Indiana University-Purdue University Indianapolis (IUPUI) / Asthma, a chronic inflammatory disease of the airways, affects nearly 25 million Americans. The vast majority of these patients suffer from exercise-induced bronchoconstriction (EIB), a complication of asthma. Although traditionally treated pharmacologically, nutritional strategies provide a promising alternative for managing EIB as the prevalence of asthma may be due in part to changes in diet. Our objective was to determine the effects of novel nutritional strategies on hyperpnea-induced bronchoconstriction (HIB) in asthmatic individuals. HIB uses rapid breathing to identify EIB in a research or clinical setting. Fish oil, a combination of the omega-3 fatty acids eicosapentaenoic acid (EPA) and docsahexaenoic acid (DHA), has been shown to be effective in suppressing EIB. However, its use in combination with other nutritional supplements, the optimal fish oil formula, and its effect on smooth muscle contractility have not been fully explored. An in vivo study (study 1) was conducted in individuals with both asthma and HIB to determine whether a combination of fish oil and vitamin C was more effective than either one alone in alleviating HIB. Pulmonary function was significantly improved with both fish oil and the combination treatment but not with vitamin C alone. In study 2, individuals with both asthma and HIB were supplemented with DHA alone since the optimal formula for fish oil has yet to be ascertained; previous in vitro studies have suggested DHA may be the more potent omega-3 fatty acid in fish oil. However, no significant changes in pulmonary function or airway inflammation were seen with DHA supplementation. For study 3, canine airway smooth muscle tissue was treated with fish oil to determine the in vitro effect of fish oil on smooth muscle contractility. Acute treatment with fish oil relaxed smooth muscle strips that had been contracted with 5-hydroxytryptamine. These minor relaxations in smooth muscle tension with fish oil may represent significant changes at the level of the smaller airways. These studies have confirmed that fish oil represents a viable treatment modality for asthmatic individuals with EIB and suggest that fish oil may influence airway smooth muscle contractility.

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In 2018, 1 in every 10 infants born in the United States was born preterm. The majority of neonatal deaths and nearly a third of infant deaths that occur are linked to preterm birth. Preterm birth is initiated when the quiescent state of the uterus ends prematurely, leading to contractions and parturition beginning as early as 32 weeks, though the origins are not well understood. Tocolytics are pharmaceuticals utilized to postpone preterm labor, but currently only manage to prolong pregnancy for up to 48 hours and have not proven effective in completely preventing preterm delivery. To enable research and discovery of therapeutics with potential to better address preterm birth, the capability to study isolated cell processes of pregnant uterine tissue in vitro is needed. Our development of an in vitro model of the myometrium utilizing uterine myocytes - uterine smooth muscle cells (uSMCs) responsible for contractions - provides a platform to examine the cellular mechanisms of late-stage pregnancy potentially involved in preterm birth. In this thesis, we discuss the optimized culture of uterine SMCs on a flexible polydimethylsiloxane (PDMS) substrate functionalized using a cationic solution, Poly-L-lysine (PLL), followed by extracellular matrix (ECM) protein coating. Using the model we developed, we then exposed this elastic substrate with uterine SMCs to different strain rates in order to investigate the impact of mechanical strain parameters on uterine SMC hypertrophy in the uterus during late-stage pregnancy. It was found that PLL and ECM protein coatings significantly impact cell morphology and density in unstrained substrates. It was also observed that when exposed to strain conditions, strain significantly increased hypertrophic morphological traits in select conditions. These results indicate that both surface and mechanical properties of in vitro systems impact uterine SMC phenotype, offering further understanding of cellular pathways involved in the uterus under mechanical load. / 2024-05-31T00:00:00Z

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13515号 / 論医博第2265号 / 新制||医||1061(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 湊谷 謙司, 教授 石見 拓, 教授 江木 盛時 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Studies on the perfused rabbit ear artery preparation showed that withdrawal of Mg 2+ from extracellular fluid potentiated the responses to histamine and ATP but not to catecholamines. Similar results were obtained in [2xCa2+] Krebs solution. Increases in [Mg 2+] decreased responses to the three agonists to a similar extent. In subsequent experiments attempts were made to alter the availability of calcium for contraction induced by these agonists either by changing the [Ca 2+] of the Krebs solution or by using Ca 2+ influx inhibitors, ouabain and ryanodine. The effects of these agonists were compared to those observed when Mg2+ was altered. In general, the results obtained in perfused rabbit ear artery supported the hypothesis that changes in extracellular [Mg2+] affect the availability of calcium for contraction but were not consistent with the suggestion that Mg2+ alters Ca2+ influx. In a second type of preparation tension responses of superfused rings of ear artery were studied. Responses to changes in extracellular [Ca2+] and[ Mg2+] were found to differ slightly from those obtained in the perfused artery. A simultaneously perfused and superfused arterial preparation showed that responses to changes in [ Mg2+] and[Ca2+] were different if the agonist was administered to the adventitial surface of the vessel rather than via the intimal surface. The effects of alterations in extracellular [Mg 2+] were studied in mesenteric arteries from weight matched normotensive and spontaneously hypertensive rats (SHR). No differences in response to NA or ATP when extracellular [Mg 2+ ] was either increased or reduced were observed in the SHR compared to the normotensive animal. However, a difference in calcium dependence was demonstrated between the two types of vessels to NA. In contrast to mesenteric arteries, experiments on aortae from normotensive rats and SHR showed no differences in the calcium dependence of NA responses between normotensive and SHR vessels, whereas, [4xMg2+ ] Krebs solution reduced the responses of normotensive aorta to NA more than SHR. These results in the rat were not consistent with the hypothesis that alteration in [Mg 2+] can be explained in terms of altered calcium availability. Attempts to increase intracellular cyclic AMP with theophylline showed that the response to ED50 NA in both mesenteric arteries and aortae from normotensive were reduced more than SHR. It is concluded that the effect of changes in extracellular [Mg2+] on the reactivity of vascular muscle varies depending on the type of vessel and species of animal from which the vessel is taken. In addition when all the experimental results are considered, it is not possible to explain all the actions of altered [ Mg2+ ] simply in terms of changed calcium availability.

Despite advancements in percutaneous coronary intervention, stents are still limited by a 2% annual rate of in-stent restenosis (ISR) related to neointimal (NI) tissue proliferation. Efforts to prevent ISR formation remain the focus of ongoing work. Adenosine (ADO) is a purine nucleoside with integral roles in vascular homeostasis, though it has limited clinical application. ADO signals primarily via four receptors with ADO receptor-A2B (ADOR-A2B) considered to play an integral role in vascular healing. Dipyridamole (DP) is a commercially approved therapy known to improve vascular events and modulate adenosine biology. Our objectives with this study included (i) assessing whether ADO could serve as a biomarker of cardiac events; (ii) determine if DP could mitigate NI formation in a pre-clinical stent model; and, (iii) quantify the mechanisms of DP-related vasculoprotection, specifically related to ADOR-A2B. We assessed the analytic and biologic variability of circulating ADO levels in humans and demonstrated that circulating ADO was not predictive of cardiac events at one year following invasive coronary angiography. We then assessed whether modulation of adenosine biology with DP had therapeutic efficacy in a pre-clinical model. Utilizing meta-analysis, we confirmed the sustained effects of DP on vascular patency rates in both pre-clinical and clinical studies. We refined a pre-clinical rabbit model of stent implantation with assessment of stent healing by intravascular optical coherence tomography – with excellent translation to clinical observations. We then assessed DP in a pre-clinical model, demonstrating reduction in ISR and improved stent healing with DP compared to control. Last, we sought to elucidate the mechanisms behind the observed DP effects, specifically related to ADOR-A2B. In vivo, DP therapy demonstrated reduced NI smooth muscle cell (SMC) content. In vitro assessment of DP demonstrated dose-dependent inhibition of SMC proliferation and migration with alteration of SMC phenotypic switching, while selective modulation of ADOR-A2B and ADOR-A2B knockdown support an ADOR-A2B-mediated component to the observed DP effects. Adenosine biology is integral to vascular homeostasis. In humans, circulating adenosine levels in humans are not predictive of one year cardiovascular events. However, DP may improve vascular healing post stent implantation and warrants clinical evaluation for stent healing. The observed DP benefits may, in part, stem from ADOR-A2B modulation. ADOR-A2B is a viable target for assessment of small molecule modulation as a novel therapeutic target to improve vascular outcomes.

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