Thesis (Ph.D.)--Ohio University, March, 2006. / Includes bibliographical references (leaves 152-160)

Le travail présenté ici porte sur l’étude de la production et du transport du monoxyde d’azote (NO) dans le poumon humain. Le NO est une molécule dont l’implication dans des processus physiologiques n’a été mis en évidence qu’en 1987. Depuis, il a été démontré que le NO joue de nombreux rôles dans le corps humain. Le NO est un gaz labile (instable) dans les conditions physiologiques, il diffuse très facilement au travers des parois et il a une grande affinité pour l’hémoglobine. La production du NO est liée à 3 isoformes différentes de la protéine appelées synthases du NO ou NO synthases. En 1991, Gustafsson et al. ont découvert du NO endogène (produit par les poumons) dans l’air exhalé chez l’homme et le cochon d’Inde. Depuis près de 15 ans, de plus en plus de groupes de recherche travaillent sur le NO pulmonaire sans s’accorder sur ses rôles exactes. Il est cependant établit que, dans les pathologies comme l’asthme, la production accrue de NO est liée aux processus inflammatoires. Le NO peut être produit par la surface épithéliale (au niveau des conduits pulmonaires) ou alvéolaire, mais les sites exactes de production sont encore débattus. De part l’impossibilité de mesures directes au-delà des premières générations de l’arbre bronchique, les modèles mathématiques sont indispensables pour interpréter les résultats de mesures de concentrations exhalées et étudier la production et le transport du NO dans le poumon. Récemment, un modèle a été proposé par notre groupe, tenant compte de la convection, de la diffusion moléculaire et de la production du NO dans le poumon. Le but de ce travail est d’utiliser et d’adapter ce modèle pour reproduire des résultats expérimentaux soit existants, soit originaux. Dans ce travail, nous montrons que l’augmentation de concentration alvéolaire chez des sujets asthmatiques bien contrôlés est liée à une augmentation de production dans les conduits et non dans les alvéoles. Nous montrons également que, sur base de résultats expérimentaux, la production bronchique dans le poumon des sujets sains doit être très hétérogène pour reproduire des résultats expérimentaux apparemment irréconciliables. Nous montrons enfin que la localisation des conduits pulmonaires subissant une constriction influence la chute de NO exhalé mesurée après cette constriction. Nous avons également participé à 2 expériences liées à la gravité qui constitueront le matériel susceptible de faire évoluer le modèle.

Endothelial progenitor cells (EPCs) have reduced neovascularization capacity in the context of coronary artery disease (CAD) or cardiac risk factors (RFs). Since, endothelial NO synthase (eNOS) is critical to normal EPC function, we hypothesized that bone marrow cells (BMCs) from rats with RFs and EPCs from humans with CAD and/or RFs show dramatically reduced neovascularization capacity in vitro and in vivo, which can be reversed by eNOS overexpression. BMCs were isolated from rat models of type II diabetes and the metabolic syndrome, and we showed a significant reduction in their ability to stimulate neovascularization in vitro and in vivo. In humans, we isolated circulating ‘early EPCs’ from healthy subjects and patients with CAD and RFs, and transduced them using lentiviral vectors containing either eNOS or GFP (sham). EPCs from patients had reduced in vitro migration in response to SDF-1 or VEGF, which was reversed by eNOS-transduction. In co-culture with human umbilical vein endothelial cells (HUVECs) on Matrigel, eNOS-transduced EPCs contributed to increased and more complex angiogenic tube formation compared to sham-transduced cells. Human EPCs from patients were ineffective in enhancing ischemic hind limb neovascularization and perfusion in a nude mouse, whereas eNOS-transduced EPCs resulted in a significant improvement compared to sham-transduced cells. In a swine model of acute myocardial infarction (MI), eNOS- and non-transfected BMCs both increased left ventricular function compared to sham. However, there was no benefit to eNOS overexpression in this model. Various differences in the models and procedures may explain the incongruous results obtained. Taken together, these results show that eNOS overexpression significantly improves the neovascularization capacity of EPCs of human subjects with CAD and RFs and could represent an effective adjunctive approach for the improvement of autologous cell therapies for cardiovascular disease.

Atherosclerosis is a leading cause of morbidity and mortality worldwide and type 2 diabetes and obesity-associated metabolic syndrome, both characterized by insulin resistance, are potent risk factors. These conditions also increase the risk for restenosis after revascularization procedures used for treatment of atherosclerosis. Studies have shown that insulin and resveratrol (RSV), a red wine polyphenol, decrease neointimal growth after vessel injury in models of restenosis, demonstrating a protective effect on the vasculature. However, oral glucose and sucrose were used in insulin studies to maintain normoglycemia, and their effect on neointimal formation was not assessed. Several studies have shown that nitric oxide (NO) production is stimulated by insulin and RSV, and since NO can decrease neointimal growth, the objective of this thesis was to address the mechanism of action of insulin or RSV to protect against restenosis, and determine whether NO production mediates these effects. To examine this, we treated rats with insulin or RSV and performed arterial balloon injury. In Study 1, insulin reduced neointimal area after injury in rats receiving oral glucose but not oral sucrose. Oral glucose alone had no effect on neointimal formation or insulin sensitivity whereas oral sucrose increased neointimal growth and induced insulin resistance. In Study 2, insulin decreased neointimal area and cell migration, and increased re-endothelialization. These effects were abolished by nitric oxide synthase (NOS) inhibition. In addition, insulin increased eNOS protein expression in the vessel. In Study 3, RSV reduced neointimal growth, cell proliferation, and migration after injury, without affecting re-endothelialization. Most of these effects were abolished by NOS inhibition, except for the decrease in cell migration. Insulin sensitivity and systolic blood pressure were not affected by RSV. Together, the results demonstrate that insulin, independent of glycemic effects, and RSV have a protective effect on the vessel against restenosis, which is mediated by NO. Since both insulin and RSV decrease neointimal formation without negatively impacting re-endothelialization, insulin or RSV treatment could provide some advantage over anti-mitogenic agents currently used in drug-eluting stents, which delay re-endothelialization. These studies suggest that insulin or RSV may have clinical potential in the prevention of restenosis after angioplasty.

The lymphatic system plays an important role in protein and solute transport as well as the immune system. Its functionality is vital to proper homeostasis and fluid balance. Lymphatic fluid (lymph) may be propelled by intrinsic (active) vessel pumping or passively. With regard to the former, nitric oxide (NO) is known to play an important role in lymphatic vessel contraction and vasodilation. Lymphatic endothelial cells (LECs) are sensitive to shear and increases in flow have been shown to cause enhanced production of NO by LECs. Additionally, high concentrations of NO have been experimentally observed in the sinus region of mesenteric lymphatic vessels. The goal of this work was to develop a computational flow and mass transfer model using physiologic geometries obtained from confocal images of a rat mesenteric lymphatic vessel to determine the characteristics of NO transport in the lymphatic flow regime. Both steady and unsteady analyses were performed. Steady models were simulated by prescribing fully developed velocity profiles ranging from 0.5 mm s^-1 to 7 mm s^-1 as the inlet boundary conditions. Unsteady simulations were generated using a velocity profile taken from experimental data from in situ experiments with rats. Production of NO was shear-dependent; basal cases using constant production were also generated. Simulations revealed areas of flow stagnation adjacent to the valve leaflets, suggesting the high concentrations observed here experimentally are due to lack of convection in this region. LEC sensitivity was found to alter the concentration of NO in the vessel, and the convective forces were found to profoundly affect the concentration of NO at a Peclet value greater than or equal to approximately 61. The quasi-steady analysis was able to resolve wall shear stress within 0.15% of the unsteady case. However, the percent error between unsteady and quasi-steady conditions was higher for NO concentration (approximately 6.7%).

Endothelial progenitor cells (EPCs) have reduced neovascularization capacity in the context of coronary artery disease (CAD) or cardiac risk factors (RFs). Since, endothelial NO synthase (eNOS) is critical to normal EPC function, we hypothesized that bone marrow cells (BMCs) from rats with RFs and EPCs from humans with CAD and/or RFs show dramatically reduced neovascularization capacity in vitro and in vivo, which can be reversed by eNOS overexpression. BMCs were isolated from rat models of type II diabetes and the metabolic syndrome, and we showed a significant reduction in their ability to stimulate neovascularization in vitro and in vivo. In humans, we isolated circulating ‘early EPCs’ from healthy subjects and patients with CAD and RFs, and transduced them using lentiviral vectors containing either eNOS or GFP (sham). EPCs from patients had reduced in vitro migration in response to SDF-1 or VEGF, which was reversed by eNOS-transduction. In co-culture with human umbilical vein endothelial cells (HUVECs) on Matrigel, eNOS-transduced EPCs contributed to increased and more complex angiogenic tube formation compared to sham-transduced cells. Human EPCs from patients were ineffective in enhancing ischemic hind limb neovascularization and perfusion in a nude mouse, whereas eNOS-transduced EPCs resulted in a significant improvement compared to sham-transduced cells. In a swine model of acute myocardial infarction (MI), eNOS- and non-transfected BMCs both increased left ventricular function compared to sham. However, there was no benefit to eNOS overexpression in this model. Various differences in the models and procedures may explain the incongruous results obtained. Taken together, these results show that eNOS overexpression significantly improves the neovascularization capacity of EPCs of human subjects with CAD and RFs and could represent an effective adjunctive approach for the improvement of autologous cell therapies for cardiovascular disease.

Atherosclerosis is a leading cause of morbidity and mortality worldwide and type 2 diabetes and obesity-associated metabolic syndrome, both characterized by insulin resistance, are potent risk factors. These conditions also increase the risk for restenosis after revascularization procedures used for treatment of atherosclerosis. Studies have shown that insulin and resveratrol (RSV), a red wine polyphenol, decrease neointimal growth after vessel injury in models of restenosis, demonstrating a protective effect on the vasculature. However, oral glucose and sucrose were used in insulin studies to maintain normoglycemia, and their effect on neointimal formation was not assessed. Several studies have shown that nitric oxide (NO) production is stimulated by insulin and RSV, and since NO can decrease neointimal growth, the objective of this thesis was to address the mechanism of action of insulin or RSV to protect against restenosis, and determine whether NO production mediates these effects. To examine this, we treated rats with insulin or RSV and performed arterial balloon injury. In Study 1, insulin reduced neointimal area after injury in rats receiving oral glucose but not oral sucrose. Oral glucose alone had no effect on neointimal formation or insulin sensitivity whereas oral sucrose increased neointimal growth and induced insulin resistance. In Study 2, insulin decreased neointimal area and cell migration, and increased re-endothelialization. These effects were abolished by nitric oxide synthase (NOS) inhibition. In addition, insulin increased eNOS protein expression in the vessel. In Study 3, RSV reduced neointimal growth, cell proliferation, and migration after injury, without affecting re-endothelialization. Most of these effects were abolished by NOS inhibition, except for the decrease in cell migration. Insulin sensitivity and systolic blood pressure were not affected by RSV. Together, the results demonstrate that insulin, independent of glycemic effects, and RSV have a protective effect on the vessel against restenosis, which is mediated by NO. Since both insulin and RSV decrease neointimal formation without negatively impacting re-endothelialization, insulin or RSV treatment could provide some advantage over anti-mitogenic agents currently used in drug-eluting stents, which delay re-endothelialization. These studies suggest that insulin or RSV may have clinical potential in the prevention of restenosis after angioplasty.

An oxidative stress in the form of H2O2 exposure previously has been shown to cause a transient increase in NO??oduction and a chronic increase in eNOS protein levels. Nevertheless, oxidative stress can cause an uncoupling of catalytic activity resulting in decreased NO??d increased O2??roduction from eNOS. This uncoupling seems to be mediated predominantly by oxidation of tetrahydrobiopterin (BH4), an eNOS required cofactor. To study how these phenomena regulate the physiological balance of reactive oxygen species (ROS), H2O2-induced NO??oduction was measured in bovine aortic endothelial cells (BAECs) using an NO??ecific electrode. Following H2O2 exposure, NO??ncentrations initially increased; however, if cells were challenged a second time with H2O2, the increase in NO??oduction was attenuated. We postulated that the decline in NO??oduction after H2O2 exposure resulted from BH4 oxidation and tested this by supplementing cells with BH4 prior to the second H2O2 exposure. This resulted in a recovery of NO??oduction. Since H2O2 also activates NADPH oxidase to produce superoxide (O2?? we tested whether the decrease in NO??oduction during the second H2O2 exposure could be explained by increased NADPH oxidase-dependent oxygen free radical production, including O2??peroxynitrite (ONOO-), and hydroxyl radicals (??. A reduction in H2O2-induced NO??lease was prevented in apocynin-and PEG-SOD-treated cells and in p47phox-knockout mouse aortic endothelial cells (MAECs), which lack a critical subunit of the NADPH oxidase. These results suggest that O2??roduced by NADPH oxidase leads to eNOS dysfunction. Scavenging ONOO- resulted in a full recovery of NO??oduction, and scavenging ??resulted in a partial recovery of NO??oduction. This implies roles for these O2??erivatives in the reduced NO??sponse to repeated H2O2 exposures.

Recent studies suggest that carbon monoxide (CO) is another gas molecule that has similar biological actions as nitric oxide (NO). The purpose of this study is to investigate the relationship between NO and CO in the survival of naïve rat pheochromocytoma PC12 cells. Western blot analysis revealed that all three isoforms of nitric oxide synthase (NOS) exhibited low expression and two isoforms of heme oxygenase (HO), especially HO-1, exhibited higher expression in PC12 cells under basal condition. Exposure of PC12 cells for 24 h to the NO scavenger, carboxy-2-phenyl-4,4,5,5,- tetramethylimidazoline-1-oxy-1-3-oxide (carboxy-PTIO, 2 £gmol) or HO inhibitor, zinc protoporphyrinIX (ZnPP, 25 nmol) resulted in a progressive reduction in mitochondria dehydrogenase activity reflected cell viability as determined by the WST-1 (4-[3-(4-lodophenyl)- 2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) assay. On the other hand, incubation with NO donors, amino-3-morpholinyl- 1,2,3-oxadiazolium chloride (SIN-1, 1 £gmol) or S-Nitroso-N-acetyl- penicillamine (SNAP, 1 £gmol), or the CO precursor, hematin (500 nmol), resulted in an elevation in cell viability. The progressive reduction in cell viability induced by carboxy-PTIO (2 £gmol) or ZnPP (25 nmol) was significantly blunted by co-treatment with SIN-1 (1 £gmol). However, incubation with the NO precursor, L-arginine (L-Arg, 2 £gmol), or the selective inhibitors for nNOS, iNOS or eNOS, N£s-propyl-L-arginine (NPLA, 100 pmol), S-methylisothiourea (SMT, 10 nmol) or N5-1-Iminoethyl-L-ornithine dihydrochloride (L-NIO, 4 nmol) did not significantly alter cell viability. Co-treatment with carboxy-PTIO (2 £gmol) and L-Arg (2 £gmol) was also ineffective. These results suggest that NO or CO contributes to the survival of naïve PC12 cells.

The objectives of this study were to 1) determine the effects of cycle-to-cycle variations (ccv) on nitric oxide (NO) emissions, and 2) determine if the consideration of ccv affects the average NO emission as compared to the mean cycle NO emission. To carry out the proposed study, an engine simulation model was used. The simulation determines engine performance and NO emissions as functions of engine operating conditions, engine design parameters, and combustion parameters. An automotive, spark-ignition engine at part load and 1400 rpm was examined in this study. The engine cycle simulation employed three zones for the combustion process: (1) unburned gas, (2) adiabatic core region, and (3) boundary-layer gas. The use of the adiabatic core region has been shown to be especially necessary to capture the production of nitric oxides which are highly temperature dependent. Past research has shown that cyclic variations in combustion cause ccv of burn duration, ignition delay and equivalence ratio. Furthermore, literature has shown that variations of these three input parameters may be approximated by a normal frequency distribution. Using the mean and standard deviation, and a random number generator, input values were tabulated for the ignition delay, burn duration and equivalence ratio. These three input parameters were then used to simulate cyclic variations in the combustion process. Calculated results show that cyclic variations of the input parameters cause the cycle-by-cycle NO emissions to increase and decrease by as much as 59% from the mean cycle NO of 3,247 ppm. The average NO emission resulting from ccv was 4.9% less than the mean cycle NO emission. This result indicates that cyclic variations must be considered when calculating the overall NO emissions.