Increased airway smooth muscle (ASM) mass contributes to airway hyperresponsiveness (AHR) in asthma and is orchestrated by growth factors, cytokines and chemokines. Airway contractile responses are influenced by neuromediators, such as acetylcholine, and glutamate released by parasympathetic and sympathetic airway nerves. Hyperactivity of these neural elements, termed neurogenic inflammation, is linked with hypercontractility and AHR. Glutamate is a non-essential amino acid derivative, and its physiological role is traditionally considered with respect to its being the primary excitatory neurotransmitter in brain, and regulation of neuronal development and memory. In allergic inflammation, immune cells including dendritic cells, neutrophils and eosinophils, constitutively synthesize and release glutamate, which signals through activation of glutamate receptors, most important among which are ionotrophic N-methyl D-aspartate receptors (NMDA-R). We hypothesized that glutamatergic signaling mediated through NMDA-Rs plays an important role in inducing functional Ca2+ responses in human (H) ASM cells that can underpin airway hypercontractility. We investigated the expression and function of NMDA-Rs in HASM cells, and assessed the effects of pro-inflammatory cytokines on NMDA-R expression and functional responses. Moreover, we measured airway responses to NMDA in mice, murine thin cut lung slice preparations, and floating collagen gels seeded with HASMs. Our data reveal that airway myocytes express multi-subunit NMDA-R complexes that function as receptor-operated calcium channels (ROCCs), mobilizing intracellular Ca2+ in ASM in vitro and airway contraction ex vivo. Individual airway myocytes treated with NMDA-R agonist exhibit disparate temporal patterns of intercellular Ca2+ flux that can be partitioned into four discrete function sub-groups. Further we show that tumor necrosis factor (TNF) exposure modulates NMDA-R subunit expression, and these changes are associated with a shift in the distribution of myocytes in individual Ca2+-mobilization sub-groups in vitro. Further, post-TNF exposure, NMDA-R agonists’ treatment induced Ca2+-dependent airway dilation in murine lung slice preparations, an effect that was prevented by co-treatment with inhibitors of nitric oxide synthase (NOS) or cyclooxygenase (COX). Taken together, we conclude that NMDA-R regulate HASM-mediated airway contraction and their role can be affected upon exposure to asthma-associated inflammatory mediators. Thus, NMDA-Rs are of relevance to mechanisms that determine airway narrowing and AHR associated with chronic respiratory diseases. / October 2015

Thesis (Ph. D.)--University of Virginia, 2007. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Indiana University-Purdue University Indianapolis (IUPUI) / Coronary artery disease (CAD) is a leading cause of death among Americans and is fueled by underlying metabolic syndrome (MetS). The prevalence and lethality of CAD necessitates rigorous investigations into its underlying mechanisms and to facilitate the development of effective treatment options. Coronary smooth muscle (CSM) phenotypic modulation from quiescent to synthetic, proliferative, and osteogenic phenotypes is a key area of investigation, with underlying mechanisms that remain poorly understood. Using a well-established pre-clinical model of CAD and MetS, the Ossabaw miniature swine, we established for the first time the time course of Ca2+ dysregulation during MetS-induced CAD progression. In particular, we used the fluorescent Ca2+ dye, fura-2, to examine alterations in CSM intracellular Ca2+ regulation during CAD progression, as perturbations in intracellular Ca2+ regulation are implicated in several cellular processes associated with CAD pathology, including CSM contractile responses and proliferative pathways. These studies revealed that the function of several CSM Ca2+ handling proteins is elevated in early CAD, followed by loss of function in severe atherosclerotic plaques. Decreased intracellular Ca2+ regulation occurred concurrently with reductions in CSM proliferation, measured with Ki-67 staining. In particular, alterations in sarcoplasmic reticulum (SR) Ca2+ store together with altered function of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) were associated with induction of proliferation. Organ culture of coronary arterial segments revealed that culture-induced medial thickening was prevented by SERCA inhibition with cyclopiazonic acid (CPA). Activation of SERCA with the small molecule activator, CDN1163, increased CSM proliferation, which was attenuated by treatment with CPA, thus establishing upregulated SERCA function as a proximal inducer of CSM proliferation. Further, we demonstrated that in vitro treatment of CSM from lean Ossabaw swine with the glucagon-like peptide-1 (GLP-1) receptor agonist, exenatide, increased SERCA function. However, in vivo treatment of Ossabaw swine with MetS with the GLP-1 receptor agonist, AC3174, had no effect on CAD progression and in vitro examination revealed resistance of SERCA to GLP-1 receptor agonism in MetS. These findings further implicate SERCA in CAD progression. Collectively, these are the first data directly linking SERCA dysfunction to CSM proliferation and CAD progression, providing a key mechanistic step in CAD progression.

Indiana University-Purdue University Indianapolis (IUPUI) / Cardiovascular disease is the number one cause of death for Americans. New treatments are needed for serious conditions like atherosclerosis, as it can lead to stroke and heart attack. Many types of cells contribute to the progression of cardiovascular disease, including smooth muscle cells that comprise the middle layers of arteries. Inappropriate growth and migration of smooth muscle cells into the lumen of arteries has been implicated in vascular diseases. Death associated protein kinase (DAPK) is a protein that has been found to regulate the survival and migration of cancer cells, but has not been well characterized in vascular cells. The objective of this work was to determine the signaling pathways that DAPK regulates in smooth muscle cells. These studies have focused on smooth muscle cells isolated from human coronary arteries (HCASM cells). We have determined that HCASM cells depleted of DAPK exhibit more rapid migration, showing that DAPK negatively regulates migration of vascular cells. Results from a focused RT-PCR array identified matrix metalloproteinase 9 (MMP9) as a gene that is increased in cells depleted of DAPK. MMP9 is an important enzyme that degrades collagen, a component of the extracellular matrix through which smooth muscle cells migrate during atherosclerosis. We found that DAPK regulates phosphorylation of the NF-kappa B transcription factor p65 at serine 536, a modification previously found to correlate with increased nuclear levels and activity of p65. In DAPK-depleted HCASM cells, there was more phosphorylation of p65, which causes increased MMP9 promoter activity. Additional experiments were conducted using transgenic mice in which the DAPK gene has been deleted. By studying these mice, we have determined that under some circumstances DAPK augments maximal MMP9 levels in mouse carotid arteries which have been injured by ligation surgery via other signaling pathways. MMP9 has been previously implicated as a protein that promotes vascular diseases such as atherosclerosis. Our research in identifying DAPK as a regulator of MMP9 expression identifies a new target for treatment of vascular diseases like atherosclerosis.

OBJECTIVE:Vascular smooth muscle cell (VSMC) hypertrophy and proliferation occur in response to strain-induced local and systemic inflammatory cytokines and growth factors which may contribute to hypertension, atherosclerosis, and restenosis. We hypothesize VSMC strain, modeling normotensive arterial pressure waveforms in vitro, results in attenuated proliferative and increased hypertrophic responses 48 hrs post-strain.METHODS:Using Flexcell Bioflex Systems we determined the morphological, hyperplastic and hypertrophic responses of non-strained and biomechanically strained cultured rat A7R5 VSMC. We measured secretion of nitric oxide, key cytokine/growth factors and intracellular mediators involved in VSMC proliferation via fluorescence spectroscopy and protein microarrays. We also investigated the potential roles of VEGF on VSMC strain-induced proliferation.RESULTS:Protein microarrays revealed significant increases in VEGF secretion in response to 18 hours mechanical strain, a result that ELISA data corroborated. Apoptosis-inducing nitric oxide (NO) levels also increased 43% 48 hrs post-strain. Non-strained cells incubated with exogenous VEGF did not reproduce the antimitogenic effect. However, anti-VEGF reversed the antimitogenic effect of mechanical strain. Antibody microarrays of strained VSMC lysates revealed MEK1, MEK2, phospo-MEK1T385, T291, T298, phospho-Erk1/2T202+Y204/T185+T187, and PKC isoforms expression were universally increased, suggesting a proliferative/inflammatory signaling state. Conversely, VSMC strain decreased expression levels of Cdk1, Cdk2, Cdk4, and Cdk6 by 25-50% suggesting a partially inhibited proliferative signaling cascade.CONCLUSIONS:Subjecting VSMC to cyclic biomechanical strain in vitro promotes cell hypertrophy while attenuating cellular proliferation. We also report an upregulation of MEK and ERK activation suggestive of a proliferative phenotype. Hhowever, the proliferative response appears to be aborogated by enhanced antimitogenic cytokine VEGF, NO secretion and downregulation of Cdk expression. Although exogenous VEGF alone is not sufficient to promote the quiescent VSMC phenotype, we provide evidence suggesting that strain is a necessary component to induce VSMC response to the antimitogenic effects of VEGF. Taken together these data indicate that VEGF plays a critical role in mechanical strain-induced VSMC proliferation and vessel wall remodeling. Whether VEGF and/or NO inhibit signaling distal to Erk 1/2 is currently under investigation.

BACKGROUND: Cell migration is a vital process for growth and repair. In vitro migration assays, utilized to study cell migration, often rely on physical scraping of a cell monolayer to induce cell migration. The physical act of scrape injury results in numerous factors stimulating cell migration - some injury-related, some solely due to gap creation and loss of contact inhibition. Eliminating the effects of cell injury would be useful to examine the relative contribution of injury versus other mechanisms to cell migration. Cell exclusion assays can tease out the effects of injury and have become a new avenue for migration studies. Here, we developed two simple non-injury techniques for cell exclusion: 1) a Pyrex® cylinder - for outward migration of cells and 2) a polydimethylsiloxane (PDMS) insert - for inward migration of cells. Utilizing these assays smooth muscle cells (SMCs) and human umbilical vein endothelial cells (HUVECs) migratory behavior was studied on both polystyrene and gelatin-coated surfaces. RESULTS: Differences in migratory behavior could be detected for both smooth muscle cells (SMCs) and endothelial cells (ECs) when utilizing injury versus non-injury assays. SMCs migrated faster than HUVECs when stimulated by injury in the scrape wound assay, with rates of 1.26 % per hour and 1.59 % per hour on polystyrene and gelatin surfaces, respectively. The fastest overall migration took place with HUVECs on a gelatin-coated surface, with the in-growth assay, at a rate of 2.05 % per hour. The slowest migration occurred with the same conditions but on a polystyrene surface at a rate of 0.33 % per hour. CONCLUSION: For SMCs, injury is a dominating factor in migration when compared to the two cell exclusion assays, regardless of the surface tested: polystyrene or gelatin. In contrast, the migrating surface, namely gelatin, was a dominating factor for HUVEC migration, providing an increase in cell migration over the polystyrene surface. Overall, the cell exclusion assays - the in-growth and out-growth assays, provide a means to determine pure migratory behavior of cells in comparison to migration confounded by cell wounding and injury.

Surgical intervention can result in endothelial denudation, driving growth factor-stimulated vascular smooth muscle cell (VSMC) migration towards the intima, leading to luminal narrowing and restenosis. Clinically approved PGI₂ analogues, including beraprost, activate the cyclic adenosine monophosphate (cAMP) signaling pathway to inhibit VSMC migration in vitro. This pathway is a potential therapeutic target, however the downstream proteins involved in the inhibitory effects of cAMP on migration remain unknown. The aims of this study were to determine the signalling pathways involved in inhibiting VSMC migration through cAMP downstream mediators, protein kinase A (PKA) and the more recently characterised exchange protein activated by cAMP (Epac), and delineate the mechanisms involved. In human saphenous vein VSMCs, Epac activation using an Epac analogue inhibited VSMC migration. Therapeutic concentrations of beraprost (1 nM) also resulted in an inhibition of VSMC migration. The use of fluorescence resonance energy transfer (FRET) confirmed 1 nM beraprost activated Epac, but not PKA. Epac is a guanine nucleotide exchange factor (GEF) for Rap1 thus Rap1 siRNA was used to inhibit the Epac pathway. This blocked the inhibitory effects of beraprost on VSMC migration. Epac1 was localised to the leading edge of migrating VSMCs. Another G-protein, RhoA, was investigated since it is essential for cell migration and is involved in several processes including actin regulation. Epac signaling inhibited PDGF-induced RhoA activation and disassembled F-actin at the leading edge, where Epac1 was previously located. This indicates that beraprost activated the Epac pathway, which inhibited RhoA to decrease VSMC migration. The clinical relevance of this study has discovered the mechanisms of Epac's inhibitory action on VSMC migration and this pathway could be targeted therapeutically to reduce restenosis. In the future the potential use of beraprost on a drug eluting stent might be beneficial to prevent restenosis formation following surgical intervention.

The 1-adrenoceptors (1-ARs) serve as an interface between the sympathetic nervous system and the cardiovascular system where they are mediators of systemic arterial blood pressure, initiators of positive inotropy, and regulators of cellular growth responses. There are three subtypes: 1A-, 1B-, and 1D-ARs. This dissertation research investigated the trafficking properties of the 1-ARs at the cellular level as well as physiological relevance of the 1-ARs at the tissue level. In vitro studies using transiently transfected 1-AR/GFP subtypes revealed distinct basal localization patterns and different agonist-mediated activation and desensitization properties. The 1A- and the 1B-AR/GFP subtypes displayed agonist-mediated receptor redistribution, in which rate and degree of redistribution differed. Additionally, redistribution of either of these two receptor subtypes required arrestin-1, a protein often associated with receptor internalization. In contrast, the 1D-AR/GFP did not require arrestin-1 for maintaining the basal receptor orientation pattern. Although these data increase our knowledge of trafficking properties of the 1-AR subtypes, it is of equal importance to determine the role(s) that each subtype contributes to cardiovascular function. The lack of subtype-selective 1-AR pharmacological agents prompted the use of genetically manipulated mouse models with a systemic overexpression of a constitutively active 1B-AR. Echocardiographic analysis of transgenic hearts indicated both an enlarged left ventricular chamber in the absence of hypertrophy and a depressed cardiac function. From isolated transgenic hearts, experimental results suggested a role for the 1B-AR in attenuating the inotropic responses. However, experiments using isolated thoracic aortae from transgenic animals suggested that the 1B-AR does not participate in vascular smooth muscle contractile responses. Additional studies investigated the role of 1D-AR in cardiovascular function by using animals systemically lacking the 1D-AR subtype. Experimental data suggested an 1D-AR participation in vascular smooth muscle function since the deficiency of the 1D-AR subtype affected vasoconstriction in the coronary arteries but not inotropy in the heart. The data presented in this dissertation research suggest subtype specific differences of 1-ARs in cellular localization, signal regulation, and trafficking. Additionally, the data provide an investigation into the physiologic significance of both the 1B- and the 1D-ARs in cardiovascular tissue.

Impaired mass transfer characteristics of blood borne vasoactive species such as ATP in regions such as an arterial bifurcation have been hypothesized as a prospective mechanism in the aetiology of atherosclerotic lesions. Arterial endothelial (EC) and smooth muscle cells (SMC) respond differentially to altered local hemodynamics and produce coordinated macro-scale responses via intercellular communication. Using a computationally designed arterial segment comprising large populations of mathematically modelled coupled ECs & SMCs, we investigate their response to spatial gradients of blood borne agonist concentrations and the effect of micro-scale driven perturbation on the macro-scale. Altering homocellular (between same cell type) and heterocellular (between different cell types) intercellular coupling we simulated four cases of normal and pathological arterial segments experiencing an identical gradient in the concentration of the agonist. Results show that the heterocellular calcium (Ca2+) coupling between ECs and SMCs is important in eliciting a rapid response when the vessel segment is stimulated by the agonist gradient. In the absence of heterocellular coupling, homocellular Ca2+ coupling between smooth muscle cells is necessary for propagation of Ca2+ waves from downstream to upstream cells axially. Desynchronized intracellular Ca2+ oscillations in coupled smooth muscle cells are mandatory for this propagation. Upon decoupling the heterocellular membrane potential, the arterial segment looses the inhibitory effect of endothelial cells on the Ca2+ dynamics of underlying smooth muscle cells. The full system comprising hundreds of thousands of coupled nonlinear ordinary differential equations simulated on the massively parallel Blue Gene architecture. The use of massively parallel computational architectures shows the capability of this approach to address macro-scale phenomena driven by elementary micro-scale components of the system.

Numerous macroscale models of arteries have been developed, comprised of populations of discrete coupled Endothelial Cells (EC) and Smooth Muscle Cells (SMC) cells, an example of which is the model of Shaikh et al. (2012), which simulates the complex biochemical processes responsible for the observed propagating waves of Ca2+ observed in experiments. In a 'homogenised' model however, the length scale of each cell is assumed infinitely small while the population of cells are assumed infinitely large, so that the microscopic spatial dynamics of individual cells are unaccounted for. We wish to show in our study, our hypothesis that the homogenised modelling approach for a particular system can be used to replicate observations of the discrete modelling approach for the same system. We may do this by deriving a homogenised model based on Goldbeter et al. (1990), the simplest possible physiological system, and comparing its results with those of the discrete Shaikh et al. (2012), which have already been validated with experimental findings. We will then analyse the mathematical dynamics of our homogenised model to gain a better understanding of how its system parameters influence the behaviour of its solutions. All our homogenised models are essentially formulated as partial differential equations (PDE), specifically they are of type reaction diffusion PDEs. Therefore before we begin developing the homogenised Goldbeter et al. (1990), we will first analyse the Brusselator PDE with the goal that it will help us to understand reaction diffusion systems better. The Brusselator is a suitable preliminary study as it shares two common properties with reaction diffusion equations: oscillatory solutions and a diffusion term.