Indiana University-Purdue University Indianapolis (IUPUI) / Spinal cord injury (SCI) is devastating and debilitating, and currently no effective treatments exist. Approximately, 12,000 new cases of SCI occur annually in the United States alone. The central nervous system has very low repair capability after injury, due to the toxic environment in the injured tissue. After spinal cord trauma, ruptured blood vessels cause neighboring cells and tissues to be deprived of oxygen and nutrients, and result in the accumulation of carbon dioxide and waste. New blood vessels form spontaneously after SCI, but then retract as the injured tissue forms a cavity. Thus, the newly formed vasculature likely retracts because it lacks a structural support matrix to extend across the lesion. Currently, in the field of spinal cord injury, combinational treatment approaches appear to hold the greatest therapeutic potential. Therefore, the aim of these studies was to transplant a novel, non-immunogenic, bioengineered hydrogel, into the injured spinal cord to serve as both a structural scaffold (for blood vessels, axons, and astrocytic processes), as well as a functional matrix with a time-controlled release of growth factors (Vascular endothelial growth factor, VEGF; Glial cell line-derived neurotrophic factor, GDNF). The benefit of this hydrogel is that it remains liquid at cooler temperatures, gels to conform to the space surrounding it at body temperature, and was designed to have a similar tensile strength as spinal cord tissue. This is advantageous due to the non-uniformity of lesion cavities following contusive spinal cord injury. Hydrogel alone and combinational treatment groups significantly improved several measures of functional recovery and showed modest histological improvements, yet did not provoke any increased sensitivity to a thermal stimulus. Collectively, these findings suggest that with further investigation, hydrogel along with a combination of growth factors might be a useful therapeutic approach for repairing the injured spinal cord.

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

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第19865号 / 生博第346号 / 新制||生||46(附属図書館) / 32901 / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 渡邊 直樹, 教授 松田 道行, 教授 根岸 学 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

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

No description available.

<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>

Background: Pregnant individuals who participate in physical activity throughout gestation have been shown to experience a wide spectrum of health benefits, along with the fetus. In nonpregnant populations, PA influences the polarization state of tissue resident macrophages, resulting in increased regulatory and decreased inflammatory profiles. The effects of PA on placenta-resident macrophages, or Hofbauer cells (HBCs), remains unknown. My thesis aimed to explore this novel area. Methods: The first objective of my thesis was to identify any associations between gestational PA and HBC polarization. PA was objectively measured in both mid (24-28 weeks) and late (34-38 weeks) pregnancy using accelerometry. Immunofluorescent localization of the panmacrophage marker CD68 and the anti-inflammatory macrophage marker CD206 was used to assess polarization states. Protein and gene expression of CD68 and CD206 were assessed using Western blot and qPCR, respectively. The second objective was to explore the relationships between gestational PA, HBC polarization, and angiogenic factors in the placenta. Western blot measured the relative protein expression of FGF2 and SPRY2, and the localization of FGF2, SPRY2, and VEGF within HBCs was explored using immunofluorescent colocalization in term placenta tissue and primary HBC cultures. Results: While there were no differences in the absolute numbers of total or CD206+ HBCs, the proportion of CD206+ HBCs was elevated in active individuals. There were no significant differences in the gene expression of CD68 or CD206, nor in the gene expression of CD206; however, CD206 protein expression was observed to be lower in active participants. Both CD206+ and CD206- HBCs expressed VEGF. Active individuals had significantly higher low molecular weight-FGF2. There were no differences in the protein expression of SPRY2, total FGF2, or high molecular weight FGF2 based on PA. HBCs both in vitro and in vivo of all polarizations expressed VEGF, SPRY2, and FGF2, and were observed to create intracellular junctions and multi-nucleated giant cells. Conclusions: In conclusion, PA was associated with a higher proportion of CD206+ HBCs and reduced levels of CD206 protein. In combination with the lack of significant difference in CD206 mRNA based on PA levels, this suggests a potential effect mediated by PA on the transcriptional regulation of CD206. HBCs were seen to express SPRY2, VEGF, and FGF2, identifying them as potential players in angiogenesis regulation in the placenta. The elevated levels of low molecular weight FGF2 in active individuals suggests the PA may play a role in the modulation of placental angiogenesis. Future research should continue to explore the relationships between PA, HBC polarization, and angiogenesis.

No description available.

In vitro and animal studies indicate that the response to heat stress is associated with beneficial adaptations that promote cell health and survival. Few studies to date have examined this finding in human subjects, and it is unclear how the adaptation compares in magnitude to exercise training. The purpose of this study was to investigate the skeletal muscle adaptations (namely mitochondrial biogenesis and capillarization) of 6 weeks of deep-muscle heat treatment relative to exercise training. We hypothesized that heat treatment (HT), applied through pulsed shortwave diathermy (2 hr, 3 days/week) over a 6-week intervention period would lead to increased mitochondrial content and capillarity within skeletal muscle, though to a lesser extent than single-leg knee extension exercise training (EX; 40 min, 3 days/week). We randomized 28 sedentary but otherwise healthy, young adults (ages 18–36; n = 13 female, n = 15 male) to receive either HT, EX, or sham heating sessions (CON; 2 hr, 3 days/week) over 6 weeks. Diathermy increased muscle temperature by 3.2 ± 0.33 C (P < 0.0001) within 20 minutes. Muscle biopsies were taken from the vastus lateralis at baseline, after 3 weeks of intervention and again after 6 weeks of intervention. Following 3 and 6 weeks of heat treatment, we did not observe significant changes in mitochondrial biogenesis or capillarization. However, exercise training was sufficient to elicit an increase in individual capillary-to-fiber ratio (P = 0.0003), capillary density (P = 0.0428), and the Capillary to Fiber Perimeter Exchange Index (P = 0.0089). Significant increases in the expression of mitochondrial protein Complexes I (P = 0.0073) and IV (P = 0.0015), were observed in the exercise group, but not the heat or control groups. These results indicate that 6 weeks of localized HT, when applied to young healthy individuals, is insufficient to induce mitochondrial biogenesis or capillarization in skeletal muscle. Additionally, our findings provide support for the extensive body of literature that connect exercise training to beneficial skeletal muscle adaptations.

Major diseases such as cardiovascular diseases, diabetes, obesity and tumor growth are known to involve inflammatory angiogenesis. MCP-induced protein 1 (MCPIP1) encoded by ZC3H12A gene, was reported to promote angiogenesis and is addressed in my dissertation as MCPIP. The mechanism/s involved in the angiogenic differentiation induced by MCPIP was however unknown. The aim of this study was to bridge this gap in our knowledge and delineate the molecular mechanisms and sequential processes involved in angiogenesis mediated via MCPIP. To determine if angiogenesis induced by inflammatory cytokines, TNF-, IL-1 and IL-8 is mediated via induction of MCPIP, knockdown of MCPIP by its specific siRNA, in human umbilical vein endothelial cells was performed. Oxidative stress, ER stress and autophagy are known to be involved in mediating inflammation. We hypothesized that MCPIP-induced angiogenic differentiation is mediated via induction of oxidative stress, ER stress and autophagy. Chemical inhibitors and specific gene knockdown approach were used to inhibit each process postulated. Oxidative stress was inhibited by apocynin or cerium oxide nanoparticles or knockdown of NADPH oxidase subunit, phox47. Endoplasmic reticulum (ER) stress was blocked by tauroursodeoxycholate or knockdown of ER stress signaling protein IRE-1 and autophagy was inhibited by the use of 3methyl adenine, or LY 294002 or by specific knockdown of beclin1. Matrigel assay was used as an in vitro tool to assay angiogenic differentiation. Inhibition of each step inhibited the subsequent steps postulated. The results reveal that angiogenesis induced by inflammatory agents is mediated via sequential induction of MCPIP that causes v oxidative and nitrosative stress resulting in ER stress leading to autophagy required for angiogenesis. MCPIP has deubiquitinase and anti-dicer RNase activities. If and how the dual enzymatic activities of MCPIP mediate angiogenesis was unknown. Our results showed that hypoxia-induced angiogenesis is mediated via MCPIP. MCPIP deubiquitinated ubiquitinated hypoxia-inducible factor (HIF-1) and the stabilized HIF-1 entered the nucleus to promote the transcription of its target genes, cyclooxygenase-2 and vascular endothelial growth factor causing the activation of p38 MAP kinase involved in angiogenesis. MCPIP expression promoted angiogenesis by inhibition of thrombospondin-1 synthesis via induction of silent information regulator (SIRT)-1 and/or via suppression of VEG-inhibitor levels caused by inhibition of NF-B activation. MCPIP inhibited the production of the anti-angiogenic microRNAs (miR)-20b and miR-34a that repress the translation of HIF-1 and SIRT-1, respectively. Cells expressing the RNasedead mutant of MCPIP, D141N, that had lost the ability to induce angiogenesis had deubiquitinase activity but did not inhibit the production of miR-20b and miR-34a. Mimetics of miR-20b and miR-34a inhibited MCPIP-induced angiogenesis. These results show for the first time that both deubiquitinase and anti-dicer RNase activities of MCPIP are involved in inflammatory angiogenesis. Results from our study delineate key processes that could be potential targets for therapeutic intervention against inflammatory angiogenesis.