytosing amyloid plaques or phosphorylated tau (p-tau). Through stereotaxic injection of adeno-associated virus expressing mutant P301L tau (AAV-P301L-tau) into the medial entorhinal cortex (MEC) of both wild-type (WT) and APPNL-G-F mice, we demonstrate how amyloid plaques exacerbate p-tau propagation to the granule cell layer (GCL) of the hippocampus. However, in mice receiving the colony-stimulating factor 1 receptor inhibitor (PLX5622), ~95% of microglia were depleted, which dramatically reduced p-tau propagation to the GCL. Although microglia depletion curtailed p-tau propagation, it also led to reduced plaque compaction and an increase in overall amyloid-beta (Aβ) plaque presence. Additionally, we found microglia depletion resulted in greater p-tau aggregation in dystrophic neurites surrounding amyloid plaques. We investigated neurodegenerative microglia (MGnD), which are activated in response to amyloid plaques, for their propensity to release extracellular vesicles in comparison to homeostatic microglia. We discovered that MGnD, identified by Clec7a or Mac2 staining, strongly express Tumor susceptibility gene 101 (Tsg101), which is an ESCRT-1 protein and a marker for extracellular vesicles (EVs). To further investigate EV release and MGnD, a novel lentivirus expressing fluorescent mEmerald conjugated to CD9 (mE-CD9) was constructed and injected into the MEC of both WT and APPNL-G-F mice which allowed for visualization of mE-CD9+ puncta around individual microglia. CD9 is a tetraspanin and also a marker for EVs. We observed that the number of mEmerald+ particles surrounding MGnD was three-fold higher compared to non-diseased, homeostatic microglia. Sequential injection of mE-CD9 and AAV-P301L-tau into the MEC revealed that microglia-derived EVs encapsulate pathologic p-tau, which is augmented by the MGnD phenotype. Taken together, these data provide strong evidence that MGnD exhibit increased secretion of tau-containing EVs, providing a possible mechanism for how amyloid deposition indirectly exacerbates tau propagation.

The thesis introduces two developed methods to quantify extracellular vesicles (EVs) isolated from cancer cell lines and healthy human saliva by using capillary zone electrophoresis. In the first chapter, the importance of EVs, as well as the existing EV isolation, characterization, and quantification methods, are described. The general principle of capillary electrophoresis (CE) is explained for a better understanding of these two methods. Chapter II describes the idea and concepts of Extracellular Vesicles quantitative Capillary Electrophoresis (EVqCE). The method evolved from the previous study carried out in our research group for the quantification of viruses. After the isolation of EVs from different cell lines, the characterization and quantification of EVs were performed using nanoparticle tracking analysis (NTA) and flow cytometry. EVqCE consists of four steps for EV quantification. In this study, EVqCE was employed to know the concentrations of EVs in unknown samples, followed by calculation of the average mass of the RNA present in EVs. In the next chapter, one of the human body fluids, i.e., saliva, was chosen for the quantification of EVs. Salivary EVqCE was developed in a similar way as EVqCE for cell lines since the general theories and procedures are practically the same. However, human saliva contains an abundant amount of viscous proteins and ribonuclease (RNase), that were the major obstacles for salivary EVs detection and quantification. The method for the isolation of EVs from the saliva was optimized, and the quantification was performed successfully. The average mass of RNA in saliva EVs was also calculated and analyzed. The concentration of saliva EVs in unknown samples were compared with the results from NTA and flow cytometry to validate the salivary EVqCE. In the last chapter, I described the application of EVqCE to study the quality control of EVs. The method calculates the degradation level of EVs samples under different conditions, providing a potential way for real-time monitoring of the EVs status in the body fluid sample.

abstract: Extracellular Vesicles (EVs), particularly exosomes, are of considerable interest as tumor biomarkers since tumor-derived EVs contain a broad array of information about tumor pathophysiology including its metabolic and metastatic status. However, current EV based assays cannot distinguish between EV biomarker changes by altered secretion of EVs during diseased conditions like cancer, inflammation, etc. that express a constant level of a given biomarker, stable secretion of EVs with altered biomarker expression, or a combination of these two factors. This issue was addressed by developing a nanoparticle and dye-based fluorescent immunoassay that can distinguish among these possibilities by normalizing EV biomarker level(s) to EV abundance, revealing average expression levels of EV biomarker under observation. In this approach, EVs are captured from complex samples (e.g. serum), stained with a lipophilic dye and hybridized with antibody-conjugated quantum dot probes for specific EV surface biomarkers. EV dye signal is used to quantify EV abundance and normalize EV surface biomarker expression levels. EVs from malignant (PANC-1) and nonmalignant pancreatic cell lines (HPNE) exhibited similar staining, and probe-to-dye ratios did not change with EV abundance, allowing direct analysis of normalized EV biomarker expression without a separate EV quantification step. This EV biomarker normalization approach markedly improved the ability of serum levels of two pancreatic cancer biomarkers, EV EpCAM, and EV EphA2, to discriminate pancreatic cancer patients from nonmalignant control subjects. The streamlined workflow and robust results of this assay are suitable for rapid translation to clinical applications and its flexible design permits it to be rapidly adapted to quantitate other EV biomarkers by the simple swapping of the antibody-conjugated quantum dot probes for those that recognize a different disease-specific EV biomarker utilizing a workflow that is suitable for rapid clinical translation. / Dissertation/Thesis / Doctoral Dissertation Biomedical Engineering 2019

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23411号 / 医博第4756号 / 新制||医||1052(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 妹尾 浩, 教授 藤田 恭之, 教授 山下 潤 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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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.