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

<p>The present thesis examined the acute and chronic (training) hemodynamic responses to the unique exercise stimulus of high-intensity "sprint" interval exercise or training (SIT). Previous research has characterized the muscle metabolic and exercise performance adaptations to both short and medium term SIT, however the cardiovascular adjustments and adaptations have not been examined. As part of this thesis two studies were designed to permit evaluations of the chronic cardiovascular responses to a six-week SIT intervention protocol, while two separate studies examined the acute impact of a sprint exercise session on indices of vascular structure and function. Comparisons were made between the SIT and traditional endurance exercise training (ET) in the two exercise training studies, while comparisons were made between a single sprint and that of multiple sprints in the acute exercise studies. The subject population examined in this research was young healthy participants.</p> <p>Our general hypothesis regarding the training adaptations was that similar changes of artery stiffness, vascular endothelial function, blood flow kinetics and oxygen uptake kinetics would occur following SIT compared to ET. Regarding the acute effects of a sprint exercise, we expected arterial stiffness to decrease in the exercising limbs and increase in the central arteries, similar to the responses observed previously immediately following endurance exercise, while we hypothesized that endothelial function would be decreased immediately following the exercise session because of the intense nature of the exercise. The overarching hypothesis guiding these specific hypothesis is that we believe that individual bouts of exercise impact on the arterial wall through the generation of a shear stimulus related to cyclic increases in blood flow and blood pressure. In the short-term the acute response of the artery depends on the composition of the arterial wall and the local stimulus. Over time, functional and structural adjustments occur to normalize the impact of shear forces.</p> <p>Training adaptations in vascular structure and function to SIT were similar to those observed with ET. Both exercise training methods stimulated improved peripheral artery stiffness and endothelial function. The rate of increase in oxygen uptake (kinetic response) was not improved with either training method. However, estimated myocardial demand was reduced with ET but not SIT, which indicates more favourable adaptation in central hemodynamics with ET.</p> <p>Acute sprint exercise markedly reduced peripheral artery stiffness in the exercised limbs well into recovery (~45 minutes), which may benefit central hemodynamics after exercise completion. Sprint exercise also acutely decreased endothelial function, likely because of high oxidative stress generated during the exercise bout and may provide the ideal stimulus for endothelial adaptation.</p> <p>In summary, this thesis highlights the chronic and acute effects of sprint interval exercise and training in young health individuals. The notion that sprint interval exercise provides equivalent benefits to the cardiovascular system as endurance exercise may be true in the peripheral circulation. However, further study focusing is required before the general acceptance of more favorable central hemodynamic effects from endurance exercise training.</p> / Thesis / Doctor of Philosophy (PhD)

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Yes / Angiography is a minimally invasive diagnostic procedure in endovascular interventions. Training interventional procedures is a big challenge, due to the complexity of the procedures with the changes of measurement and visualization in blood flow rate, volume, and image contrast. In this paper, we present a novel virtual reality-based 3D interactive training platform for angiography procedure training. We propose a multi-fluid flow approach with a novel corresponding non-slip boundary condition to simulate the effect of diffusion between the blood and contrast media. A novel syringe device tool is also designed as an add-on hardware to the 3D software simulation system to model haptics through real physical interactions to enhance the realism of the simulation-based training. Experimental results show that the system can simulate realistic blood flow in complex blood vessel structures. The results are validated by visual comparisons between real angiography images and simulations. By combining the proposed software and hardware, our system is applicable and scalable to many interventional radiology procedures. Finally, we have tested the system with clinicians to assess its efficacy for virtual reality-based medical training. / National Natural Science Foundation of China grant number 61402278, the Shanghai Natural Science Foundation of China grant number 14ZR1415800, Research Program of Shanghai Engineering Research Center of Motion Picture Special Effects grant number 16dz2251300, Shanghai University Film Peak Discipline, and Shanxi Natural Science Technology Foundation grant number 2016JZ026.

Stroke is a leading cause of morbidity and long-term neurological disability in the U.S. Ischemic stroke, which accounts for approximately 90% of all strokes, is the result of an occlusion in the arteriole cerebrovascular network. No effective treatment options exist to provide neuroprotection from occlusion, and limited success has been seen clinically when attempting to restore blood flow to vulnerable neural tissue regions. Enhancement of pial collateral remodeling (Arteriogenesis) has recently been shown to improve blood flow and mitigate neural tissue damage following stroke (1-3). Arteriogenesis is the remodeling of pre-existing arteriole vessel which are able to re-route blood to blood-deprived regions of tissue. Arteriogenesis requires endothelial cell (EC) and smooth muscle cell proliferation, extracellular matrix degradation and recruitment of circulating bone marrow-derived cells (4-6). Unlike spouting angiogenesis, which requires weeks following occlusion to develop, arteriogenesis begins as early as 24-48hrs post-stroke (7, 8) and can expeditiously enhance blood flow to ischemic regions, making it an attractive target for therapeutic intervention. Our preliminary studies, in an EphA4 global knockout mouse model, indicated that EphA4 receptor tyrosine kinase severely limits pial arteriole collateral formation. The preliminary work also showed that activation of EC EphA4 receptor in vitro inhibited vascular formation. Additionally, ECs lining the collateral vessel have been shown to play a role in collateral remodeling (9). Taken together, the objective of this dissertation was to elucidate the cell autonomous role of the EphA4 receptor and given the central role of the EC in collateral remodeling, we postulated that EphA4 receptor on ECs the limits pial collateral formations. Using a cell-specific loss-of-function approach, we tested the hypothesis that EC-specific EphA4 plays an important role in pial collateral development and remodeling after induced stroke. The results from this dissertation show that (1) EphA4 expression on ECs suppress the formation of pial collaterals during development and limits EC growth via suppression of p-Akt in vitro (2) EC-specific EphA4 ablation leads to increased collateral remodeling, enhanced blood flow recovery, tissue protection and improved neurological behavioral outcomes after stroke and (3) Mechanistically, EphA4 limits pial collateral remodeling via attenuation of the Tie2/Angiopoietin-2 signaling pathway. The work presented in this dissertation demonstrate that EphA4 can be targeted therapeutically to increase pial collateral remodeling to alleviate neurological deficits after ischemic stroke. / Doctor of Philosophy / Stroke is the fifth leading cause of death in the United States. Ischemic stroke is the most common type of stroke and occurs when blood flow to part of the brain is impeded. Lack of blood results in cell death and tissue damage in the brain. In an effort to restore blood flow, specialized blood vessels in the brain called collaterals remodel and become larger to allow re-routed blood to the blood-deprived region of the brain. The duration it takes to remodel these remarkable blood vessels and re-route blood varies in humans, and sometimes is not able to prevent adequate tissue damage. The current work explores novel therapeutic targets to accelerate collateral remodeling in an effort to reduce tissue loss after stroke. We present studies which show that a protein called EphA4, found on endothelial cells restricts remodeling, and when inhibited in the brain can increase collateral remodeling and reduced adverse effects after ischemic stroke.