Doctor of Philosophy (PhD) / It is well recognised that both genetic and environmental factors determine an individual’s predisposition to asthma. In recent years, the airway smooth muscle (ASM) cell has come to the attention of researchers to, not merely be a contractile cell of the airway, but one that orchestrates events affecting airway remodelling and proliferation. Experiments described in this thesis have, for the first time, examined genes that are associated with various aspects of the pathogenesis of asthma by using the candidate gene approach and a genome wide search. Genes have not only been identified to be differentially expressed in ASM cells derived from asthmatic and non-asthmatic participants, but have also been linked with a functional consequence of asthma. The three genes found to be differentially regulated between ASM cells derived from asthmatic and non-asthmatic participants were Peroxisome Proliferator-Activated Receptor- gamma (PPARγ), mimecan and fibulin-1. Expression of the anti-proliferative transcriptional factor PPARγ, found by the candidate gene approach, was elevated in ASM cells derived from asthmatic participants. Whilst elevated, the anti-proliferative effect of PPARγ was absent in ASM cells derived from asthmatic participants. By microarray analysis, mimecan, an anti-proliferative agent was identified. Mimecan levels, although not different basally in ASM cells, were upregulated by transforming growth factor β (TGFβ) only in asthmatic derived ASM cells. Silencing mimecan, by the use of specific oligonucleotides, increased proliferation of ASM cells. This suggested that by increasing mimecan expression, the proliferation of ASM cells may be halted. Fibulin-1, also found by microarray analysis and the final gene examined in this thesis, was found in elevated levels in BAL fluid, serum and ASM cells obtained from asthmatic participants. In addition, ASM cells derived from asthmatic participants, for the first time were shown to have faster wound healing rates compared with nonasthmatics. The elevated fibulin-1 levels in ASM cells derived from asthmatic participants, in the presence of TGFβ, were demonstrated to contribute to this increased wound healing. Specifically, fibulin-1 was found to affect wound healing by increasing proliferation rather than migration. The current available treatments for asthma, target the contractility and inflammatory conditions in the airway. Through this thesis, novel genes discovered to be associated with proliferation may be potential therapeutic targets to treat asthma. In particular, the fibulin-1 gene is outstandingly promising, as it was shown that silencing fibulin-1 resulted in slower wound healing rates through decreased cell proliferation, to possibly inhibit the airway remodelling observed in asthma, and furthermore, corticosteroid therapy was demonstrated not to affect to this gene.

TTAGGG repeat factor 2 (TRF2) is a protein that plays an important role in capping telomere ends from DNA damage responses. Telomere DNA consists of double strand repeats of the TTAGGG sequence ending with a 3'single-stranded overhang of the guanine strand (the G-strand overhang). TRF2 protects telomeres from being recognized as double-stranded breaks. It is thought that this protection is performed through the formation of T-loop structures and recruitment of proteins into a complex called shelterin. The exact mechanism of T-loop formation is unknown. I show with in vitro biochemical studies that TRF2 specifically interacts with telomeric ss/ds DNA junctions and binding is sensitive to the sequence of the G-strand overhang and double-stranded DNA sequence at the junction. Binding assays with TRF2 truncation mutants suggest that TRF2 interacts with both the double-stranded DNA through the C-terminal DNA binding domain and the G-strand overhang through the N-terminus. Mobility shifts and atomic force microscopy with truncation mutants bound to telomeric DNA also show that a previously uncharacterized "linker" region within TRF2 is involved in DNA-specific TRF2 oligomerization. From these observations, I suggest that TRF2 forms protective loops by oligomerizing through both a previously characterized dimerization domain and the linker region. I propose that loop formation involving the telomere ends is accomplished through direct interactions between TRF2 and the G-strand overhang. In addition to DNA protection, a new role has emerged for TRF2 in sensing DNA damage. TRF2 can be phosphorylated within its dimerization domain by ATM and recruited to DNA damage foci in cells. The inhibition of TRF2 function alone has been shown to induce senescence and apoptosis in vascular endothelial cells. Since the common stimuli for a senescence phenotype is activation of a DNA damage response, I studied the relationship between DNA damage and TRF2 phosphorylation. Ex-vivo characterization of DNA damage-induced changes in vascular smooth muscle cells (VSMC) was undertaken. VSMC treated with H202 induced an increase in reactive oxygen species (ROS), and 8-oxo-guanine accumulation resulting in cell cycle arrest, chromatin condensation and a senescent phenotype. Interestingly phosphorylated TRF2 and ATM were also up regulated. Balloon injury was used to test the connection between phosphorylated TRF2 and senescence during vascular remodeling in rat arteries. Vascular remodeling as judged by neointima formation was associated with accumulation of 8-oxo-guanine, DNA damage signaling, including phosphorylated TRF2, an increase in cell cycle inhibitors and senescence. These events were exaggerated in aged animals and are consistent with a role in telomere dysfunction, and age related diseases.

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.

The effect of Ddr1 deletion on the expression of genes involved in atherosclerotic vascular remodeling and on the development of atherosclerotic calcification Pamela J. Ahmad, PhD Institute of Medical Science, 2008 During atherosclerosis, collagen molecules, which are abundant in the healthy vessel, are extensively degraded, re-synthesized or newly synthesized, and remodeled to induce profound changes in VSMCs as they colonize and expand atherosclerotic lesions. The central theme of this thesis was to investigate the effect of genetic deletion of a collagen receptor, DDR1, on VSMC processes during atherosclerosis. In the first study, we demonstrated a role for DDR1 as an important regulator of gene expression in synthetic VSMCs. We have profiled the expression of vascular collagen matrix molecules, MMPs and TIMPs in synthetic VSMCs and we have demonstrated that deletion of Ddr1 is sufficient to accelerate ECM remodeling in synthetic VSMCs, which may influence cell migration during atherosclerosis. Moreover, we have extended our knowledge of DDR1 function in synthetic VSMCs, by demonstrating that DDR1 limits VSMC proliferation in a complex matrix microenvironment representative of the ECM produced in the vessel wall during vascular disease. In the second study, we investigated the role of DDR1 in atherosclerotic calcification, a feature of advanced atherosclerotic disease. Here, we demonstrated that intimal calcification in Ldlr-/- mice fed a high-fat/ high-cholesterol diet may be mediated through the initiation of a chondrogenic transcriptional regulatory program and that deletion of Ddr1 significantly attenuated the frequency and extent of atherosclerotic mineralization in vivo, as well as the ability of vascular smooth muscle cells to calcify in vitro, suggesting an important role for DDR1 in VSMCs as a positive regulator of this pathological process. In our third study, we provided evidence of a biochemical association between MMP-2 and DDR1b in VSMCs, which involves a direct interaction between MMP-2 and the extracellular region of the DDR1 receptor. In addition, we reported an association between endogenous MMP-2 and Stat1 in VSMCs, providing a platform for future research to investigate the functional consequences of these novel interactions.

Previous studies have shown that L-tryptophan treatment has been known to reduce blood pressure (BP) in hypertensive rats. L-tryptophan is converted to serotonin (5-HT), a potent vasoconstrictor agonist. The direct vascular effects of L-tryptophan, an essential amino acid, and the mechanism that contributes to the fall in BP have not been fully explored. The present study aims to examine the direct vascular responses to both D- and L- tryptophan using perfused mesenteric vascular bed, an ex-vivo preparation that represents the resistance function of circulation. Perfusion was maintained at a constant flow rate (5 mL/min) with Krebs buffer (pH 7.4, 37˚C) after isolation from 12 to 14 week old male Sprague-Dawley rats. The basal perfusion pressure (PP) (mean ± SEM) was 27 ± 3 mmHg. Inclusion of D- and L-isomers in the perfusion medium led to concentration-dependent increase in PP. While the maximal response (Emax) was similar, D-tryptophan (EC50: 0.25 ± 0.12* µmol; Emax: 128 ± 8 mmHg) was more potent (lower EC50 value; *p < 0.01) than L-tryptophan (EC50: 0.79 ± 0.30 µmol; Emax: 141 ± 7 mmHg). Inclusion of increasing concentrations (2, 5 and 10 nM) of the 5-HT2A selective antagonist, ketanserin, led to parallel right-ward shifts in the concentration-response curves to D- and L-tryptophan with restoration of their Emax. In contrast, the α1 selective agonist, methoxamine (30 µM), constricted preparations, both D- (IC50: 0.94 ± 0.30* µmol; Imax: 96 ± 2%) and L-tryptophan (IC50: 2.8 ± 1.0 µmol Imax: 88± 1%) evoked concentration-dependent vasodilatation, an effect that was resistant to blockade by either ketanserin or other 5-HT antagonists. Again, D-tryptophan was more potent than L-tryptophan in the presence of 5-HT antagonist (*p < 0.05). Neither the removal of endothelium nor incubation with selective inhibitors of dilatory mediators released from the endothelium, failed to alter the vasodilator responses to D- and L-tryptophan. In potassium chloride depolarized preparations, L-tryptophan evoked an additive vasoconstrictor response. The vasodilator responses to L-tryptophan persisted in the presence of glibenclamide, a KATP channel inhibitor, or tetraethyl ammonium, a BKCa channel inhibitor, or BaCl2, a Kir channel inhibitor, or ouabain, a Na+-K+-ATPase pump inhibitor. These data confirm that the essential amino acid, L-tryptophan, as well as its D-isomer, evoke a biphasic vasoconstrictor and vasodilator responses in the resistance type mesenteric vascular bed. While the vasoconstrictor responses are mediated by activation of vascular 5-HT receptors, the endothelium-independent vasodilator responses are not linked to activation of vascular 5-HT receptors, vascular potassium channels, Na+-K+-ATPase pump or via inhibition of voltage-operated Ca2+-channels. Plasma concentration of L-tryptophan is about 90 - 120 µM. The endothelium/5-HT independent direct vasodilator responses characterized here for the first time could account for the antihypertensive/ BP lowering effect of L-tryptophan reported earlier by other laboratories.

Methylglyoxal (MG) is a reactive á-oxoaldehyde and a glucose metabolite. Previous studies in our laboratory have shown that MG induces the production of reactive oxygen species (ROS), such as superoxide (O2.-), nitric oxide (NO) and peroxynitrite (ONOO-), in vascular smooth muscle cells (VSMCs, A-10 cells). However, the effect of endogenous MG and mechanisms of MG-induced oxidative stress have not been thoroughly explored. The present study investigated fructose (a precursor of MG)- induced ONOO- formation in A-10 cells and whether this process was mediated via endogenous MG formation; roles of MG in regulating mitochondrial ROS (mtROS) production and mitochondrial functions in A-10 cells; and effect of MG on neutrophils in patients with type 2 diabetes mellitus (T2DM). Fructose induced intracellular production of MG in a concentration- and time- dependent manner. A significant increase in the production of NO, O2.−, and ONOO− was observed in the cells exposed to fructose or MG. Fructose- or MG-induced ONOO− generation was significantly inhibited by MG scavengers and by O2.− or NO inhibitors. The data showed that fructose treatment increased the formation of ONOO− via increased NO and O2.− production in A-10 cells, and this effect was directly mediated by an elevated intracellular concentration of MG. By inhibiting complex III and manganese superoxide dismutase activities, MG induced mitochondrial overproduction of O2.-, and mitochondrial ONOO- further. MG also reduced mitochondrial ATP synthesis, indicating the dysfunction of mitochondria. In addition, MG increased plasma NO levels in patients with T2DM, which reflected the oxidative status in those patients. MG-induced oxidative stress in patients with T2DM significantly enhanced levels of cytokines released from neutrophils. Moreover, the neutrophils from T2DM patients showed a greater proclivity for apoptosis, which was further increased by in vitro MG treatment. Our data demonstrate that MG-induced oxidative damage, particularly ONOO- production, contributes to the pathogenesis of T2DM and its vascular complications.

Increased intracellular calcium (Ca2+) is required for vascular smooth muscle cell (VSMC) proliferation through mechanisms that are not well-known. Preventing calmodulin (CaM)-cyclin E interaction with a synthetic peptide inhibits VSMC proliferation in a cyclin E-dependent manner, without increasing de-differentiation or cell death, or affecting re-endothelialization or collagen deposition. Moreover, in situ Ca2+-sensitive phosphorylation and degradation of the cell cycle inhibitor p27Kip1 (p27) in VSMC is specific to G1 and dependent on camodulin kinase-II (CaMK-II) and the proteasome, but not MEK. Lastly, IQGAP1 binding to CaM increases during G1 with no change in total IQGAP1 expression across the cell cycle. Therefore, we determined the clinical potential of an established mechanism (CaM/cyclin E), the existence of a putative mechanism (CaMK-II/p27), and a target novel mechanism (CaM-IQGAP1). Characterization of calcium-sensitive mechanisms of VSMC cycle control could form the basis for new drug-eluting stent agents that have increased selectivity for rapidly dividing VSMC.

Increased intracellular calcium (Ca2+) is required for vascular smooth muscle cell (VSMC) proliferation through mechanisms that are not well-known. Preventing calmodulin (CaM)-cyclin E interaction with a synthetic peptide inhibits VSMC proliferation in a cyclin E-dependent manner, without increasing de-differentiation or cell death, or affecting re-endothelialization or collagen deposition. Moreover, in situ Ca2+-sensitive phosphorylation and degradation of the cell cycle inhibitor p27Kip1 (p27) in VSMC is specific to G1 and dependent on camodulin kinase-II (CaMK-II) and the proteasome, but not MEK. Lastly, IQGAP1 binding to CaM increases during G1 with no change in total IQGAP1 expression across the cell cycle. Therefore, we determined the clinical potential of an established mechanism (CaM/cyclin E), the existence of a putative mechanism (CaMK-II/p27), and a target novel mechanism (CaM-IQGAP1). Characterization of calcium-sensitive mechanisms of VSMC cycle control could form the basis for new drug-eluting stent agents that have increased selectivity for rapidly dividing VSMC.

ATP-sensitive potassium (K+) channels (KATP) in vascular smooth muscle cells (VSMC) play a major role in the regulation of vascular tone by coupling cell contractility and K+ fluxes to cellular metabolism. They are composed of the regulatory sulphonylurea receptors (SUR) and the pore-forming inwardly rectifying K+ (Kir) channels. SUR subunits interact closely with Kir subunits by conferring their sensitivity to nucleotide or sulphonylurea. However, the modulatory mechanisms of KATP channels in VSMC are largely unknown. In particular, the effects of hydrogen sulfide (H2S) and hydroxylamine (HA) on KATP channels and underlying mechanisms have not been addressed in VSMC of resistance arteries. The combined approaches including molecular biology, biochemical assays, and patch-clamp techniques were applied. The electrophysiological and pharmacological features of native KATP channels in VSMC and cloned KATP channels in HEK-293 cells, and the modulation of KATP channels by H2S and HA in single freshly isolated VSMC from rat mesenteric arteries were characterized. In the present study, only small conductance KATP channels of 13 pS were found in rat mesenteric artery VSMC. The recorded macroscopic and unitary KATP currents were activated by nucleoside diphosphate in the presence of magnesium and K+ channel openers, inhibited by a specific KATP channel blocker glibenclamide, but were insensitive to ATP inhibition. The reversal potential shifted rightward in response to the elevation of extracellular K+ and matched the calculated K+ equilibrium potential, indicating the basal currents in both VSMC and HEK-293 cells are carried by K+ ions. Heterologous expression of Kir6.1 with SUR2B in HEK-293 cells formed functional channels and elicited whole-cell K+ currents, which shared some similar biophysical characteristics of native KATP channels in VSMC. Basal KATP currents and resting membrane potential in VSMC were reduced by glibenclamide, demonstrating that KATP channels contribute to background K+ conductance and in the setting of resting membrane potential in this resistance artery. Exogenous H2S enhanced macroscopic and unitary KATP currents with an EC50 of 116 ± 8.3 µM and hyperpolarized membrane potential. H2S activated KATP channels by increasing the open probability of single channels, but not single channel conductance. The reduced endogenous H2S production by D, L-propargylglycine resulted in the attenuation of KATP currents. H2S-induced activation of KATP channels and resultant hyperpolarization were not mediated by cGMP signaling pathway. HA enhanced reversibly KATP currents in a dose-dependent fashion with an EC50 of 54±3.4 µM and also hyperpolarized the cell membrane. HA-stimulated KATP currents were blocked by free radical scavengers (superoxide dismutase and N-acetyl-L-cysteine), and KATP channels were stimulated by a free radical generating system (hypoxanthine/xanthine oxidase), indicating the involvement of superoxide (O2-) in HA effects. Sodium nitroprusside and 8-Br-cGMP did not affect basal KATP currents and HA-stimulated KATP currents, disproving the involvement of NO-sGC-cGMP-mediated signaling pathway in the HA effects. Therefore, HA-induced KATP channel activation and hyperpolarization are likely due to the generation of O2-. In conclusion, KATP channels in resistance artery VSMC serve as the regulatory targets of H2S and HA. These two endogenous molecules modulate KATP channels via different mechanisms. H2S may directly act on KATP channel proteins while HA oxidized them via the formation of O2-, leading to the activation of KATP channels.