Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / "August 2000." Typescript. Vita. Includes bibliographical references (leaves 186-210). Also available on the Internet.

Thesis (Ph. D.)--University of Nevada, Reno, 1999. / Includes bibliographical references. Online version available on the World Wide Web.

Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / "May 2000." Typescript. Vita. Includes bibliographical references (leaves 152-178). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri--Columbia, 2003. / "May 2003." Typescript. Vita. Includes bibliographical references (leaves 121-137).

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.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Morbidity and mortality attributable to asthma arise mainly from contraction of airway smooth muscle (ASM) and resulting bronchospasm. Bronchospasm that is induced in the laboratory is easily reversed by a spontaneous deep inspiration (DI) whereas bronchospasm that occurs spontaneously in asthma is not. In response to a spontaneous DI, contracted ASM fluidizes rapidly and then resolidifies slowly, but molecular mechanisms accounting for these salutary bronchodilatory responses -and their dramatic breakdown in asthma- are unknown. Using a multi-scale approach, I show here that both the baseline contractile force and the fluidization response of ASM are independent of the cytoskeletal protein zyxin, whereas the resolidification response is zyxin-dependent. At the levels of the stress fiber, the isolated cell, and the integrated airway, zyxin acts to stabilize the contractile apparatus and promote the resolidification response. More than just the motor of contraction, ASM is thus viewed in the broader context of a self-healing active material wherein resolidification and its molecular determinants contribute to the biology of bronchospasm.

The pathophysiology of asthma is characterized by airway inflammation, remodeling and hyper-responsiveness. Phenotypic changes in airway smooth muscle cells (ASM) play a pivotal role in the pathogenesis of asthma. ASM cells promote inflammation and are key drivers of airway remodeling. While airway hyper responsiveness and inflammation can be managed by bronchodilators and anti-inflammatory drugs, ASM remodeling is poorly managed by existing therapies. Therefore, targeting ASM remodeling remains a challenge, and a deeper understanding of the molecular mechanism that regulates ASM phenotypes in asthma pathogenesis will facilitate the search for next-generation asthma therapy. MicroRNAs are small yet versatile gene tuners that regulate a variety of cellular processes, including cell proliferation and inflammation - two phenotypes that are often altered in asthmatic ASM. We thus hypothesized that microRNAs regulate ASM phenotypes in asthma and represent new targets for future therapy. In this thesis, we used a genomic approach that combined next-generation sequencing with functional cellular assays to characterize the role of microRNAs in regulating airway smooth muscle function and drug response to conventional therapies. In Chapter 2, we identified miR-10a as the most abundant microRNA expressed in the primary human airway smooth muscle (HASM) cells. Using an unbiased target identification approach, we identified several novel potential targets of miR-10a, including the catalytic subunit alpha of PI3 kinase (PIK3CA)--the central component of the PI3K pathway. We demonstrated that miR-10a directly suppresses PIK3CA expression by targeting its 3' Untranslated region (3'-UTR). Inhibition of PIK3CA by miR-10a reduced AKT phosphorylation and blunted the expression of cyclins and cyclin-dependent kinases that are required for HASM proliferation. In Chapter 3, we examined the effect of conventional asthma therapies on miRNA expression. While we did not find significant changes in miRNA levels, it remains to be determined whether microRNAs play a role in ASM tissue response to asthma therapy. Our study is the first to examine the role of microRNAs in ASM proliferation. Results from our study identified a novel microRNA-mediated regulatory mechanism of PI3K signaling and ASM proliferation. They suggest further that miR-10a is a potential therapeutic target to treat airway remodeling in asthma.

Τα αγγειακά λεία μυικά κύτταρα (ΑΛΜΚ) αποτελούν το κυρίαρχο στοιχείο του μέσου χιτώνα των αιμοφόρων αγγείων, ενώ συμμετέχουν ενεργά και στο σχηματισμό και την ωρίμανση του καρδιαγγειακού συστήματος. Η δομή τους εξυπηρετεί την εκτέλεση της σημαντικότερης λειτουργίας τους, που είναι η συστολή. Αξιοσημείωτο χαρακτηριστικό των αγγειακών λείων μυικών κυττάρων αποτελεί η φαινοτυπική τους πλαστικότητα, δηλαδή η ικανότητα στροφής από το συσταλτικό σε έναν περισσότερο συνθετικό φαινότυπο, που λαμβάνει χώρα υπό προϋποθέσεις. Οι αλληλεπιδράσεις με τα υπόλοιπα κυτταρικά στοιχεία του τοιχώματος των αρτηριών και των έμμορφων συστατικών του αίματος, αλλά και η φαινοτυπική πλαστικότητα καθιστούν καθοριστικό το ρόλο των αγγειακών λείων μυικών κυττάρων στην παθογένεια της αθηροσκλήρωσης. / Vascular smooth muscle cells (VSMCs) comprise the main element of the tunica media of blood vessels, while they actively participate in the formation and maturation of the cardiovascular system. Their structure serves their basic function, which is contraction. An interesting feature of vascular smooth muscle cells is their phenotypic plasticity, the ability to shift from a contractile to a more synthetic phenotype, under certain conditions. The interaction with other cellular elements within the vascular wall or in the bloodstream, as well as their phenotypic plasticity, give vascular smooth muscle cells a decisive role in the pathogenesis of atherosclerosis.

Calcific aortic valve sclerosis is characterized by focal lesions in the valve leaflet. These lesions are rich in myofibroblasts that express α-SMA and cause fibrosis. Lesions tend to occur in regions of the leaflet that are subjected to large bending loads, suggesting a mechanobiological basis for myofibrogenic differentiation and valve pathogenesis. In this thesis, a bioreactor was developed to study the effect of physiological loading on myofibrogenic differentiation of valve interstitial cells. Cyclic loading of native porcine aortic valve leaflets ex vivo resulted in increased α-SMA expression, predominantly in the fibrosa and spongiosa (similar to sclerotic leaflets). Cofilin, an actin-binding protein, was also upregulated by loading, suggesting it plays a role in mechanically-induced myofibrogenesis. Similarly, loading of a tissue engineered aortic valve leaflet model resulted in increased α-SMA transcript and protein expression. These data support an integral role for mechanical stimuli in myofibrogenic differentiation and sclerosis in the aortic valve.