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.

Heart valve disease represents the second most common indication for cardiac surgery in the US, yet no therapy exists to slow down or revert its progression. Pharmacologic treatments are greatly in need for aortic and mitral valve disease and require a greater understanding of the underlying cellular mechanisms. Serotonin (5HT) dysfunction has been associated with heart valve disease, clinically observed in carcinoid syndrome or with the use of medications, such as the diet drug, Dexfenfluramine, a 5HT transporter (SERT) inhibitor and 5HT receptor (HTR) 2B agonist. Concurrently, Angiotensin II (AngII) and the renin-angiotensin system (RAS) greatly contribute to cardiac/valvular diseases. This dissertation explores the intersecting mechanisms by which 5HT and AngII contribute to aortic and mitral valve disease with an attempt to develop therapies to mitigate their progression. Three murine models were extensively utilized, mice lacking SERT (SERT KO) and wild-type mice receiving AngII infusion or Fluoxetine, a SERT inhibitor, with/without 5HTR2B inhibitors, to characterize histopathological, hemodynamic, and cellular level changes. Valvular interstitial cells (VICs) isolated from murine and human healthy valves were treated with various stressors known to be involved in valvular remodeling/5HT signaling, including 5HT, TGFβ1, AngII, and H2O2. Patients undergoing AV and MV surgery were prospectively enrolled in our study, with their valves isolated, genotyped for SERT promotor polymorphism, and studied for 5HT-related gene expression changes. We demonstrate that pathological, fibrotic thickening occurs to the AV and MV in response to AngII infusion, lack or inhibition of SERT in mice. AngII mice developed increased velocities and gradients across their AVs, a marker of hemodynamic compromised, and the cellular changes involved 5HT, TGFβ1 and other inflammatory pathways. Concurrent HTR2B blockade mitigated many of these changes. Most notably the MV in SERT KO mice demonstrated HTR2B upregulation and increasing levels of COL1A1. Both murine and human MVICs exposed to 5HT or TGFβ1 upregulated COL1A1, ACTA2 and/or HTR2B. Human AVICs treated with AngII in the setting of SERT downregulation displayed markers of osteogenic transdifferentiation, with these changes mitigated again by HTR2B blockade. Finally, patients with aortic stenosis and aortic insufficiency have lower levels of SERT than healthy patients, along with upregulation of various 5HT receptors. Presence of LL SERT promotor polymorphism is associated with faster progression of AI. We then further investigated the mechanisms by which SERT downregulation may enhance mitral and aortic valvulopathy by studying the activation of mechanically sensitive calcium channel, Piezo1, and the role of mechano-transduction. ScRNAseq results of both MR and MV cells demonstrated the presence of Piezo1 expression in different cell types on the mitral valve. Mitral valve interstitial cells in culture demonstrated Piezo1 Ca++ channel activity following administration of Yoda1, a Piezo1 agonist, with associated significant downregulation of SERT and diminished SERT function and upregulation of HTR2B. Taken together, this dissertation provides a novel and promising therapeutic target to mitigate aortic and mitral valve disease. Dysregulated AngII and 5HT, with SERT, HTR2B, and Piezo1 signaling, contribute to pathological remodeling to both valves, and preventing this signaling through Piezo1/HTR2B inhibition can prevent these changes.