Nox1 is of considerable importance because of its involvement in a wide variety of pathologies. Activation of Nox1 induces generation of reactive oxygen species (ROS) and cell migration, events critical for the pathogenesis of cardiovascular disease, amyotropic lateral sclerosis, gastrointestinal disease, immunological disorders, and multiple forms of cancer [1-8]. In order to best determine how to treat Nox1-mediated disease, we must gain a better understanding of the mechanisms that control Nox1 activation. Within the last decade, many studies have found that protein phosphorylation and protein trafficking are critical regulatory mechanisms that control the activation of multiple Nox proteins. Yet, to date, no studies have characterized Nox1 phosphorylation or trafficking. We hypothesized that the activity of Nox1 is controlled by its phosphorylation at specific residues and by its sub-cellular localization; and that modifying Nox1 phosphorylation or localization will alter Nox1-dependent signaling. To test this hypothesis, we utilized both in vivo and in vitro approaches. We found that phosphorylation of Nox1 is significantly increased under pathological conditions in three in vivo models: (1) in atherosclerotic vs. normal aorta from monkey, (2) in neointimal vascular smooth muscle cells (VSMCs) vs. medial VSMCs from rat following aortic balloon injury, and (3) in ligated vs. normal carotid from mouse. Studies using mass spectroscopy, pharmacological inhibition, siRNA, and in vitro phosphorylation identify PKC-βI as a kinase that mediates Nox1 phosphorylation and subsequent ROS production and VSMC migration. Site-directed mutagenesis of predicted Nox1 phospho-residues revealed that cells expressing mutant Nox1 T429A have a significant decrease in TNF-α-stimulated ROS production, VSMC migration and Nox1 NADPH oxidase complex assembly compared to cells expressing wild-type Nox1. Isothermal calorimetry (ITC) revealed that a peptide containing the Activation Domain of NoxA1 (LEPMDFLGKAKVV) binds to phosphorylated Nox1 peptide (KLK-phos-T(429)- QKIYF) but not non-phosphorylated Nox1 peptide. These findings indicate that phosphorylation of Nox1 residue T429 by PKC-βI promotes TNF-α-induced Nox1 NADPH oxidase complex assembly, ROS production, and VSMC migration. Nox1 localization and trafficking studies reveal that Nox1 endocytosis is necessary for TNF-α-induced Nox1 ROS production; and that mutation of a Nox1 VLV motif inhibits Nox1 endocytosis and ROS production. These studies have provided new evidence that phosphorylation and sub-cellular localization are involved in the regulation of Nox1 ROS production and cell migration and offer new insights as to how Nox1 activity can be targeted for the purpose of treating Nox1-mediated diseases.

The skeletal muscle fibre is a syncitium where each myonucleus regulates the gene products in a finite volume of cytoplasm i.e., the myonuclear domain (MND). A novel image analysis algorithm applied to confocal images, analyzing MND size and myonuclear spatial distribution in 3-dimensions in single skeletal muscle fibres has been used in this project. The goal was to explore the modulation of myonuclei count and MND size in response to muscle adaptation processes. The effects of ageing, gender, hormones, muscle hypertrophy and body size were investigated on MND size. A strong linear relationship was found between MND size and body size in the muscle fibres from mammals representing a 100,000-fold difference in body size. Independent of species, MND size was highly dependent on MyHC isoform type and mitochondrial contents of skeletal muscle fibres. In hypertrophic mice, a significant effect of MND size on specific force and myosin content was observed. This effect was muscle fibre type-specific and shows that the bigger MNDs in fast-twitch EDL muscle fibres are optimally tuned for force production while smaller MNDs in slow-twitch soleus muscle fibres have a much more dynamic range of hypertrophy without functional compromise. This indicates a critical volume individual myonuclei can support efficiently for a proportional gain in muscle fibre force and size. In human muscle fibres, spatial organization of myonuclei was affected by both ageing and MyHC isoform expression. In fibres expressing type I MyHC isoform, an increased MND size variability and myonuclear aggregates were observed in old age although average MND size was unchanged. In contrast, in type IIa fibres, the average MND size was smaller reflecting smaller size of muscle fibres. Those changes may influence the transcriptional activity per myonucleus and/or local cooperatively of myonuclei in a gender and muscle fibre-type specific manner. Finally, hormone replacement therapy was shown to negate menopause-related functional impairment in skeletal muscle fibres. The positive effect on force was due to quantitative effect in fibres expressing fast myosin isoform while the effect was both quantitative and qualitative in fibres expressing slow myosin isoform. The effect on MND size was fibre type dependent and was achieved by significantly reducing domain size in slow- but not the fast-twitch muscle fibres. Together, our data suggest that modulation of myonuclei count and MND size is a mechanism contributing to remodelling of skeletal muscle in muscle adaptation process. These findings should be considered when developing therapeutic approaches towards restoring muscle mass and strength in muscle wasting conditions.

Despite efforts by clinicians and scientists world-wide, coronary artery disease remains to be the leading cause of morbidity and mortality in industrialized nations. Development of a tissue engineered coronary by-pass graft with low thrombogenicity and immune responses, suitable mechanical properties, and a capacity to remodel to their environment could have a significant impact on the treatment of coronary artery disease. While many methods for the tissue engineering of blood vessels have been developed, one promising approach is the self-assembly method. Using autologous cells that produce an endogenous extracellular matrix (ECM), the potential for therapeutic success is high due to biocompatibility. However, despite these advantages, improvements can be made which will give the grafts an even higher rate of patency. This dissertation presents a study of the characterization of the biaxial mechanical properties of self-assembly tissue engineered blood vessels (SA-TEBV), as well as developing a framework for fabrication strategies of SA-TEBV. Native arteries are exposed to multiaxial mechanical loads, including (a pulsatile) blood pressure that causes the vessel to cyclically distend circumferentially, blood flow that induces a shearing load along the luminal surface, and an axial extending load; the latter is relieved upon excision, causing the vessel to retract. These mechanical loads introduce intramural wall stresses and flow induced wall shear stresses that play a key role in mechano-biological signaling and tissue homeostasis. Until now, the mechanical properties of SA-TEBV have only been characterized in the circumferential direction (i.e. burst pressure and circumferential elastic modulus). The objective of this work is to characterize the biaxial mechanical properties of SA-TEBV to quantify their mechanical behavior and local intramural stresses under physiological loading. The work will show that while the global mechanical response of the SA-TEBV is similar to that of native arteries (and potentially sufficient), the local intramural stresses (using the current fabrication techniques) differ greatly from native coronary arteries. Therefore, a novel approach to fabricate the self-assembly derived tissue sheets is developed and tested which utilizes biaxial mechanical stimulation to alter the microstructure, thereby controlling their mechanical response.

The cystic fibrosis transmembrane conductance regulator (CFTR) was identified to be involved in acidosis-induced ATP release from skeletal myocytes in vitro and from contracting muscle in vivo. My PhD studies aimed to investigate the underlying mechanism and identify the pathway for ATP release in acidosis-induced CFTR-regulated ATP release. Lactic acid (10 mM) decreased the intracellular pH of L6 skeletal myocytes to 6.87 ± 0.12 after 3 hours, and the lowered pH resulted in the elevation of ATP release from skeletal myocytes. The acidosis-induced ATP release was totally abolished by GlyH-101 (40 μM), an open-channel CFTR blocker, suggesting that CFTR was involved. The cAMP/PKA signaling pathway was involved in the CFTR-regulated ATP release from skeletal myocytes: 1). Forskolin increased the extracellular ATP and the phosphorylation of CFTR; IBMX, a phosphodiesterase inhibitor, further enhanced the forskolin-induced extracellular ATP and phosphorylation of CFTR; 2). Inhibition of PKA by its selective inhibitor KT-5720 abolished the acidosis-induced ATP release and the forskolin-induced phosphorylation of CFTR. In addition, the inhibition of Na+/H+ exchanger (NHE) by amiloride, or inhibition of Na+/Ca2+ exchanger (NCX) by its specific inhibitors SN-6 and KB-R7943 abolished the lactic-acid-induced ATP release from skeletal myocytes, indicating that NHE and NCX might be involved. Previous studies demonstrated that Connexin hemichannels and Pannexin channels were able to conduct ATP in response to stimuli. This study found that connexin 43 (Cx43) was strongly expressed on skeletal myocytes, while Pannexin 1 (Panx1) showed a strong expression in gastrocnemius muscle. Investigation of the role that Cx43 may play in acidosis-induced cAMP/PKA-activated CFTR-regulated ATP release from myocytes showed that: 1). Cx43 was immunoprecipitated with CFTR suggesting a physical interaction; 2). The opening of Cx hemichannels was increased by lactic acid and this lactic-acid-induced opening was inhibited by CFTRinh-172, suggesting the mediation of CFTR; 3). Inhibition of Cxs and Panxs with carbenoxolone abolished the acidosis-induced ATP release; moreover, specific silencing of the Cx43 gene using siRNA decreased both basal and acidosis-induced ATP release, suggesting that Cx43 was involved; 4). Overexpression of CFTR alone did not elevate the acidosis-induced ATP release, while overexpression of Cx43 alone doubled the acidosis-induced ATP, and co-overexpression of CFTR and Cx43 further elevated the acidosis-induced ATP release, supporting the concept that Cx43 functionally interacted with CFTR to induce the acidosis-induced ATP release. Panx1 was studied in native skeletal muscle, and found to be coimmunoprecipitated with CFTR. Inhibition of Panxs with gadolinium or probenecid abolished the muscle-contraction-induced ATP release, while inhibition with carbenoxolone or quinine reduced it to less than 10% of control, suggesting that Panx1 may be involved in the acidosis-induced ATP release during muscle contraction. All the in vitro and in vivo studies suggested that Cxs and Panx were involved in the acidosis-induced CFTR-regulated ATP release from skeletal myocytes and skeletal muscle. / published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

In normal adult physiology, intestinal smooth muscle cells (ISMC) are characterized as contractile and non-proliferative. Inflammation induces permanent changes to the intestine including hypertrophy of the smooth muscle layer largely due to smooth muscle cell (SMC) proliferation. While the consequences of this hyperplasia are largely unknown, increased muscularis mass may present permanent challenges to organ motility. Similar SMC hyperplasia is observed in other inflammatory pathologies including atherosclerosis and pulmonary arterial hypertension (PAH) where SMC de-differentiate into a ‘synthetic’ phenotype and the mitogens responsible for hyperplasia have been well studied. However, there are limited investigations of SMC mitogens in intestinal inflammation. The identification of these factors may be of critical importance in the case of intestinal strictures, whereby recurring inflammation can lead to bowel obstruction requiring surgical intervention. A novel, primary rat ISMC model was developed to identify the factors responsible for ISMC proliferation in vitro. Primary ISMC cultures are likely more representative of SMC in vivo than the commonly used late-passage cultures. As such, this primary ISMC model is valuable in the evaluation of mitogens involved in the onset of proliferation. This primary ISMC model was used to conduct a comprehensive evaluation of potential mitogens including basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1) and platelet-derived growth factor-BB (PDGF-BB. This work identified IGF-1 and PDGF-BB as ISMC mitogens. However, multiple lines of evidence indicated that PDGF-BB was a more potent mitogen and the involvement of PDGF-BB was subsequently examined in vivo using the trinitrobenzenesulfonic acid (TNBS) model of rat intestinal inflammation. While control ISMC lacked expression of the PDGF-BB receptor (PDGF-Rβ), robust expression was observed within only 6 hr following the induction of TNBS inflammation. By Day 2, when ISMC proliferation in vivo is maximal, freshly isolated ISMC showed on-going PDGF-Rβ activation that was further increased by exogenous PDGF-BB. Taken together, the conclusions from this work in vitro identify PDGF-BB as a potent ISMC mitogen in vivo. Further, this work establishes PDGF-BB and its receptor as potential targets in the medical treatment of intestinal stricture formation. / Thesis (Ph.D, Biology) -- Queen's University, 2012-09-24 19:26:57.201

The neuron-derived orphan receptor 1 (NOR1) belongs to the NR4A nuclear receptor subfamily. As an immediate early response gene, NOR1 is rapidly induced by a broad spectrum of physiological and pathological signals. Functional studies demonstrate NOR1 as a constitutively active ligand-independent nuclear receptor whose transcriptional activity is dependent on both expression level and posttranslational modifications. To date, an increasing number of studies have demonstrated a pivotal role of NOR1 in the transcriptional control of metabolism and the development of cardiovascular diseases. In this dissertation, we demonstrate NOR1 expression in endothelial cells and sub-endothelial cells of human atherosclerotic lesions. In response to inflammatory stimuli, NOR1 expression is rapidly induced in endothelial cells through an NF-κB-dependent signaling pathway. Functional studies reveal that NOR1 increases monocyte adhesion by inducing the expression of adhesion molecules VCAM-1 and ICAM-1 in endothelial cells. Transient transfection and chromatin immunoprecipitation assays identify VCAM-1 as a bona fide NOR1 target gene in endothelial cells. Finally, we demonstrate that NOR1-deficiency reduces hypercholesterolemia-induced atherosclerosis formation in apoE-/- mice by decreasing the macrophage content of the lesion. In smooth muscle cells (SMC), NOR1 was previously established as a cAMP response element binding protein (CREB) target gene in response to platelet-derived growth factor (PDGF) stimulation. CREB phosphorylation and subsequent binding of phosphorylated CREB to the NOR1 promoter play a critical role in inducing NOR1 expression. In this dissertation, we further demonstrate that histone deacetylase (HDAC) inhibition potentiates and sustains PDGF-induced NOR1 mRNA and protein expression in SMC. This augmented NOR1 expression is associated with increased phosphorylation of CREB, recruitment of phosphorylated CREB to the NOR1 promoter, and trans-activation of the NOR1 promoter. Additionally, HDAC inhibition also increases NOR1 protein half-life in SMC. Collectively, these findings identify a novel pathway in endothelial cells underlying monocyte adhesion and expand our knowledge of the epigenetic mechanisms orchestrating NOR1 expression in SMC. Finally, we establish a previously unrecognized atherogenic role of NOR1 in positively regulating monocyte recruitment to the vascular wall.

Collagens are abundant components of the extracellular matrix in the atherosclerotic plaque. In addition to contributing to lesion volume and mechanical stability, collagens can influence the behavior of macrophages and smooth muscle cells (SMCs) and have profound effects on both inflammation and fibrosis during lesion development. The aim of this thesis was to define a functional role for the discoidin domain receptor 1 (DDR1), a collagen receptor tyrosine kinase, in murine models of atherogenesis. In our first study, using Ddr1+/+;Ldlr-/- and Ddr1-/-;Ldlr-/- mice fed a high fat diet, we identified DDR1 as a novel positive regulator of atherogenesis. Targeted deletion of DDR1 attenuated atherosclerotic plaque development by limiting inflammation and accelerating matrix accumulation and resulted in the formation of macrophage poor, matrix rich lesions. In the second study, we used bone marrow transplantation to generate chimeric mice with a deficiency of DDR1 in bone marrow derived cells and reveal a central role for macrophage DDR1 in atherogenesis. Deficiency of DDR1 in bone marrow derived cells reduced lesion size by limiting macrophage accumulation in the developing plaque. Moreover using BrdU pulse labeling, we demonstrated reduced monocyte recruitment into the early fatty streak lesions of Ddr1-/-;Ldlr-/- mice. In our third study, we again utilized bone marrow transplantation to generate mice with deficiency of DDR1 in the host derived tissues such as the vessel wall and uncovered a distinct role for DDR1 expressed on resident vessel wall smooth muscle cells in the regulation of matrix accumulation and fibrous cap formation during atherogenesis. Deficiency of DDR1 in vessel wall cells resulted in robust accumulation of collagen and elastin and resulted in the formation of larger atherosclerotic plaques, with thick fibrous caps. Taken together, these studies support a critical role for DDR1 in the development of the atherosclerotic plaque. We demonstrate that DDR1 exerts distinct and opposing effects on lesion size by regulating both monocyte recruitment and matrix accumulation. These studies underscore the importance of collagen signaling during atherogenesis, and identify DDR1 as a key transducer; providing signals that regulate both inflammation and fibrosis during atherogenesis.

Cardiovascular disease, which involves the cardiac, cerebrovascular and peripheral vascular system, is the major cause of morbidity and mortality in the western world. Changes in the vascular microenvironment trigger cascades of molecular events involving altered signaling, transcription and translation of a gene. The aim of this thesis was to increase our understanding on the molecular regulation of activated vascular smooth muscle cells. The first study looking at PDGF-D expression provides new insights into the regulatory mechanisms controlling the phosphorylation of Sp1. Studies performed identified three amino acids in Sp1 (Thr668, Ser670 and Thr681) that is phosphorylated by PKC-zeta activated by AngII. In the second study, the translational regulatory role of a novel gene YrdC induced by injury was investigated. Current knowledge of translational regulators controlling altered gene expression is little and studies in this thesis shows a splice variant of YrdC playing an important role in controlling mRNA translation and thus protein synthesis in the context of injury. The final study investigated in this study was the increased expression of the apoptotic FasL by the activation of GATA6. Although FasL has been extensively studied over the years, this is the first study linking a GATA factor with FasL in any cell type and provides key insights into the transcriptional events underpinning FasL-dependent SMC apoptosis following exposure to AngII.

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "December 2007" Includes bibliographical references.

Thesis (doctoral)--Lund University, 1994. / Added t.p. with thesis statement inserted.