Role of Cell-specific Inducible Nitric Oxide Synthase in a Mouse Model of Chronic Intermittent Hypoxia

Abstract

Obstructive sleep apnea syndrome (OSAS) is a prevalent breathing disorder during sleep. It is considered a serious disease and has a high impact on quality of life of the affected people. OSAS can lead to cardiovascular and pulmonary complications including pulmonary and systemic hypertension. So far, the mechanisms involved in pathogenesis of OSAS are not quite clarified. The available treatment methods, such as continuous positive airway pressure, surgical intervention and oral appliances which keep the upper airways open during sleep, improve symptoms and decrease the incidence of cardiovascular diseases resulted from OSAS. However, these treatments are not tolerated by all patients. Moreover, there is no effective pharmacological treatment for OSAS. For simulation of OSAS several different animal models are available. In the current thesis the chronic intermittent hypoxia (CIH) mouse model was used which is appropriate to investigate the pathophysiological mechanisms of OSAS. The results of previous studies and also investigations performed in the CIH mouse model, from the laboratory where the current work was performed, have shown that the inducible nitric oxide synthase (iNOS) plays an important role in the pathogenesis of OSAS and its associated cardiovascular and pulmonary consequences. Wild type (WT) mice and iNOS knockout (KO) mice were investigated in a mouse model of CIH. The mice lacking iNOS showed less systemic hypertension, pulmonary hypertension (PH), and pulmonary vascular remodeling compared to WT mice in the CIH model. The main aim of this study was to further investigate the role of iNOS and reactive oxygen species (ROS) in the pathogenesis of systemic and pulmonary hypertension in the CIH model of OSAS. The objective was to clarify whether iNOS is originated from the cells that are derived from bone marrow or from other cell types (non-bone marrow-derived cells). For this purpose, iNOS KO and WT mice were irradiated at the beginning of the experiment until complete bone marrow suppression, and were transplanted with either iNOS KO or WT bone marrow cells. By using bone marrow transplantation (BMT), four different groups of chimeric mice were created namely, WT(WT), irradiated WT mice that received bone marrow cells from WT mice; KO(KO), irradiated iNOS-/- mice that received bone marrow cells from iNOS-/- mice; WT(KO), irradiated WT mice that received bone marrow cells from iNOS-/- mice; and KO(WT), irradiated iNOS-/- mice that received bone marrow cells from WT mice. The chimeric mice were exposed to either CIH or normoxia-normoxia cycles (NOX). Daily blood pressure measurements, final invasive hemodynamic measurements, determination of different heart ratios, echocardiographic studies and histological determination of degree of muscularization of pulmonary vessels were used to assess the development of systemic and pulmonary hypertension. Superoxide anion production was measured in lung homogenate and blood samples of chimeric mice by electron spin resonance spectroscopy. Regarding all the readouts, the three-way interactions were not significant, indicating the data did not demonstrate that the CIH-related responses depend on the iNOS genotype combination of non-bone marrow-derived cells and bone marrow-derived cells. The two-way interactions of “WT(WT) NOX and CIH vs. KO(KO) NOX and CIH” for the echocardiographically measured cardiac output, cardiac index, and tricuspid annular plane systolic excursion (indicators of pumping function of the heart) were significant, indicating the cardioprotective effects of iNOS deletion in CIH model. CIH-exposed WT(WT) and WT(KO) chimeric mice showed significant higher right ventricular systolic pressure (RVSP) compared to respective NOX-exposed control mice. However, the degree of muscularization of the pulmonary vessels did not show significant change in any of the chimeric mice compared to their corresponding NOX groups. No statistically significant changes were observed in systemic blood pressure as a result of CIH in chimeric mice. Measurement of concentration of superoxide anion in the lung homogenate samples revealed slight but significant difference in response to CIH exposure in two-way interaction of “WT(WT) NOX and CIH vs. KO(KO) NOX and CIH”. No significant difference was observed in the concentration of superoxide anion of the blood samples as a result of CIH in any of the chimeric groups. As it has been already shown that CIH leads to systemic hypertension and higher degree of muscularization of the pulmonary vessels in WT mice, it was unexpected that CIH had no effect on these readouts in WT(WT) mice. The unexpected observations of the current study regarding some of the readouts (e.g. systemic blood pressure, degree of muscularization of pulmonary vessels, and etc.) can be associated to the effects of irradiation and/or BMT on CIH mouse model which should be investigated further.