Carotid body (CB) receptor type I cells transduce blood-borne chemical stimuli into electrical signals and release the excitatory neurotransmitter ATP onto afferent terminals that project to the breathing centre located in the brainstem. Within the CB, type I cells are ensheathed by glial-like processes of type II cells. Recently, it was hypothesized that type II cells have a paracrine function in CB chemotransduction by acting as an ATP amplifier and enhancing chemoexcitation (Zhang et al. 2012). Given this recent development, the primary goal of this thesis was to further elucidate the paracrine function of type II cells and characterize the signalling mechanisms involved in type I and type II cell interactions. Ratiometric calcium imaging was used to investigate type II cell sensitivity to two prominent CB neuromodulators, angiotensin II (ANG II) and 5-HT, in rat CB cultures. Both ANG II and 5-HT elicited large rises in intracellular Ca<sup>2+<sup> that were present in the absence of extracellular Ca<sup>2+<sup> and were inhibited by intracellular store depletion agents. ANG II and 5-HT acted on their respective G-protein coupled receptors, AT<sub>1<sub> receptor and 5-HT<sub>2A<sub> receptor, to initiate these Ca<sup>2+<sup> responses presumably via a PLC-IP<sub>3<sub> mediated mechanism. Interestingly, these Ca<sup>2+<sup> responses were required to activate pannexin-1 channels (Panx-1), a channel that has been previously shown to be a conduit for ATP in type II cells (Zhang et al. 2012). We were also interested in determining whether type II cells were capable of indirectly responding to a chemostimulus such that the stimulus would elicit neurosecretion from type I cells and result in a secondary Ca<sup>2+<sup> responses in type II cells. Isohydric hypercapnia and a depolarizing stimulus (30 mM KCl saline) were capable of eliciting indirect Ca<sup>2+<sup> responses in type II cells. These secondary Ca<sup>2+<sup> responses in type II cells were partially inhibited by suramin, a purinergic P2Y2 receptor antagonist, suggesting that ATP was the predominant neurotransmitter responsible for type I to type II crosstalk. Similarly, a selective agonist for type II cells, UTP, evoked indirect Ca<sup>2+<sup> responses in nearby type I cells. Type II to type I cell communication was dependent on Panx-1 channels since the secondary Ca<sup>2+<sup> responses in type I cells were inhibited by the Panx-1 blocker, carbenoxolone (5 µM). UTP-evoked indirect Ca<sup>2+<sup> in type I cells were partially inhibited by adenosine A<sub>2<sub> receptor antagonists suggesting that the neuromodulator, adenosine, governs cross-talk between type II and type I cells. This study elucidates the importance of purinergic signalling in the bi-directional cross-talk between receptor type I cells and glial-like type II cells. / Thesis / Master of Science (MSc)

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Food insecurity is a growing issue in the United States. Iron deficiency is the most common form of nutritional deficiency in patients with endothelial dysfunction and vascular-related diseases. This preliminary study lays the groundwork for the “Nutrient deficiency-on-a-chip” model. Endothelial cells are cultured on mechanically tunable, enzymatically cross-linked gelatin and treated with deferoxamine, an iron chelator, or angiotensin II were used to simulate a nutrient deficient and diseased environment, respectively. As oxidative stress and disturbed barrier function are the most prevailing mechanism of angiotensin II and iron deficiency induced endothelial dysfunction, to test our model we investigated the changes in reactive oxygen species production and VE-cadherin expression in engineered endothelium. Both angiotensin II and deferoxamine treated engineered endothelium showed an increase in oxidative stress and disturbed barrier function. This in vitro model can be a useful tool to better understand disease mechanisms associated with nutrient deficiency and identify novel therapeutics.

The Renin Angiotensin System (RAS) plays a vital role in the regulation of blood pressure and fluid homeostasis. RAS is regulated via the hormone Angiotensin II through an association with the Na+/H+ exchanger NHE6. Here, NHE6 was found to be activated by Angiotensin II through the Angiotensin II AT1 receptor. Furthermore, it was shown that NHE6 requires phosphorylation for activation and this phosphorylation signaling mechanism does not involve phospholipase C. The elucidation of the signaling pathway associated with NHE6 and AT1 allows for the greater understanding of function and regulation of the NHE6 protein. The Angiotensin receptor AT2 is a G-coupled protein receptor (GPCR) that is highly expressed in infant neural tissue. The S1 subunit of the pertussis toxin can inhibit GPCR signaling via ADP-ribosylation of the cognate Gi protein, suggesting that the S1 subunit may interfere with AT2 signaling. In order to observe whether S1 associates with AT2, Chinese hamster ovary cells were transfected with plasmids expressing AT2 or mutants of AT2. The lysates of these cells were incubated with His-tagged S1 subunit and it was observed that only the wild-type AT2 co-immunoprecipitated with S1. These results imply that there is a direct interaction between the S1 subunit and AT2. Municipal wastewater can be considered as an effective growth medium for the cultivation of microorganisms due to organic material found in the water. Oleaginous microorganisms produce large amounts of triacylglycerols (TAGs) when cultivated on medium containing high sugar content and low nitrogen. These TAGs can then be converted into biodiesel. To determine if the oleaginous yeast Rhodotorula glutinis could survive and synthesize lipids using wastewater as a cultivation medium, R. glutinis was inoculated into primary effluent wastewater supplemented with glucose. Results indicated that R. glutinis was able to survive and synthesize lipids in the wastewater which is suggestive that R. glutinis can successfully compete with indigenous microorganisms in the wastewater.

This study helps to fill a remaining knowledge gap surrounding the mechanisms and pathways that contribute to cardiomyopathies in SCD. A better understanding of the pathophysiological mechanisms could lead to more accurate therapeutic targets to improve quality of life as well as life expectancy. In this study I recapitulate cardiac dysfunction in vitro by exposing engineered mouse cardiac tissues to ANG II or the sickled microenvironment. Experimental results include gene expression profiles and oxidative stress generation. Gene expression profiles in the ANG II treated tissues indicated a pathological state with upregulation in biomarkers for inflammation, cell adhesion, wall stress and ECM related genes. Further research is being conducted using insights gained from this study which will lead to a broader understanding of the biological processes involved and potentially identify novel therapeutic targets that may ultimately improve patient outcomes.

Calcific aortic valve disease (CAVD) is the most common cause of aortic valve failure and replacement in the elderly population, affecting 25% of the population over 65 years of age. Current pharmacological approaches for preventing the onset and progression of calcific aortic valve disease have not shown consistent benefits in clinical studies. Differentiation of valvular interstitial cells (VICs) into osteoblast–like cells is an integral step in the calcification process. Although clinical evidence suggests hypertension as a potential candidate contributing to the development of CAVD, the underlying molecular mechanisms that cause de-differentiation remain unclear. The present study investigates the role of elevated cyclic pressure in modulating osteoblast differentiation pathways in VICs in vitro. We used a combination of systems biology modeling and pathway-based analyses to identify novel genes and molecular mechanisms that are activated in valve tissue during exposure to elevated pressure conditions. Our results show that elevated pressure induces a gene expression pattern in valve tissue that is considerably similar to that seen in CAVD, underlining the key role of hypertension as an initiating factor in the onset of pathogenesis. In addition, our analysis revealed a set of genes that was not previously known to be regulated in valve tissue in a pressure dependent manner. Currently, the molecular mechanisms involved in CAVD and their associations with changes in local mechanical environment are poorly understood, and thus a better understanding of the cell based process mediating CAVD progression will improve our ability to develop potential medical therapies for this disease.

No description available.

Abdominal aortic aneurysm (AAA) is a major cardiovascular disease and involves enhancement of renin-angiotensin system and recruitment/activation of inflammatory factors such as matrix metalloproteases (MMP's). Caveolae has been shown to play a role in a number of different cardiovascular diseases through different mechanisms including regulation of oxidative stress, inflammation and degradation of extracellular matrix components through MMP's. In addition, endothelial cell caveolae are known to localize the Ang-II (AT1) receptor and regulate renin-angiotensin signaling. Based on these findings, we evaluated the role of caveolae in AAA formation in the murine model. Here, eight week old mice were co-infused with Ang-II and BAPN, a lysyl oxidase inhibitor, to induce AAA. We found that mice lacking the main structural protein of caveolae, caveolin-1, did not develop AAA compared to WT animals in spite of hypertensive blood pressures measured by telemetry in both groups. This finding suggests that intact Ang-II signaling remains in place in caveolin-1 knockout mice. To begin to address the underlying mechanism by which caveolae contributes to AAA, we measured the level of oxidative stress and MMP's in aneurysms. We found an increased expression of MMP-2 and MMP-9 in vessels of WT mice displaying aneurysms. This increase in expression was not observed in Cav-1 knockout mice. Furthermore, KO mice showed less oxidative stress then their WT counterparts as assessed by anti-nitrotyrosine staining. Next we examined the characteristics of early AAA formation in wild-type mice. We found caveolae associated proteins, endothelial nitric oxide synthase (eNOS) and NADPH oxidase 2 (Nox2), were upregulated in early AAA formation, particularly in the endothelium. Also, Vascular Cell Adhesion Molecule (VCAM) was upregulated in the endothelium. However, macrophage infiltration and MMP-2 activation was not observed in early AAA development. In order to elucidate the role of endothelial caveolae in the formation of AAA, we induced AAA, as previously described, in endothelial specific cav-1 knockout mice. Preliminarily findings show endothelial specific knockout mice do not form AAA as compared to their WT littermates. In conclusion, caveolae appears to play a critical role in the formation of AAA in mice via oxidative stress, and recruitment and/or activation of MMPs, specifically MMP-2 and MMP-9. Early markers of AAA formation include VCAM, NOX2, eNOS, and protein nitration. Also, preliminary results indicate that endothelial specific knockout mice do not develop AAA. / Cell Biology

No description available.

Neuere Untersuchungen legen eine kardioprotektive Rolle für den Angiotensin AT2-Rezeptor nahe, welcher die Postinfarkt-Entzündungsreaktion vermindert, wobei der zelluläre Mechanismus noch wenig verstanden ist. Das Ziel dieser Arbeit war es deshalb, die potentielle Rolle des AT2-Rezeptors in der zellulären Immunantwort auf ischemische Herzverletzungen zu ergründen. Sieben Tage nach myokardialem Infarkt in Ratten wurde der AT2-Rezeptor mittels Immunfluoreszenzfärbung von Gewebeschnitten in einer CD8 T-Zellfraktion detektiert, die das Peri-Infarkt-Myokard infiltiert hatte. Wir haben eine Methode entwickelt, die es mittels kombinierter MACS und FACS Technilogie ermöglicht, CD8+AT2R+ T-Zellen aus dem Myokard zu isolieren und zu analysieren. Im Gegensatz zu den CD8+AT2R- T-Zellen, die in Kultur sowohl auf adulte als auch auf fötale Kardiomyozyten stark zytotoxisch wirkten, zeigten die CD8+AT2R+ T-Zellen keinerlei Zytotoxizität. Die CD8+AT2R+ T-Zellen zeigten eine erhöhte Expression von IL-10 und eine geringere mRNA Expression von IL-2 und IFN-gamma im Vergleich zu CD8+AT2R-T-Zellen. Weiterhin konnten wir zeigen, dass in vitro Stimulation des AT2-Rezeptors zur Hochregulation der IL-10-Expression von CD8+ T-Zellen führt. Entsprechend führt die in vivo Aktivierung des AT2-Rezeptors zur Vergrößerung der CD8+AT2R+ T-Zellpopulation und erhöhter IL-10-Produktion im ischemischen Myokard. Diese CD8+AT2R+ T-Zellen konnten auch in humanem periphärem Blut detektiert werden. Wir haben eine CD8+AT2+T-Zellpopulation definiert, welche sich während ischemischer Herzverletzung vergrößert und das Kardiomyocytenüberleben mittels kardioprotektivem IL-10 aufrechterhält. Somit konnten wir einen neuartigen AT2-Rezeptorvermittelten zellulären Mechanismus aufdecken, welcher die adaptive Immunantwort im Herzen moduliert. / One important aspect of cardiac remodeling after myocardial infarction is the activation of an immune response, which removes death cardiomyocytes and initiates scar formation. On the other hand, activation and infiltration of immunocompetent cells are responsible for augmenting damage in non-infarcted areas. Emerging evidence suggests a cardioprotective role of the angiotensin AT2R by attenuating this post-infarct inflammatory reaction, albeit the underlying cellular mechanisms are not well understood. We aimed here at elucidating a potential role of the cardiac angiotensin AT2R in regulating the cellular immune response to ischemic heart injury. Seven days after myocardial infarction in rats, immunofluorescence staining of tissue sections showed that AT2R was detected in a fraction of CD8+ T cells infiltrating the peri-infarct myocardium. We developed a method that allowed the isolation and characterization of CD8+AT2R+ T cells infiltrating the myocardium via combined MACS and FACS technology. While the CD8+AT2R- T cells exhibited potent cytotoxicity to both adult and fetal cardiomyocytes in vitro, the CD8+AT2R+ T cells were non-cytotoxic to these cardiomyocytes. The CD8+AT2R+ T cells were characterized by upregulated IL-10 and downregulated IL-2 and INF-gamma gene expression when compared to CD8+AT2R- T cells. We further showed that IL-10 gene expression was enhanced in CD8+ T cells upon in vitro AT2R stimulation. In addition, in vivo AT2R activation leads to an increment of the CD8+AT2R+ T cells and IL-10 production in the ischemic myocardium. Moreover, the CD8+AT2R+ T cell population was also detected in human peripheral blood. We have defined a CD8+ T cell population that expresses AT2R and increases during ischemic heart injury. This population sustains cardiomyocyte viability by providing cardioprotective IL-1 via a novel AT2R-mediated cellular mechanism for modulating adaptive immune response in the heart.