In living systems, ion channels are membrane transport proteins that provide pathways for the passive diffusion of ions through lipid membranes. The flow of ions across membranes is the basis of many important physiological processes, including but not limited to the regulation of membrane potential, transepithelial transport and cell volume. While many efforts have been made to understand the biological roles of natural ion channels, the biological activities of artificial ion channels remain largely unknown. Recently, it was reported that a small molecule 1, which forms synthetic chloride (Cl–) channels in membranes via self-assembly, is capable of modulating vascular functions. In this thesis, novel small molecules that are structurally similar to 1 are shown to form artificial ion channels in membranes. Together with 1, the effects of these small molecules on the contractile activities of smooth muscles and epithelial ion transport are explored. The therapeutic implications of the findings are also discussed. A collection of small molecules was screened using liposome-based fluorescence assays. In these assays, the ability of the synthetic compounds to modulate membrane potential was monitored. The screening yielded compound 3 that formed synthetic potassium (K+) channels in liposomal membranes, although the liposome-based fluorescence experiments suggested that 3 also transported Cl–. Two derivatives of 3, namely, compounds 2 and 4 were also examined. Single-channel recording experiments suggested that 2 forms synthetic Cl– channels in liposomal membranes. The effects of compounds 2 and 3 on the functions of the vascular smooth muscle are explored. Using confocal imaging, it was shown that both 2 and 3 counteracted the effects of high-K+ depolarizing solution on membrane potential and intracellular Ca2+ concentration ([Ca2+]i) in cultured vascular smooth muscle cells. 2 and 3 also relaxed mice aortic rings pre-contracted with high-K+ solution. These observations can be explained in terms of the Cl– transporting functions of 2 and 3. To determine the potential for developing the compounds into bronchodilators, the effects of compounds 1 and 3 on the contractile activities of the airway smooth muscle (ASM) were explored using organ bath technique. The contractile activities of the trachea isolated from Sprague-Dawley (SD) rats were first characterized. Among the contractile agents used, only potassium chloride (KCl), cholinergic agonists, serotonin and endothelin-1 were contractile to the SD rat trachea. 1 and 3 relaxed the ASM pre-contracted with KCl, whereas the contractions induced by other agonists were not affected. The ability of compounds 2, 3 and 4 to modulate ion transport across cultured epithelia was tested by the short-circuit current measurement technique. It was shown that the compounds were capable of inducing Cl– secretion when applied to the apical side of airway and colonic epithelia. Importantly, the synthetic compounds induced apical Cl– secretion in immortalized cystic fibrosis (CF) bronchial epithelia. This suggests that the synthetic compounds may be used to correct the anion transport defect in CF epithelia. In summary, the small-molecule based synthetic ion channels demonstrated two important general functions of natural ion channels, namely, the regulation of membrane potential and epithelial ion transport. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Native vascular tissue functions are highly dependent on structural organization at the super-cellular, cellular, and sub-cellular spatial scales. We hypothesized that the structure-function relationship of vascular tissues in vivo can be leveraged to engineer vascular tissues in vitro by prescribing the shape of constituent cells and their assembly into organized three-dimensional structures. To this end, we first asked if vascular smooth muscle cell shape influences cellular contractility. We engineered human vascular smooth muscle cells to assume similar shapes to those in elastic and muscular arteries and then measured their contraction while stimulating with endothelin-1. We found that vascular smooth muscle cells with elongated shapes exhibited lower contractile strength but a greater percentage increase in contraction after endothelin-1 stimulation, suggesting that elongated vascular smooth muscle cell shape endows the muscular artery with greater dynamic contractile range. Next, we sought to assemble cells into tissues by employing a three-dimensional cellular patterning strategy based on the folding of porous, thin polymer films. We assembled different three-dimensional endothelial and vascular smooth muscle organizations by patterning two-dimensional poly(lactic-co-glycolic) acid and collagen thin films with cell suspensions at prescribed locations. The films were subsequently folded following Miura-ori geometry guidelines and the matrices were embedded subcutaneously in immunodeficient mice in order to assess the vascularization of the implanted constructs. We found that spatial organization that allowed endothelial and vascular smooth muscle cells to interact adjacent to each other laterally in the same folding plane created the densest vascularized network, suggesting that three-dimensional structural organization of vascular cells can influence the formation of vascularized networks. Taken together, our result shows that functional vascular tissues in vitro can be engineered by encoding structure cues in their design and assembly. / Engineering and Applied Sciences

published_or_final_version / Pharmacology / Master / Master of Philosophy

No description available.

Chronic oxidative injury by allylamine induces proliferative vascular smooth muscle cell (vSMC) phenotypes in the rat aorta similar to those seen in rodent and human atherosclerotic lesions. In this study, we evaluate the potential role of cyclin dependent kinase inhibitors, p21 and p27, and extracellular regulated kinases (ERK1/2) to mediate the proliferative advantage of oxidatively stressed (i.e. allylamine injured) vSMC. Isolated rat aortic SMC from allylamine treated and control rats were cultured on different extracellular matrix (ECM) proteins. Following mitogen restriction, cultures were stimulated with serum with or without inhibitors of NF-kB or MEK. Western blot analysis was performed to identify protein differences between treatment groups. Basal levels of p21 were 1.6 fold higher in randomly cycling allylamine cells than control counterparts seeded on a plastic substrate, a difference lost when cells were seeded on collagen. p27 levels were comparable in both cell types irrespective of substrate. Basal levels of p21 and p27 were 1.4 fold higher in G0 synchronized allylamine cells compared with G0 synchronized control cells seeded on a plastic substrate. Following cell cycle progression, differences in protein levels were not detected. Treatment with 100 nM pyrollidine dithiocarbamate (PDTC) resulted in significant decreases in p21 and p27 in allylamine cells versus control cells following serum stimulation for 9 hours. This decrease was even greater for p21 in allylamine cells when grown on collagen relative to control cells. Alterations in peak and temporal activation of ERK1/2 were observed in allylamine cells seeded on a plastic substrate as compared to control cells, following serum stimulation. Seeding on collagen decreased the enhanced peak phosphorylation of ERK1/2 and increased the sustained activity in allylamine cells compared with control counterparts. Inhibition of ERK1/2 activity resulted in reduced p21 expression in both cells types, but the response was markedly enhanced in allylamine cells, and preferentially observed on a restrictive collagen substrate. We conclude that induction of proliferative (i.e. atherogenic) phenotypes following repeated cycles of oxidative injury involves ERK1/2 activity and modulation of the cyclin dependent kinase inhibitors, p21 and p27, in a matrix-dependent manner.

NF-κB activity is critical in the regulation of atherosclerotic vascular smooth muscle cell (vSMC) phenotypes induced following oxidative injury by allylamine. The present studies were designed to detail dysregulation of NF-κB activity in these altered phenotypes, and to assess the importance of NF-κB in the regulation of osteopontin, a cytokine which modulates atherosclerosis. Increased degradation of IκBα was observed in allylamine-induced atherosclerotic vSMC phenotypes (henceforth referred to as allylamine cells). Enhanced phosphorylation of I-κ-kinases was observed by Western immunoblotting. NF-κB DNA binding activity as assessed by electrophoretic mobility shift assay demonstrated changes in the kinetics and magnitude of induction of binding. Enhancement of NF-κB binding activity was evident in allylamine cells compared to controls when seeded on plastic, fibronectin, and laminin, but not collagen I. Posttranscriptional alterations in Rel protein expression and nuclear localization partly account for changes in NF-κB DNA binding activity. Promoter-specific NF-κB binding profiles suggest altered dimer prevalence as a consequence of the changes in Rel protein expression. The expression of NF-κB regulated genes osteopontin and MMP-2 was enhanced in allylamine-treated aortas, while cyclin D1 and MMP-9 were unchanged. As the importance of osteopontin in atherosclerosis has been described in several models, subsequent studies were designed to assess osteopontin promoter activity. Activity of the osteopontin promoter was significantly reduced in allylamine cells compared to controls as assessed using a luciferase reporter. Deletion analysis suggested the presence of inhibitory cis-acting elements in the regulatory region of the gene. Mutation of these elements, including VDRE, AP-1, NF-κB, and USF1, indicated that NF-κB and USF1 mediate suppression of osteopontin promoter activity in allylamine cells. Decreased serine phosphorylation of immunoprecipitated RelA/p65 was observed in allylamine cells, indicating decreased ability of this protein to transactive gene promoters. NF-κB was found to play a role in suppression of osteopontin promoter activity by collagen I-mediated integrin signaling. These findings suggest that enhancements in NF-κB activity suppress osteopontin promoter activity in oxidant-activated vSMC cultures. Dysregulation of NF-κB activity occurs as a result of altered matrix and intracellular signaling upstream of the nucleus and possibly differential dimer assembly leading to cell-specific profiles of NF-κB-dependent gene regulation.

During the development of atherosclerosis, contractile vascular smooth muscle cells (VSMCs) change to cells capable of migrating and proliferating to mediate repair, where the responses may be adaptive or mal-adaptive in effect. Cyclic adenosine monophosphate (cAMP)-elevating agents have been shown to inhibit migration of VSMC. cAMP activity within the cell is known to be ubiquitous and dynamic, requiring control through signal termination mechanisms for cellular homeostasis. Phosphodiesterase (PDE) enzymes are central to this critical regulatory process catalyzing the hydrolysis of cAMP. A great deal of insight into the role of PDEs in defining compartmentalization of cAMP signaling has arisen predominately from recent studies on the cAMP-specific PDE4 family. Compartmentalization of PDE4 is mediated by their unique N-terminal domains, which have been proposed to provide the “postcodes/zipcodes” for cellular localization. PDE4D isoforms vary widely, yet their conservation over evolutionary time suggests important non-redundant roles in distinct cellular processes. To study the potential role of individual PDE4D isoforms we seek to utilize the unique N-terminal targeting domains that are proposed to be responsible for their protein-protein interactions and site-directed localization. Herein, we report on the expression, targeting and localization of five “long” PDE4D isoforms and the impact on cell morphology of certain amino-terminal domains of individual PDE4D constructs expressing green fluorescent protein (NT-PDE4D/GFP) in human aortic smooth muscle cells (HASMCs). Through the development of engineered NT-PDE4D/GFP expression plasmids, we were able to study the cell biological impacts associated with the overexpression of individual PDE4D amino-terminal variants in HASMCs. We show that NT-PDE4D5/GFP and NT-PDE4D7/GFP expressing cells exhibited an elongated cell morphology, where this effect was much more marked in NT-PDE4D7/GFP expressing cells, exhibiting multiple leading edge structures and highly elongated “tails”. We identify a potential role for PDE4D7 targeting in the regulation of cell polarity and migration. Our results suggest the novel idea that PDE4D7, rather than the four other long PDE4D isoforms (PDE4D3, PDE4D5, PDE4D8, or PDE4D9), represents the dominant PDE4D variant involved in controlling cAMP-mediated effects on cell tail retraction dynamics. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2014-03-13 13:00:31.684 / Video I: Time-lapse video of GFP-expressing cell migration in HASMC. GFP expressing cells did not differ in cell migration or morphology compared to non-injected control cells. HASMCs were microinjected with GFP construct. Representative images of micoinjected GFP cells were taken 24 h post-injection overnight at 30min intervals using a Zeiss Axiovert S100 microscope and processed as described in Materials & Methods. (10X) / Video II: Time-lapse video of NT-PDE4D7/GFP-expressing cell migration in HASMC. NT-PDE4D7/GFP expressing cells exhibit elongated tail and decrease in cell migration compared to non-injected control cells. HASMCs were microinjected with NT-PDE4D7/GFP construct. Particle tracking of NT-PDE4D7 cells showed cleaving and full detachment of elongated tail. Representative images of micoinjected NT-PDE4D7 cells were taken 24 h post-injection overnight at 30min intervals using a Zeiss Axiovert S100 microscope and processed as described in Materials & Methods. (10X)

The lower esophageal sphincter (LES) is characterized by basal tone and appropriately timed neurogenic relaxation. The physiological mechanisms underlying these crucial LES functions remain poorly understood. The current studies were designed to characterize the electrophysiological properties and neural regulation of LES circular smooth muscle (CSM), and to determine whether interstitial cells of Cajal (ICC) play a role in neurotransmission. Conventional intracellular recordings were performed in CD1, nNOS knock-out, eNOS knock-out and W/Wv mutant mice. Mouse LES consists of “sling” and “clasp” smooth muscle, which were studied separately in CD1 mice. In subsequent studies of mutant mice and respective controls, only the clasp muscle was examined, Immunohistochemical c-Kit staining of ICC was performed in wild-type and W/Wv mutant mice that were first characterized electrophysiologically. The smooth muscle of the LES clasp and sling displayed unitary membrane potentials with a resting membrane potential (RMP) of ~ -43 mV. Spontaneous nifedipine-sensitive action potentials superimposed on the unitary potentials were usually recorded in the LES clasp, but not sling muscle. A monophasic inhibitory junction potential (IJP) was recorded in sling CSM, whereas a biphasic IJP consisting of an initial IJP, followed by long-lasting slow IJP (LSIJP) was recorded in clasp. Further pharmacological studies using control and various knockout mice suggest that: 1. the CSM of the mouse LES is innervated by cholinergic, nitrergic and purinergic nerves; 2. the LSIJP is mediated entirely by nitrergic nerves, whereas purinergic and nitrergic nerves produce the monophasic IJP in the LES sling and initial phase of biphasic IJP in the LES clasp; 3. Ca2+/CaM-kinase II is involved in the regulation of the nitrergic IJPs; 4. TREK-1 K+ channels are not involved in the nitrergic IJP; 5. purinergic and cholinergic neurotransmission is intact in LES CSM of W/Wv mutant mice, whereas nitrergic neurotransmission is impaired in about half of the animals. In animals in which nitrergic neurotransmission was intact, ICC-IM were markedly deficient immunohistologically, suggesting that ICC are not required for nitrergic neurotransmission; 6. impaired nitrergic neurotransmission in W/Wv mutant mice is associated with dysfunction of a Ca2+-dependent signaling cascade primed by spontaneous Ca2+ release from the sarcoplasmic reticulum. / Thesis (Ph.D, Physiology) -- Queen's University, 2008-01-24 15:54:52.175

An important part of repair processes activated by vascular injury is the recruitment of vascular smooth muscle cells (SMC) from the existing contractile coat. Phenotypic modulation of SMCs enables these cells to proliferate and migrate into the vessel intima. Despite its importance in vessel repair, this plasticity of SMCs can also promote both the pathogenesis of atherosclerosis as well as neointimal formation following revascularization- induced injury. Vascular growth factors are major contributors to the migratory and proliferative responses to injury. IGF-1 is one such growth factor that elicits a response via its receptor, the IGF-1R, a classical tyrosine kinase receptor. However, it has been suggested that the IGF-1R may also be coupled to a heterotrimeric G protein and can thus initiate cellular responses via this alternate pathway. The objective of this study was to investigate the structural aspects of IGR-1R coupling to a heterotrimeric G protein in SMCs, as well as the contribution of this pathway to the cellular responses. In a porcine primary SMC culture model, IGF-1R co-precipitated with both the α- and β-subunits of a G protein, with the latter demonstrating activation dependent precipitation. The specific Gα class activated by IGF-1R was Gαi, in a manner that was independent of the activity of the tyrosine kinase. Both Gαi1 and Gαi2 directly interacted with the receptor. Gβγ mediated the activation of MAPK and its inhibition was sufficient to attenuate both the proliferation and migration of SMCs in vitro. In contrast, the contribution of Gαi was related to regulation of protein translation and histone modification. The data supports the conclusion that IGF-1 regulates the phenotype of vascular SMCs at least partially via a non-classical G protein-coupled receptor. Investigation into the individual subunits of the G protein complex led to the elucidation of a model in which both components play an integral role in the IGF-1 response, independent of the receptor tyrosine kinase activity. In one case, an interplay of specific Gαi-subunits leads to modulation of the VSMC translational and transcriptional responses, while in the other, release of the Gβγ-subunit activated the MAPK response in a manner that significantly contributes to both the migration and proliferation of SMCs.

The prevalence and economic burden of chronic airway disorders such as asthma is on the rise annually. Allergic asthma is characterized by chronic airway inflammation, airway hyper-responsiveness (AHR), and structural airway remodeling due to increased smooth muscle mass. Most allergic asthma occurs due to the overproduction of immunoglobulin E (IgE) antibodies against common allergens. Classically, IgE has been shown to modulate airway smooth muscle (ASM) contraction/relaxation which is believed to be the underlying cause of airway hyperreactivity. However, the molecular mechanisms underlying IgE effects on ASM cell are not established. Recently, the high-affinity Fc receptor for IgE (FcεRI) has been identified in human ASM cells in vitro and in vivo within bronchial biopsies of allergic asthma patients. However, it is unknown whether FcεRI activation on ASM can modulate the immune response within the airways. We hypothesized that the IgE-FcεRI interaction plays a key role in inducing phenotypic and functional changes in ASM cells that eventually contributes to the establishment of airway inflammation, AHR, and remodeling. We sought to investigate the regulation, effector functions, and underlying mechanisms of FcεRI activation in ASM cells. Our work shows that the proinflammatory tumor necrosis factor (TNF) and T helper type 2 (Th2) cytokine interleukin (IL)-4 enhanced the FcεRI abundance and amplified the IgE-induced chemokine (eotaxin-1/CCL11, RANTES/CCL5, IL-8/CXCL8, and IP-10/CXCL10) release in ASM cells via transcriptional mechanisms. Both TNF and IgE induced a novel, Th2-favoring cytokine thymic stromal lymphopoietin (TSLP) through the activation of spleen tyrosine kinase (Syk), and nuclear factor kappa B (NF-κB) and activator protein-1 (AP-1). In addition, IgE induced de novo DNA synthesis and ASM cell proliferation via mitogen-activated protein kinases (MAPKs) and signal-transducer and activator of transcription 3 (STAT3) activation. Collectively, our data suggest that the IgE-induced FcεRI activation leads to the expression of multiple chemokines in ASM which may indirectly recruit inflammatory cells and promote allergic airway inflammation; IgE induces TSLP which can promote the Th2 immune responses within the airways; and IgE may potentially induce airway remodeling by directly inducing ASM cell proliferation. Therefore, targeting the IgE-FcεRI network on ASM may offer a novel therapeutic strategy in allergic asthma.