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Endothelial dysfunction in mice subjected to intermittent hypoxia and fed a high fat diet Badran, Mohammad Salah El Deen 2013

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!!Endothelial dysfunction in mice subjected to intermittent hypoxia and fed a high fat diet  by   Mohammad Salah El Deen Badran B.Pharm, Applied Science University, Jordan, 2009  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science in The Faculty of Graduate Studies (Pharmacology and Therapeutics)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)   July 2013   ? Mohammad Salah El Deen Badran, 2013 ii!!Abstract   Background: Obstructive sleep apnea (OSA) and obesity are major independent risk factors for cardiovascular disease; nearly 70% of OSA patients are obese. This thesis examines the vascular effects of sleep disordered breathing in a mouse model with diet- induced obesity. Markers of oxidative stress and inflammation were also determined in these mice.   Methods: Mice were divided into four groups for 6 weeks: i) mice subjected to intermittent hypoxia alone (IH) ii) mice fed a high fat (60%) diet alone (HFIA), iii) mice subjected to intermittent hypoxia and a high fat diet and (HFIH) iv) mice subjected to intermittent air  (IA) alone. Vasodilatory and contractility responses were examined in isolated aortas to assess changes in endothelial function.  Markers of oxidative stress (MDA and TAC) and inflammation (CRP) levels were measured and finally, eNOS expression in the aortas was evaluated.   Results: Only mice subjected to intermittent hypoxia and a high fat diet showed endothelial dysfunction. Intermittent hypoxia alone increased plasma oxidative stress and inflammatiory markers but adding high fat diet further increased those markers. High fat diet alone did not increase oxidative stress or inflammation. Total antioxidant capacity and aortic eNOS expression were not significantly different between the groups.  iii!!Conclusion: there is a synergistic effect between intermittent hypoxia and high fat diet since only their combination of caused endothelial dysfunction. These data suggest that aortic endothelial dysfunction may be mediated by increased oxidative stress and inflammation.                                     iv!!Preface  The animal care and experimental protocols described in this thesis were approved by the Animal Care Committee at the University of British Columbia (Animal Care Certificate A06-0308).  Table 1-1: Selected animal models of sleep apnea with some of the related benefits and disadvantages, was published in a review article: S Golbidi, M Badran, N Ayas and I Laher. 2012. Cardiovascular consequences of sleep apnea. Lung 190, no. 2: 113-132.  I was responsible for designing the study, conducting the vascular functional experiments and measuring the biochemical parameters. Expression of eNOS was done in Dr. Angela Devlin?s lab in the Child and Family Research Institute. Dr Saeid Golbidi helped with the removal and cleaning of the aorta.!!As for the rest of the work presented in this thesis, I was responsible for the experimental design, data collection, analysis and presentation in addition to the writing. !!!!! v!!Table of contents Abstract.......................................................................................................ii Preface........................................................................................................iv Table of contents........................................................................................v List of tables?.????............................................................................ix List of figures??..???????????????????????..x List of abbreviations..................................................................................xi Acknowledgments...................................................................................xiii 1- Introduction????????????????????????..?.1 1-1 Obstructive sleep apnea???????????????????????....1 1-2 OSA and cardiovascular disease???????????????????....2 1-2-1 Hypertension ?????????????????????????????????...?3 1-2-2 Coronary artery disease?????????????????.???????????..?.4 1-2-3 Stroke?????????????????????????????????????.....5 1-2-4 Heart failure???????????????????????????????????...5 1-3 Oxidative stress in OSA???????????????????????.?6 1-3-1 Reactive oxygen species and their sources????????????????????....7 vi!!1-3-2 Evidence of oxidative stress in OSA???????????????????????..?8   1-4 Inflammation in OSA?????????????????????????..11   1-5 Endothelial dysfunction in OSA????????????????????..14   1-6 Obesity and OSA???????????????????????????.17   1-7 Animal models of sleep disordered breathing ?????????????..20   1-8 Research objectives and experimental design?????????????...26 2-Materials and methods????????????????????..28   2-1 Animal model?????????????????????????????28   2-2 Plasma and tissue collection?????????????????????...29   2-3 Assessment of vascular function???????????????????...30      2-3-1 Assessment of endothelium-dependent and independent vasodilatation??????.?31       2-3-2 Assessment of basal NO????????????????????????????.?.31   2-4 Measuring plasma variables??????????????????????32   2-5 Real-time PCR????????????????????????????..32   2-6 Statistical analysis??????????????????????????..33   2-7 Drugs, reagents and solutions?????????????????????34 3-Results???????????????????????????..35   3-1 Increased body weight only in mice fed high fat diet???????????35 vii!!   3-2 Impaired endothelial function in mice subjected to intermittent hypoxia and         high fat diet?????????????????????????????...36   3-2-1 Attenuated ACh-induced endothelial vasodilatation????????????????.....36   3-2-2 Impaired basal NO production?..?????????????????????????..38   3-3 Increased oxidative stress in mice subjected to intermittent hypoxia and high         fat diet????????????????????????????????.40     3-3-1 Increased MDA levels???????????????????????????????..40    3-3-2 Total antioxidant capacity was not changed..???.????????????????.?42   3-4 Increased inflammation in mice subjected to intermittent hypoxia and high          fat diet????????????????????????????????.43     3-4-1 Increased CRP levels?????????????????????????????.??..43   3-5 Expression of aortic eNOS was not changed?????????????.....45 4-Discussion?????????????????????...46   4-1 Effects of intermittent hypoxia and high fat diet on body weight?????.46   4-2 Endothelial dysfunction in intermittent hypoxia and diet induced obesity?47 4-3 Oxidative stress and inflammation in intermittent hypoxia and diet induced        obesity????????????????????????????????49 viii!!  4-4 Expression of eNOS in intermittent hypoxia and diet induced obesity ?.....52 5- Summary?????????????????????????.?53 6- Limitation of the animal model and study?.??????????55 7- Future directions?????????.?????????????.56 References?????????????????....??????.??57 ix!!List of tables Table 1-1: Biology of some adipokines and their levels in OSA patients??????..19 Table 1-2: A selected animal model of sleep apnea with some of the related benefits and disadvantages?????????????????????????????.21 Table 3-1: Body weight differences between groups????...??????????35           x!!List of figures Figure 1-1: Oxidative stress and inflammation in Obstructive sleep apnea.???.?..16 Figure 1-2: Research design and methods............................................................?....27 Figure 3-1: Cumulative concentration response curves of Acetylecholine (upper panel) and Sodium nitroprusside (lower panel) in phenylephrine constricted aorta of wild type mice???????????????????????????????????..37 Figure 3-2: Cumulative concentration response curves to Phenylephrine before and after adding L-NAME for HFIA and HFIH. Area under the curve calculated for the contraction response to Phenylephrine after adding L-NAME??.??????..........39 Figure 3-3: Plasma Malondialdehyde levels as marker of oxidative stres???.?..?41 Figure 3-4: Plasma total antioxidant capacity???????????????..?.?42 Figure 3-5: Plasma C-reactive protein levels as marker of inflammation?????....44 Figure 3-6: Endothelial nitric oxide synthase expression in aorta?????????.45 Figure 5-1: Intermittent hypoxia and diet-induced obesity lead to endothelial dysfunction????????????????????????????????..54  xi!!List of abbreviations ACh                                            acetylcholine AHI                                             apnea/hypopnea index AUC                                           area under the curve BH4                                               tetrahydrobiopterin BMI body mass index CRP                                          C-reactive protein DIO diet induced obesity eNOS                                        endothelial nitric oxide synthase IH                                              intermittent hypoxia IL-6                                           interleukin-6 L-NAME                                    N?-Nitro-L-arginine methyl ester  M                                              Molar MDA malondialdehyde NADPH oxidase                       nicotinamide adenine dinucleotide phosphate oxidase xii!!Nf-kB                                        nuclear factor-kappaB NO                                           nitric oxide  O2-                                           superoxide anion ONOO-                                                       peroxynitrite PE phenylephrine ROS                                        reactive oxygen species RT-PCR   reverse transcriptase-polymerase chain reaction TAC   total antioxidant capacity SNP                                        sodium nitroprusside TNF-alpha                              tumor necrosis factor-alpha      xiii!!Acknowledgments Throughout my short journey, I would like to thank my supervisor Dr. Ismail Laher who has been more than a supervisor to me and have a special place in my heart. His generosity and humbleness are limitless, I would love to thank him for accepting me as a graduate student in his lab and for having enough confidence in me to design my experiments and direct the path of my study. Dr. Saeid Golbidi has been a mountain of help, I owe him a lot since he taught me everything about vascular experiments and treated me like his little brother. I would like also to thank Dr. Najib Ayas, an amazing and kind doctor who showed a lot of support and dedication to the lab and the project. My gratitude to the chairman of my committee: Dr.Bhagavatula Sastry. Also Dr. James Wright and Dr. Kim Basset for accepting to be on my committee. I would like to thank them all for showing their support and kindness. I also want to thank all the people in the Department of Anesthesiology, Pharmacology and Therapeutics for being so supportive and my staying very comfortable.1!!1- Introduction 1-1 Obstructive sleep apnea Obstructive sleep apnea (OSA) is characterized by momentary cessations in breathing (apnea) or significant reductions in breathing amplitude (hypopnea) caused by an obstructed or collapsed upper airway; both conditions can cause significant arterial hypoxemia and hypercapnia. The apnea/hypopnea index (AHI) describes the total number of apnea/hypopnea episodes per hour of sleep, which is usually less than 5 in normal individuals. AHI scores of 5?15, 15?30, and >30 categorize patients with sleep apnea as mild, moderate, and severe, respectively (Golbidi et al., 2012). An obstructed airway increases resistance to airway flow that results in a greater breathing effort and intrathorcic pressure, resulting in disruption of sleep, arousal and reopening of the airway (Gleeson et al., 1990).   At least 2% of women and 4% of men are diagnosed with OSA and its characteristic symptoms. based on the average of prevalence estimates from many clinical studies, it is estimated that nearly 1 of every 5 adults have at least mild OSA and that 1 of every 15 have at least moderate OSA; moderate OSA occurs predominantly at body mass index (BMI) values of 25 to 28 (Young et al., 1993; Bixler et al., 2001; Duran et al., 2001).  Despite numerous advancements in the medicine, the majority of those affected with OSA remain undiagnosed (Punjabi, 2008). OSA is suspected in people who are obese, hypertensive, hypersomnolent and are habitual snorers (Netzer et al., 1999). 2!!Polysomnography is the main method for assessing patients with suspected sleep apnea (1997). Sleep stages are recorded along with oxyhemoglobin saturation, breathing and airflow. In addition, limb and eye movements and the electrocardiogram are also monitored (1999).  OSA creates a huge economic burden when compared to other chronic diseases. In 2000, OSA-related automobile collisions alone attributed to 1400 fatalities and a total cost of 15.9 billion dollars in the United States. Treatment with continuous positive airway pressure (CPAP) resulted in saving 7.9 billion dollars and 1000 lives (AlGhanim et al., 2008).  It is well established that the outcomes of OSA can lead to serious vascular disease. Data from different studies implicate OSA in the development of hypertension, and to some extent cardiac ischemia, congestive heart failure, arrhythmias, and also to cerebrovascular disease and stroke (Shamsuzzaman et al., 2003). Many intermediary mechanisms, such as sympathetic activation, endothelial dysfunction, vascular oxidative stress, inflammation, increased coagulation and metabolic dysregulation, link OSA to vascular disease (Kapur, 2010).  1-2 OSA and cardiovascular disease Evidence that relate OSA directly to vascular disease comes from small longitudinal studies of incident cardiovascular disease and studies assessing the outcomes of CPAP 3!!intervention. Nevertheless, many studies can only indirectly implicate OSA in the etiology of cardiovascular disease, mainly because of the cost of establishing the diagnosis of OSA in large population samples, which means that most large scale epidemiologic studies do not monitor OSA. Another important reason is that patients with OSA also have coexisting morbidities such as hypertension or obesity, making the independent risk of OSA on vascular disease more difficult to assess.  1-2-1 Hypertension About 50% of OSA patients have hypertension while 30% of hypertensive patients who have OSA are undiagnosed (Fletcher et al., 1985; Silverberg et al., 1998). The Wisconsin sleep cohort reports that in patients with AHI of 15 or higher have a 3 fold increased risk of developing hypertension during this 4 years study (Peppard et al., 2000). In a more recent study, OSA (apnea-hypopnea index: >15 events per hour) was the most common condition associated with resistant hypertension (64.0%), followed by primary aldosteronism (5.6%), renal artery stenosis (2.4%), renal parenchymal disease (1.6%), oral contraceptives (1.6%), and thyroid disorders (0.8%) in 125 patients with resistant hypertension (Pedrosa et al., 2011). These data suggest that essential hypertension might be secondary to OSA. Studies in rats and mice also show that chronic intermittent hypoxia increases blood pressure (Allahdadi et al., 2005; Dematteis et al., 2008). OSA is now included as one of the main 4!!causes of hypertension in the sixth report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (Chobanian et al., 2003).   1-2-2 Coronary artery disease OSA is also related to coronary artery disease and stroke, since the prevalence of OSA among hospitalized men with acute myocardial infarction is nearly 70% (Konecny et al., 2010). Intermittent hypoxia, sympathetic vasoconstriction and changes in intrathorasic pressure can all contribute to cardiac ischemia. A study of 200 patients without a history of coronary artery disease shows that the median coronary artery calcification score (Agatston units) was 9 in OSA patients and 0 in non-OSA patients. This was measured by electron beam computed tomography on these patients within 3 years of polysomnography (Sorajja et al., 2008). The median calcification score increased with the severity of OSA. A recent study of more than 500 subjects showed that OSA patients are more likely to have a family history of premature death from coronary artery disease than non-OSA patients. The results were independent of BMI, gender and personal history of coronary artery disease (Gami et al., 2007).  A five-year follow up of 62 patients with coronary artery disease reported a higher mortality rate in OSA patients (38%) compared to non-OSA patients (9%) (Peker et al., 2000).  5!!1-2-3 Stroke In a cross-sectional study of over 6,000 subjects from the Sleep Heart Health Study, the odds ratio of prevalent stroke was higher (1.58) in patients with moderate sleep apnea (AHI ?11) (Shahar et al., 2001). Other studies report that the risk of stroke or death increases with the severity of sleep apnea independently of age, sex, BMI, diabetes, hypertension and dyslipidemia. Investigators found that the hazard ratio for patients with severe OSA was 3.3 (Yaggi et al., 2005). Redline et al. followed a total of 5,422 participants without a history of stroke at the baseline examination and untreated for OSA for a median of 8.7 years. Around 193 strokes were detected. A significant positive association between ischemic stroke and AHI was observed in men (P = 0.016), such that those in the highest AHI quartile (>19) had an adjusted hazard ratio of 2.86. In the mild to moderate range (AHI, 5-25), each one-unit increase in OAHI in men was estimated to increase stroke risk by 6% (Redline et al., 2010). However, the relationship between OSA and stroke remains circumstantial. The population of patients who are at risk of OSA is demographically similar to the patients who are at risk of stroke. The fact that only survivors of strokes are tested complicates the causal association between stroke and OSA (Shamsuzzaman et al., 2003).   1-2-4 Heart failure It is estimated that 37% of 450 patients with systolic heart failure were diagnosed with sleep apnea after polysomnography. The prevalence was greater in men (38%) with obesity being the main risk factor while the prevalence in women was 31% with the 6!!main risk factor being old age (Sin et al., 1999). Gottlieb et al. reported that obstructive sleep apnea predicted incident heart failure in men but not in women (adjusted hazard ratio 1.13 per 10-unit increase in AHI). Men with AHI > or =30 were 58% more likely to develop heart failure than those with AHI <5 (Gottlieb et al., 2010). OSA has also been noticed in more than fifty percent of heart failure patients with preserved systolic function (Chan et al., 1997). OSA can lead to heart failure through many mechanisms including increasing blood pressure, increasing left ventricular afterload and by increasing the risk of myocardial infarction (Marin et al., 2005). It is clear that OSA is related to many cardiovascular diseases and its treatment is a necessity.  1-3 Oxidative stress in OSA Oxidative stress results from an imbalance in oxidant production and antioxidant defense mechanisms; this means that either the overproduction of reactive oxygen and nitrogen species (ROS/RNS) and / or a decreased antioxidant capacity leads to oxidative stress (Valko et al., 2007). Free radicals play an important role in regulating cellular function and signal transduction. However, overproduction of free radicals can affect many cellular and physiological mechanisms by damaging lipids, proteins and DNA. Recent studies show important links between the formation of hypoxia-related free radicals related oxidative stress and cardiovascular disease in OSA patients. Pathways linking OSA/IH and oxidative stress and endothelial dysfunction are summarized in Figure 1-1. 7!!1-3-1 Reactive oxygen species and their sources in OSA ROS are atoms or molecules that posses one or more unpaired electrons in their outer orbit and thus are chemically unstable and highly reactive. When two radicals react, the product is a non-radical, but when radicals react with non-radicals the product is a new radical and therefore, the radical chain reaction propagates. ROS are by-products of oxygen metabolism that are generated during normal cellular respiration and their elimination occurs through enzymatic and non-enzymatic anti-oxidant systems. When ROS generation exceeds the capacity of the antioxidant systems, oxidative stress and damage to tissues and cells ensue. This mechanism can eventually contribute to the pathological conditions of cardiovascular disease. One of the most abundant ROS is the superoxide anion; although it?s a weak radical, it reacts to give rise to other potent oxidants such as hydroxyl radical and peroxynitrite. The latter is a RNS, which has a major role in causing endothelial dysfunction since it is the product of superoxide anion reacting with nitric oxide. As a result, nitric oxide bioavailability decreases and the relaxing ability of the blood vessels is compromised.  Free radical leakage from mitochondria during oxidative phosphorylation is the major source of superoxide anions. It is estimated that at least 3-5% of the oxygen that is consumed is converted to superoxide anion during aerobic respiration. During hypoxia, ROS production is elevated due to excessive mitochondrial reduction (Duranteau et al., 1998; Valko et al., 2007). Phagocytes also generate large amounts of ROS when activated. Phagocytes are the immune system?s first line of defense against pathogens, 8!!and ROS production is one of their mechanisms for destroying invading pathogens. They generate superoxide anion through NADPH oxidase and other ROS (H2O2, HCLO and NO) through other enzymes (Babior, 2000).  Although this mechanism of producing ROS can kill pathogens, it can also cause damage to surrounding tissues. NADPH oxidase is also found in non-phagocytic cells where it generates less superoxide anion for different purposes such as signaling (Mohazzab et al., 1994). For example, vascular cells express NADPH oxidase where superoxides play an essential role in vascular cell growth, migration and alterations of extracellular matrix (Griendling et al., 2000).   1-3-2 Evidence of oxidative stress in OSA Many studies confirm that OSA is associated with oxidative stress that is due to ROS generation and confirmed by measurements of oxidative stress markers. A study by Schulz et al. reports increased production of ROS in stimulated neutrophils and monocytes from OSA patients (Schulz et al., 2000), while others report that ROS production was also significantly higher in non-stimulated monocytes of OSA patients (Dyugovskaya et al., 2002; Dyugovskaya et al., 2008). Rats subjected to intermittent hypoxia for two weeks have increased vascular production of ROS (Troncoso Brindeiro et al., 2007). IH-induced pulmonary hypertension in mice is associated with increased lung levels of the NADPH oxidase subunits NOX4 and p22phox, indicating that NADPH oxidase-derived ROS contributes to the development of pulmonary hypertension caused by chronic intermittent hypoxia (Nisbet et al., 2009). NADPH oxidase is 9!!activated in many animal models of intermittent hypoxia in tissues such as the myocardium, brain, carotid body and liver (Zhan et al., 2005; Jun et al., 2008; Peng et al., 2009).  Oxidative stress markers of lipid peroxidation, protein carbonylation and DNA oxidation are increased in OSA patients. Lipid peroxidation is an important marker of oxidative stress since lipids are easily oxidized. Many studies show that lipid peroxidation increases in OSA patients. In an overnight study of OSA patients with and without cardiovascular disease, thiobarbituric acid (TBARS) were significantly increased (Lavie et al., 2004). In another study, fourteen males with severe OSA fasted all night and TBARS levels were measured in the next morning. TBARS levels in those patients were significantly higher (28.1 nmol LDL protein) when compared to thirteen healthy age matched controls (20.0 nmol LDL protein) (Barcelo et al., 2000).  Oxidized LDL is also increased in OSA, as shown in a prospective study of 37 subjects who have OSA and 38 who do not, where plasma levels of oxidized LDL were higher in OSA patients (43.6 U/L) compared to control (32.3 U/L) (Kizawa et al., 2009). Protein carbonylation is increased as well in OSA patients, since a study of 17 moderate to severe OSA patients, protein carbonyl levels were significantly higher (1.11 ?mol/g protein) when compared to matched control (0.99 ?mol/g protein). On the other hand, the increase was not significant in mild OSA patients (1.03 ?mol/g protein) (Vatansever et al., 2011). 8-hydroxyl-2?deoxyguanosine (8-OHdG), a marker of DNA oxidation, is also elevated in OSA patients.  Urinary excretion of 8-OHdG significantly correlates with the severity of OSA (Yamauchi et al., 2005).  10!!In animals, many chronic intermittent hypoxia studies show significant increases in oxidative stress markers. One month of intermittent hypoxia significantly increased MDA levels in mice (Liu et al., 2010). This is in accordance with another study where Polotsky et al. found that serum MDA levels increased 4 fold in mice subjected to chronic intermittent hypoxia for 6 months when compared to control (Savransky et al., 2007a). Oxidative stress markers are also elevated in tissues such as the liver and brain (Xu et al., 2005; Rosa et al., 2011). Antioxidant capacity is impaired in OSA patients. Although the antioxidant capacity in OSA subjects and controls did not differ in their study, Christou et al. showed a linear negative relationship between antioxidant capacity and apnea/hypopnea index  (R=-0.551, p=0.041) (Christou et al., 2003). When Barcelo et al. tested total antioxidant status in OSA patients, they found that it is significantly decreased (1.4 mmol/L) when compared to healthy subjects (1.5 mmol/L, p=0.0001). They also noticed lower levels of vitamin A (64 ?g/dL) and vitamin E (1525 ?g/dL) when compared to control (74 and 1774 ?g/dL, respectively) (Barcelo et al., 2006), while Katsoulis et al. reported some unexpected results where they found that total antioxidant status before and after sleep was significantly lower in OSA patients with AHI<30 (1.73 vs. 1.65 mmol/L, p=0.01) but not in severe OSA patients with AHI>30 (1.64 vs. 1.58 mmol/L, p=0.07). a possible explanation could be the difference between acute effect of hypoxia immediately resulting from apneic sleep and any chronic state of oxidative stress that may be sustained in severe OSA patients even during daytime (Katsoulis et al., 2011). 11!!Combined, animal and clinical studies indicates that OSA is a condition of increased oxidative stress.   1-4 Inflammation in OSA Inflammation is a process of dynamic complex cytologic changes, mediator release and cellular infiltration that occurs to the blood vessel and adjacent tissue in response to an injury or abnormal stimulation. Inflammation plays an important role in the pathogenesis of atherosclerosis, where levels of inflammatory circulatory markers are associated with cardiovascular risk. Inflammatory cascades involve nuclear transcription factors where the primary target is nuclear factor kappa B (NF-kB) while the markers include adhesion molecules, C-reactive protein, tumor necrosis factor alpha and others. Expression of adhesion molecules and inflammatory cytokines facilitate the recruitment of macrophages loaded with oxidized lipids known as foam cells. The accumulation of foam cells eventually leads to formation of atherosclerotic plaque, which is destined to rupture at one point, and lead to cardiovascular complications (Lusis, 2000). Evidences from clinical studies show that inflammatory markers are elevated in OSA patients. Intermittent hypoxia in animals and cell cultures also demonstrate activation of inflammatory pathways (McNicholas, 2009). Inflammatory pathway behind OSA/IH is explained in Figure1-1. 12!!ROS, hypoxia, cytokines, bacterial and viral products and many other mediators can activate NF-kB. Once activated, it translocates to the nucleus and binds to promoters of specific genes initiating transcription of many products such as IL-1, TNF-alpha, COX-2, adhesion molecules and acute phase proteins (Valen et al., 2001).  Nine patients with OSA showed significantly higher levels of p65 (a marker of Nf-kB activation) when compared to seven healthy matched subjects (median, 0.037 ng/?l vs. 0.013 ng/?l) (Yamauchi et al., 2006). Htoo et al. showed that neutrophils from patients with mild to moderate and severe OSA had 4.8-fold and 7.9-fold increases in NF-kB binding activity compared to control group (Htoo et al., 2006). CRP is an acute-phase reactant secreted by the liver, adipose tissue and other cell types, and is considered one of the main markers of inflammation with a strong predictor-value of coronary artery disease and cardiovascular events (Libby et al., 2004; Willerson and Ridker, 2004). CRP potentially links OSA to inflammation, oxidative stress and atherosclerosis. CRP has pro-oxidant effects when added to cultured coronary artery smooth muscle cells. Moreover, vascular smooth muscle cells and macrophages obtained from vulnerable plaque of coronary artery patients undergoing atherectomy   co-express CRP protein and its mRNA with NADPH oxidase (Kobayashi et al., 2003). In addition to that, CRP induces adhesion molecule expression in cultured endothelial cells (Pasceri et al., 2000). It is clear that CRP contributes to cardiovascular complications via inflammatory and oxidative stress processes. In OSA, plasma CRP levels in 22 patients were significantly higher (0.33mg/dL) when compared to 20 control subjects 13!!(0.02 mg/dL), but importantly, CRP levels were independently associated with OSA severity (p=0.032) (Shamsuzzaman et al., 2002). A recent study reports that CRP levels are significantly increased in OSA patients (4.03 mg/L) compared to the control group (2.41 mg/L) (Guven et al., 2012). These data suggests that OSA is associated with elevated levels of CRP, a marker of cardiovascular risk. Circulating leukocytes and platelets are the main source of inflammatory cytokines and adhesion molecules. Under physiological conditions, these cells express low levels of intracellular inflammatory cytokines and adhesion molecules, but when activated by different stimuli including hypoxia/reoxygenation or OSA, expression levels increase dramatically. This inflammatory pathway also occurs in endothelial cells which since they are also capable of expressing inflammatory cytokines and adhesion molecules (Lavie, 2012).  Endothelial cells provide a vascular permeability barrier, regulate vascular tone and maintain an anti-thrombotic and anti-inflammatory phenotype in their non-activated state. Endothelial cells resist adhesion to platelets, RBCs and adhesion molecules. However, in response to injury such as hypoxia/reoxygenation, increased expression of adhesion molecules is triggered and the interaction between the endothelium and circulating cells is mediated (Gavins et al., 2007; Packard and Libby, 2008). Many studies show that the expression of adhesion molecules is increased in OSA patients. Ursavas et al. measured the levels of ICAM-1 and VCAM-1 in 39 OSA patients and found that both ICAM-1 (480.1 vs. 98.6 ng/ml) and VCAM-1 (1,156.6 vs. 878.8 ng/ml) were significantly increased in the OSA group compared to their matched controls (Ursavas et al., 2007). There is also increased in vivo endothelial cells 14!!activation in OSA patients, which is a procoagulant and proinflammatory state where there is an increase white blood cells interaction with endothelial cells leading eventually to atherosclerosis (Jelic and Le Jemtel, 2008).    1-5 Endothelial dysfunction in OSA Diminished endothelial function is an important consequence of OSA, and is frequently measured as impaired endothelium dependent vasodilatation (Kato et al., 2000). Different studies show lower levels of circulating NO in OSA, for example by the reduced levels of serum nitrite/nitrate (byproducts of normal NO metabolism) in OSA subjects (38.9 ?M vs 63.1 ?M in controls) (Ip et al., 2000). This was confirmed in other studies where nitrate/nitrite levels were significantly lower in OSA patients (35.6 ?M) when compared to control (72.6 ?M) (Teramoto et al., 2003). Many mechanisms have been suggested for endothelial dysfunction due to OSA or IH including (1) interaction on NO and ROS forming peroxynitrite, (2) uncoupling of eNOS, (3) decreased endothelial expression of eNOS and increased levels of endogenous eNOS inhibitors (Schulz et al., 2001). Due to its short half-life and large volume of distribution, peroxynitrite is hard to measure and these factors explain the lack of difference in nitrotyrosine levels between OSA and healthy subjects (Tabrizi-Fard et al., 1999; Svatikova et al., 2004). However, Jelic et al. found an increased expression of nitrotyrosine in endothelial cells derived from OSA patients (Jelic and Le Jemtel, 2008). 15!!In all the forms of nitric oxide synthase, including the endothelial one, enzymatic activity requires five cofactor groups to incorporate oxygen into the amino acid L-argenine to produce NO. Those cofactors include:  flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), heme, tetrahydrobiopterin (BH4), and Ca2+-calmodulin. If nitric oxide synthase lacks L-arginine or any of these co-factors, it will produce superoxide anion instead of NO through an uncoupled state of nitric oxide synthase (Yung et al., 2006). Antoniades et al. showed that increased ROS production during hypoxia could lead to BH4 oxidation and increased levels of arginase II that degrades L-arginine, leading to further eNOS uncoupling (Antoniades et al., 2006). Patients with OSA have increased levels of asymmetrical dimethylargenine (ADMA), a competitive inhibitor of NOS (Ohike et al., 2005). Studies by Tanaka et al. suggest that eNOS activation is sensitive to regulation by redox status and that oxidative stress leads to decreased eNOS phosphorylation, so reducing its activity (Tanaka et al., 2005), while Jelic et al. supported the latter findings when they reported decreased ratios of total to phosphorylated eNOS in endothelial cells from OSA (Jelic and Le Jemtel, 2008).  16!! Figure 1-1: Oxidative stress and inflammation in OSA. ROS formation induced by intermittent hypoxia activates an inflammatory cascade via activation of nuclear factor kappa-b, which influences the transcription of inflammatory cytokines and adhesion molecules. Those in turn activate different blood cells and transcription factors. Activated platelets and leukocytes generate greater amounts of ROS, pro-inflammatory cytokines and adhesion molecules, thus exacerbating the inflammatory and oxidative cycle leading eventually to endothelial dysfunction and promoting cardiovascular disease.  17!!1-6 Obesity and OSA Overweight and obesity are considered the biggest health burden throughout the world, nearly affecting every aspect of life and remains a great challenge to medical practice. It is estimated that there are 1.6 billion overweight adults [body mass index (BMI) > 25 kg/m2] and nearly 400 million obese (BMI >30 kg/m2) in the world (WHO 2005). One of every three adults in the United States is obese (Fakhouri et al., 2012). Almost 20% of individuals in Western countries are obese and 1?2% are morbidly obese (BMI >40 kg/m2). It appears that the prevalence of morbid obesity is increasing rapidly (Fakhouri et al., 2012). Furthermore, it has been predicted that life expectancy for obese adults may decrease (Finkelstein et al., 2010). In the U.S.A., health care costs are already over 30% higher for obese subjects as compared to those with normal BMI. In Europe, obesity is estimated to be responsible for 2?8% of health costs and 10?13% of deaths (Withrow and Alter, 2011). Obesity is strongly associated with OSA, with nearly 60-90% of OSA patients being obese (Young et al., 2002). Thus, gaining weight worsens the severity of OSA, while losing weight improves it (Loube et al., 1994). But the association between obesity and OSA seems to be bi-directional, meaning that also OSA can also lead to weight gain (Wolk et al., 2003; Pillar and Shehadeh, 2008). Localization of excess adipose tissue, for eaxmple around the neck, affects pharyngeal neural and mechanical mechanisms that mediate airway collapsibility and so promoting OSA (Schwartz et al., 2010). Obesity can also narrow the upper airway and reduce chest wall compliance (Horner et al., 18!!1989). Obesity itself is considered a cardiovascular risk factor and similar to OSA, it is associated with male gender, cardiovascular morbidity, insulin resistance, hypertension, type 2 diabetes and stroke (Kopelman, 2000). Oxidative stress is thought to be a unifying mechanism leading to the development of co-morbidities in obesity. Evidence suggests that the sources of oxidative stress in obesity come from a variety of sources including hyperleptinemia, hyperglycemia, decreased antioxidant capacity, increased ROS formation, increased tissue lipids levels, enzymatic sources within the endothelium and chronic inflammation (Vincent and Taylor, 2006). Furukawa et al. showed that fat accumulation in non-diabetic subjects closely correlates with markers of oxidative stress (Furukawa et al., 2004). The Framingham Heart Study also demonstrates that BMI, diabetes and smoking are independently and significantly associated with oxidative stress markers (Keaney et al., 2003).  Unfortunately, OSA is not commonly considered in studies investigating oxidative stress in obese subjects and the contribution of OSA to oxidative stress cannot thus be excluded. In animal models of obesity, oxidative stress was mediated through the development of metabolic syndrome via dysregulated adipokine production. The mechanisms involved in increased oxidative stress include decreased expression of antioxidant enzymes in adipocytes and the upregulation of NADPH oxidase (Furukawa et al., 2004).   Adipocytes exposed to hypoxia show some dysregulation of some adipokines such as TNF alpha and leptin (Hosogai et al., 2007). It seems that activation of nuclear factor 19!!kappa B by hypoxia and/or by increased adipokines and free fatty acids released by excess adipose tissue is the final common inflammatory pathway linking obesity, OSA and the metabolic syndrome both individually and, in many cases, synergistically (Alam et al., 2007). Table 1-1 explores the biological effects of some adipokines and their levels in OSA patients. Table 1-1: Biology of some adipokines and their levels in OSA patients Adipokine Function Levels in OSA Leptin 1) Satiety signal with direct effects on the hypothalamus 2) Stimulates lipolysis  3) Inhibits lipogenesis  4) Improves insulin sensitivity 5) Increases glucose metabolism 6) Stimulates fatty acid oxidation  Increased  (Tokuda et al., 2008) (Phillips et al., 2000) Resistin 1) Induces severe hepatic insulin resistance-increased rate of glucose production in rat  2) Functions controversial in humans  Unchanged (Cherneva et al., 2013) (Ursavas et al., 2010) Adiponectin 1) Increases fatty acid oxidation with reduction in plasma fatty acid levels  2) Decreases plasma glucose levels 3) Increases insulin sensitivity  4) Anti-inflammatory 5) Antioxidant 6) Antiatherogenic   7) Anticancer properties through the inhibition TNF-?-mediated of NF-?B pathway  Decreased  (Zhang et al., 2006) (Vatansever et al., 2011)  Unchanged  (Tokuda et al., 2008) (Ursavas et al., 2010)  20!!1-7 Animal models of sleep apnea OSA patients usually have comorbidities such as obesity, diabetes or hypertension that likely will affect cause-effect relationships. Creating animal models of OSA would minimize the influence of comorbidities and behavioral variables common in humans. Using animal models also permits the use of pharmacological agents to study the pathological mechanisms under a well-controlled environment. Ideally, animal models should mimic OSA in humans in at least three ways: (a) share aspects of the underlying pathophysiology, (b) have similar symptoms and the spectrum of disease severity that occur in humans, and (c) respond to treatment modalities that are useful in humans. Furthermore, a short life span (to allow for the unveiling of a wide range of disease-related complications within a reasonable time period), routine availability, cost effectiveness, and availability of disease-free littermates add to the usefulness of animal models. There are additional considerations when using animals that need to be considered for sleep-related research. For instance, rodents are nocturnal animals that sleep in the prone position; there is not sufficient data to evaluate how these differences relate to sleep apnea studies undertaken in humans. Table 1-1 explores various animal models for sleep disordered breathing with some of the advantages and disadvantages.   Animal models for studying sleep-disordered breathing should address at least one (or a combination) of the three main injurious consequences of sleep apnea: intermittent hypoxia/hypercapnia, strained breathing due to mechanical obstruction, and sleep fragmentation. In this regard, rodents are amenable to genetic manipulation suitable for 21!!the production of phenotypes that may characterize OSA in humans. One advantage of using rodent models to examine neurophysiological aspects of sleep apnea in humans is the high degree of similarity between the structures of the nervous systems of rodents, such as rats and mice, and humans.  Table 1-2: Benefits and disadvantages of selected animal models of sleep apnea  Model Advantages Disadvantages  Models of spontaneous sleep apnea ! ! !English Bulldog ! No intervention needed for inducing sleep apnea! Significant hypoxia occurs only during REM sleep!! Shows both central and peripheral aspects of sleep apnea!Milder non-rapid eye movement (NREM) disease (apnea) in bulldogs (AHI < 15) compared to humans!! Similar pathogenesis to human disease in terms of chronicity and progression!Apnea is not related to obesity!! Usually genetically more uniform in terms of genetic variability and disease co-morbidities Large animals are expensive to use in research!22!!Model  Advantages Disadvantages ! Similar polysomnographic structure during NREM sleep in bulldogs and patients with upper airway resistance syndrome!Sleep position different from humans!Obese Yucatan miniature pigs! Apnea is obesity-related! Majority of apnea/hypopnea events occur during REM sleep! Shows both central and peripheral aspects of sleep apnea Not well studied Obese Vietnamese pot-bellied pig SDB is obesity-related Apneas and hypopneas are not a feature of breathing during sleep in these animals  These animals show features of increased airflow resistance (snoring, inspiratory flow limitation, sleep fragmentation, and daytime sleepiness) that are similar to OSA in humans  Zucker obese rat Apnea is obesity-related Not suitable models for genetic studies 23!!Model Advantages Disadvantages  Impaired baroreflexes make them suitable for studying the relationship between sleep apnea and blood pressure  C57BL/6J mice They show two types of central apnea, post-sigh and spontaneous apnea There are several differences between rodents and humans, e.g., rodents are nocturnal animals, which sleep in a prone position; not known how these differences are relate to sleep apnea in humans  Genetic engineering easily applied to mice   Post-sigh apnea is sleep stage-dependent, similar to human children and adolescents, which makes it suitable for studying SIDS   Transgenic mice can provide valuable information about molecular targets of sleep apnea  Model Advantages Disadvantages 24!!Model Advantages Disadvantages Induced airway obstruction   Induction of obstruction through endotracheal tube Allows evaluation of the effects of varying degrees of oxygen desaturation Technical issues due to using small animals   The degree of obstruction is adjustable Need for surgical intervention  PaCO2 can be adjusted to mimic human sleep apnea  Applying a latex collar around the neck No need for surgical intervention Restriction of animal movement     The degree of obstruction is adjustable Imposed stress  Easy to use in rodents  Injection of liquid collagen in pharynx region of monkeys Similarity of upper airway anatomy in monkeys and humans Presence of obstruction during waking hours (even though limited data suggest that diurnal ventilation is not affected)  Relatively noninvasive method Problems of using large animals, especially primates, for medical research purposes 25!!Model Advantages Disadvantages  Possibility of animal training to evaluate neurocognitive consequences of sleep apnea  Intermittent hypoxia   Induction of hypoxia by endotracheal tube or mask Allows evaluation of the effects of varying degrees of oxygen desaturation Needs surgical intervention or animal restraint  Can compare the contribution role of hypoxia distinct from CO2 No airway obstruction and strenuous breathing  Evaluation of different patterns of hypoxic cycle (intermittent vs. sustained) and exposure duration (acute vs. chronic)  Induction of hypoxia by hypoxic tent or cage Same benefits as above model with added feature of being free of surgical intervention Does not allow for the study of potential strenuous breathing, making it unsuitable for studying the pathogenesis of OSA   26!!1-8 Research objectives and experimental design OSA causes cardiovascular diseases due to endothelial dysfunction and atherosclerosis. Moreover, obesity is a major risk factor for cardiovascular disease, and since a large percentage of OSA patients are obese, I wanted to study the vascular effects of intermittent hypoxia in a mouse model of diet induced obesity to mimic the physiological changes in obese OSA patients. The main hypothesis of my thesis work is that intermittent hypoxia causes endothelial dysfunction in wild type mice fed high fat diet. Endothelial function was also examined also in mice subjected to intermittent hypoxia alone, high fat diet alone or normal mice. I also examined oxidative stress, inflammation and eNOS gene expression.   The experiments explored the role of NO under basal conditions and following vasodilatory response to acetylecholine. Markers of oxidative stress (malondialdehyde) and inflammation (C-reactive protein) were measured along with total antioxidant capacity. I also used real time PCR to examine changes in eNOS gene expression. The general framework of my experiments is shown in Figure 1-2.      27!!      Figure 1-2: Research design and methods  Biochemical!(plasma):!1=!Oxidative!stress:!MDA!2=!Inflammation:!CRP!3=!Total!antioxidant!capacity!Vascular!function!(aorta):!1=!Endothelium!dependent!vasodilatation!2=!Basal!NO!production!Molecular!(aorta):!1=!eNOS!gene!expression!28!!2- Materials and methods  2-1 Animal model  Male WTC57BL/6 mice that were 8-10 weeks old were purchased from Jackson Laboratory (n = 5-10 per group; Bar Harbor, ME) and housed in the University Animal Facility with a 12-h: 12-h dark/light cycle and allowed free access to water and food. The animals were either fed a high-fat diet or a low-fat control formula. The high fat diet consisted of 60% kcal of fat (225 kcal soybean oil and 2205 kcal lard), 20% carbohydrates (500 kcal maltodextrin 10 and 275 kcal sucrose) and 20% protein (800 kcal casein and 12 kcal L-cystine) with a total of 4057 kcal/773gm. The control diet consisted of 10% fat (225 kcal Soybean and 180 kcal lard), 70% carbohydrates (1260 kcal corn starch, 140 kcal maltodextrin 10 and 1400 kcal sucrose) and 20% protein (800 kcal casein and 12 kcal L-cystine) with a total of 4057 kcal/1055gm. The diet was purchased from Research Diets (New Brunswick, NJ). Animals were divided in groups where they were exposed to IH, high fat diet, or a combination of the two. This gives four groups: 1) IA, 2) IH, 3) HFIA and 4) HFIH (see Figure 1-2).  For the IH protocol, we chose a murine model of intermittent hypoxia created by Polotsky and Tagaito (Polotsky et al. 2003). We selected this model because its profile represents extreme physiological changes occurring during sleep-related IH. Mice were housed in customized cages to deliver either an intermittent hypoxic stimulus or an 29!!intermittent room air control. Ports evenly spaced near the bottom of the cages allowed the gas to enter from four sides at the level of the bedding material. A gas control delivery system regulated the flow of room air, N2, and O2 into the customized cages housing the mice. Programmable solenoids and flow regulators controlled the manipulation of inspired O2 fraction (FIO2) levels in each cage over a wide range of IH profiles. During the 12-h light cycle, FIO2 was reduced from 20.9 to 5.0% over a 30-s period and rapidly reoxygenated to room air levels using a burst of 100% O2 from a medical air compressor in the following 30-s period. During the 12-h dark cycle, a constant flow of room air was delivered to the cages. The use of multiple inputs into the cage produced a uniform nadir FIO2 level throughout the cage. The fluctuating FIO2 levels were monitored with an O2 analyzer (model OM11, Sensor Medics, Yorba Linda, CA). Mice were exposed for 6 wk in either chronic IH or intermittent air (control). In mice exposed to the chronic IH protocol, an initial titration period was necessary over the first two days to allow the mice to adapt to IH. Initially, the nadir FIO2 was set to 12% and then gradually reduced over a 8-h periods to 10%, 8% then to the experimental level of nadir FIO2 of 4.8?5%. The control groups of mice were exposed to chronic intermittent air with flow rates and timing of solenoid valves identical to the IH group.  2-2 Plasma and tissue collection  Pentobarbital (100 mg/kg) was used to anesthetize the mice. Blood samples were withdrawn from the inferior vena cava using heparinized syringes and then transferred 30!!to Eppendorf tubes. The blood was then centrifuged (10 min at 4oC, 1000g) for plasma separation and the collected plasma was stored at -76oC in aliquots after snap freezing in liquid nitrogen. Mice were euthanized after blood collection by removing the heart. Aortic blood vessels were dissected and immersed in ice-cold physiologic solution (PSS). The connective tissue around the blood vessels was removed using microsurgery equipment and aided by a dissecting microscope. Dissected blood vessels were either preserved in RNAlater, snap frozen in liquid nitrogen and stored at -76oC, or mounted on the wire myograph for functional studies.   2-3 Assessment of vascular function  The cleaned aortas were cut into equal 2mm long rings and mounted on a wire myograph DMT 620M (Danish Myotechnology, Aarhus, Denmark) for measuring isometric force (Mulvany and Halpern, 1977). Each chamber in the wire myograph was filled with 6 ml PSS gassed with 95% O2 and 5% CO2. To maintain physiologic conditions, the PSS was kept at pH 7.4 and 37Co. The aortic rings were stretched to their optimal tension (5.5 mN) according to preliminary studies where the resting tension exhibiting the maximum force in response to 60 mM KCl was considered the optimal resting tension. The PSS was replaced every 30 minutes during the adjusting and equilibration periods. At the resting tension, the aortic rings were allowed to equilibrate for 20 minutes and then challenged twice with 80 mM KCl before creating the concentration response curves. 31!! 2-3-1 Assessment of endothelium-dependant and-independent vasodilatation  The aortic rings were constricted with a submaximal concentration (producing 60%-80% of the maximum response to 80 mM KCl) of the alpha1 adrenoceptor agonist phenylephrine (PE). After a stable contraction was obtained, ACh (10-9to 10-5M) was cumulatively added in half-log increments to examine endothelium-dependent vasodilatation. After a 30-minute washout period, the arterial rings were reconstricted with phenylephrine, and sodium nitroprusside (SNP), a direct NO donor (10-10 to 10-5M at half-log increments in a cumulative manner) was added to examine endothelium-independent vasodilatation. Responses to vasodilators (ACh and SNP) were calculated as the percent decrease in force with respect to the initial PE induced constriction (% relaxation).   2-3-2 Assessment of basal NO  PE concentration response curves were constructed in the absence and the presence of L-NAME (10-4M). By inhibiting NOS, L-NAME decreases basal NO production and thus eliminates its background vasorelaxant effect. Therefore, L-NAME is expected to increase PE-induced contractions. The area under the curve (AUC) for PE concentration response curve obtained in the presence of L-NAME was calculated, and divided by the AUC obtained in the absence of L-NAME. The increase in AUC due to 32!!the presence of L-NAME is calculated and used as a measure of basal NO release. The higher the level of basal NO, the larger the increase in PE contractions induced by L-NAME.  2-4 Measuring plasma variables  MDA levels were assayed using colorimetric lipid peroxidation assay (GenWay Biotech, California, USA) while CRP levels were measured using mouse C-reactive protein enzyme linked immunoassay (GenWay Biotech, California, USA). Some of the plasma samples were diluted to fall within the standard curve provided by the kit manufacturer.  2-5 Real-time PCR   When animals were sacrificed, pieces of the extracted aorta were preserved in RNAlater to stabilize RNA and prevent its degradation for gene expression analysis. All samples were stored at -20C. For RNA isolation, aortic pieces were ground using Tissuelyser;  RNeasy fibrous mini kit was then used to extract total RNA. The kit uses proteinase K to digest the fibrous tissue and DNAse to remove unwanted genomic DNA. Eventually, total RNA bound to the column is eluted using RNase free water. The total RNA volumes were then determined by the absorbance at 260nm.  Synthesis of cDNA was done using High Capacity Archive Kit (Applied Biosystems, Ontario, Canada). Aortic RNA samples were incubated in 1X Multiscribe Reverse Transcriptase, 1X 33!!random primer mix, 1X dNTP mix, 1X Reverse Transcription Buffer in a final reaction volume of 40Ul. Real-Time PCR using CT method of relative quantification quantified Gene expression of eNOS.  The primers used were TaqMan MGB probes (FAM-fluorescin dye-labeled) from Applied Biosystems specific for eNOS (Mm00435217_m1). B-Actin was used as an endogenous control. Samples were heated at 50?C for 2 min followed by incubation at 95?C for 10 min and 40 cycles of 95?C for 15 s and 60?C for 1 min in a 7500 Real Time PCR System (Applied Biosystems, Ontario, Canada). Quantification of relative changes in expression was determined following the manufacturer?s suggested protocol for a ??CT assay (an assay that involves comparing the Ct values of the samples of interest with a control or calibrator such as a non-treated sample or RNA from normal tissue). The Ct values of both the calibrator and the samples of interest are normalized to an appropriate endogenous housekeeping gene), and data were analyzed using the 7500 System Sequence Detection software, version 1.2.3 (Applied Biosystems, Ontario, Canada). Each sample was run in duplicate.  2-6 Statistical analysis All values are expressed as means ? SE. The sample size of each group is specified as (n) in the footnote for each table or figure. Vascular function data were recorded and analyzed by Powerlab 4/25 and Labchart 7 reader (AD instruments, Australia). Statistical analysis was performed using Prism version 6.0 (GraphPad software, 34!!California, USA) using different statistical tests as indicated below each table or figure. The statistical significant difference was set at p<0.05.  2-7 Drugs, reagents and solutions  All drugs were obtained from Sigma-Aldrich (Missouri, USA) and RT-PCR reagents were purchased from AB Laboratories (Ontario, Canada). PSS was prepared according to the following composition (in mM):  NaCl (119), KCl (4.7), KH2PO4 (1.18), MgSO4 (1.17), NaHCO3  (24.9), EDTA (0.023), CaCl2 (1.6) and dextrose (11.1). High KCl solution was prepared by equimolar substitution of NaCl in PSS.             35!!3- Results!3-1 Increased body weight only in mice fed high fat diet  Mice were weighed before starting the experiment to determine the effect of IH and high fat diet on body weight after 6 weeks of feeding. We found no differences in mice subjected to IH or IA with normal diet, but mice subjected to IH and IA with high fat diet showed a significant weight increase when we compared the weight between day 0 and day 44 (Table 3-2).  !Table 3-2: Weight changes between different mice groups after 44 days. ! Group! Baseline!(g)! Day!44!(g)! P!value!HFIH!(n=10)! 27.59!?!0.48! 34.42!?!0.55***! P<0.0001!HFIA!(n=10)! 27.60!?!0.51! 32.66!?!0.60***! P<0.0001!IH!(n=5)! 26.43!?!0.69! 27.01!?!0.66! P=!NS!IA!(n=5)! 26.76!?!0.92! 28.65!?!1.16! P=!NS!! 36!!3-2 Impaired endothelial function in mice subjected to intermittent hypoxia and fed high fat diet  3-2-1 Attenuated ACh-induced endothelial vasodilatation  After mounting the aortic rings on the wire myograph, ACh was added to stimulate relaxation through NO production (Figure 3-1). Maximum ACh induced relaxation was not attenuated in mice fed high fat diet, either subjected to IH or control. (Emax: HFIA 95 ? 0.7%, IH 94 ? 2%, IA 97 ? 1%) but it was significantly attenuated in mice fed high fat diet and subjected to IH when compared to all other groups (Emax: 78 ? 2%, p<0.0001).  No changes were seen in half-maximal effective concentration of ACh between all groups (log EC50: HFIH 7.4 ? 0.1, HFIA -7.4 ? 0.07, IH -6.9 ? 0.07, IA -7.0 ? 0.05M, p = NS). To confirm that the change in relaxation is endothelial-dependent, I measured endothelial?independent relaxation using sodium nitroprusside and I found no differences between the groups (Emax: IH: 98.7 ? 0.3, IA: 98.3 ? 0.5, HFIH: 98.9 ? 0.3, HFIA: 98.6 ? 0.5, p = NS).   37!!  Figure 3-1: Cumulative concentration response curves of ACh (upper panel) and SNP (lower panel) in phenylephrine constricted aortae of wild type mice. Values are displayed as mean + S.E. and represent n= 5-8 mice Statistical analysis was done using two-way repeated measures ANOVA followed by Bonferroni post-test *denotes p<0.05 vs. HFIA, IH and IA aorta. ACh= acetylcholine, SNP= sodium nitroprusside 38!!3-2-2 Impaired basal NO production Basal nitric oxide production from the vascular endothelium maintains an underlying vasodilatory tone. Basal NO production was impaired in HFIH group when compared to HFIA. Figure 3-2 shows that after incubating the aorta in L-NAME, HFIA group show augmented PE contraction response (Emax: 179 ? 10% in the presence of L-NAME) more effectively than HFIH group (Emax: 149 ? 11% in the presence of L-NAME). Basal NO production is significantly higher in HFIA group when compared to HFIH (AUC= 183.4 ? 7.7 vs. 145.7 ? 9.9, p<0.05)         39!!                        40!! Figure 3-2: A, B) Cumulative concentration response curves to Ph before and after adding L-NAME for HFIA and HFIH. C) Area under the curve calculated for the contraction response to Ph after adding L-NAME. Values are displayed as mean + S.E. and represent n= 5 mice AUC was compared using unpaired, two-tailed t-test. *denotes p<0.05 vs. HFIH. L-NAME = N?-Nitro-L-arginine methyl ester, PE = Phenylephrine  3-3 Increased oxidative stress in mice subjected to intermittent hypoxia and high fat diet   3-3-1 Increased MDA levels MDA is an oxidative stress marker that is produced as a byproduct of polyunsaturated fatty acid peroxidation and arachidonic acid metabolism. Figure 3-3 shows that MDA levels were increased significantly in mice subjected to IH when compared to IA (0.68 ? 0.04 vs. 0.41 ? 0.03?M, p<0.05) but in HFIH group, MDA levels were increased even further when compared to all groups (0.83 ? 0.08?M, p<0.05). High fat diet by itself (HFIA group) had no significant effect on MDA levels (0.45 ? 0.03?M. p = NS). !!!!41!!!  Figure 3-3: Plasma MDA levels as markers of oxidative stress Values are displayed as mean + S.E. and represent n= 6 mice Statistical analysis was done using two-way repeated measures ANOVA followed by Bonferroni post-test *denotes p<0.05 vs. IA normal chow, #denotes p<0.05 vs. IA high fat. MDA= malondialdehyde.   42!!3-3-2 Total antioxidant capacity was not changed Figure 3-4 shows that there was no change in antioxidant capacity between all groups. (IH: 4.19 ? 0.09mM, IA: 4.10 ? 0.09mM, HFIA: 4.26 ? 0.14mM, HFIH: 4.02 ? 0.05mM, p = NS)   Figure 3-4: Plasma total antioxidant capacity. Values are displayed as mean + S.E. and represent n= 6 mice Statistical analysis was done using two-way repeated measures ANOVA followed by Bonferroni post-test 43!!3-4 Increased inflammation in mice subjected to intermittent hypoxia and high fat diet  3-4-1 Increased CRP levels CRP is an important marker of inflammation and is a strong predictor cardiovascular events, at least in humans. Figure 3-5 shows CRP levels were higher in IH group when compared to IA (10.39 ? 0.38 vs. 8.70 ? 0.21?g/ml, p<0.05); adding a high fat diet to intermittent hypoxia (HFIH group) increased CRP levels even more (11.87 ? 0.31?g/ml, p<0.05). High fat diet alone did not have a significant effect on CRP levels compared to control (9.96 ? 0.37?g/ml, p = NS).        44!! Figure 3-5: Plasma CRP levels as marker of inflammation. Values are displayed as mean + S.E. and represent n= 6 mice Statistical analysis was done using two-way repeated measures ANOVA followed by Bonferroni post-test *denotes p<0.05 vs. IA normal chow, #denotes p<0.05 vs. IA high fat, $denotes p<0.05 vs. IH normal chow. CRP= C-reactive protein.  45!!3-5 Expression of aortic eNOS was not changed In Figure 3-6, expression of eNOS (relative quantity value) did not significantly change in any of groups of mice studied (IH: 1.26 ? 0.13, IA: 1.10 ? 0.10, HFIH: 1.25 ? 0.19, HFIA: 1.18 ? 0.21).      Figure 3-6: Endothelial nitric oxide synthase expression in aorta. Values are displayed as mean + S.E. and represent n= 6 mice Statistical analysis was done using two-way repeated measures ANOVA followed by  Bonferroni post-test 46!!4- Discussion  My study investigated the effects of intermittent hypoxia and high fat diet on aortic endothelial function, oxidative stress and inflammation in male WTC57BL/6 mice. The major finding of this study is that intermittent hypoxia combined with high fat diet caused endothelial dysfunction (impaired ACh-induced vasodilation and reduced basal NO production) after 6 weeks in wild type mice, while neither intermittent hypoxia nor high fat diet alone had an effect on endothelial function. My study also demonstrates that: 1) Intermittent hypoxia causes oxidative stress but the effect is exacerbated when high fat diet is added. 2) Intermittent hypoxia leads to general inflammation but adding high fat diet aggravates inflammation. 3) Intermittent hypoxia and high fat diet had no effect on antioxidant capacity or eNOS gene expression.  4-1 Effects of intermittent hypoxia and high fat diet on body weight High fat diet increased weight significantly regardless the hypoxic events. Yang et al. support this data where they found that 4 weeks of intermittent hypoxia did not affect body weight. They suggested that the unchanged weight was related to increased circulating levels of leptin, an adipocyte-secreted hormone inducing satiety and metabolic rate (Friedman, 1998; Breslow et al., 1999), which has a feed-back effect to prevent weight gain and even causing weight loss (Polotsky et al., 2003; Li et al., 2005; Yang et al., 2011).  In contrast, less severe hypoxia (10% oxygen) did not increase serum leptin and also had no effect on weight (Li et al., 2007).  47!!Patients with OSA have high leptin levels (hyperleptinemia) but tend to gain weight (Phillips et al., 2000). Hyperleptinemia in the presence of obesity per se has been explained by ?leptin resistance? (Caro et al., 1996), and may therefore predispose patients with sleep apnea to weight gain. This may explain the difficulty in weight management in this population, inasmuch as patients with sleep apnea may already be predisposed to weight gain secondary to reduced physical activity resulting from tiredness and daytime somnolence.  4-2 Endothelial dysfunction in intermittent hypoxia and diet induced obesity Endothelium dependent relaxation was only compromised when both intermittent hypoxia and high fat diet were combined, but there was no effect on endothelium dependent dilation when either was used alone; this suggests a synergistic effect. Yang et al. used a similar model of intermittent hypoxia and showed that mice exhibit endothelial dysfunction after 14 days of IH only (Yang et al., 2011), while a different study shows that 35 days of intermittent hypoxia had no effect on ACh-induced endothelial vasodilatation (Dematteis et al., 2008). My study shows that intermittent hypoxia alone for 6 weeks had no effect on the ACh-induced vasodilatation or basal NO production. There could be a number of fators contributing to the discrepancy between the results in different studies although the same model and methods were used; one factor could be that male wild-type C57BL/6J mice are usually resistant to atherosclerosis (Paigen et al., 1987).  Another factor could be the time of sacrifice, since 48!!sacrificing mice after 12 hours of intermittent hypoxia may have different physiological and pathological effects than sacrificing mice after 12 hours of intermittent air.  Obesity is a polygenic disease that is usually caused by increased calorie intake from especially saturated fats (Caro et al., 1996). Endothelial dysfunction, a hallmark of atherosclerosis, is frequently associated with obesity (Avogaro and de Kreutzenberg, 2005). Obesity promotes endothelial dysfunction through many metabolic disorders such as accumulation of adipose tissue, dyslipidemia, elevated blood pressure and diabetes, which are linked to vascular oxidative stress and inflammation (Van Gaal et al., 2006). In a recent study, obesity and endothelial dysfunction were induced after feeding mice a high fat diet for 100 days. Diet induced obesity resulted in uncoupling eNOS through loss of BH4 which in turn is related to imbalance between NO and superoxide production (Ketonen et al., 2010). In my study, we fed mice a high fat diet for only 6 weeks, which could explain why we did not observe endothelial dysfunction when mice were fed a high fat diet alone. Combining IH and high fat diet synergistically affected not only stimulated NO production but also basal levels since HFIH group showed weak contractility response to phenylephrine after incubating aortic rings in L-NAME when compared to HFIA (which had a greater response to phenylephrine). Our results are consistent with other reports that mice subjected to intermittent hypoxia or high fat/cholesterol diet alone did not demonstrate signs of atherosclerosis, but when combined for 12 weeks, mice developed atherosclerotic lesions in the aortic origin and descending aorta (Savransky et al., 2007b). 49!!4-3 Oxidative stress and inflammation in intermittent hypoxia and diet induced obesity Oxidative stress is features in intermittent hypoxia; my results show that MDA levels are increased in the mice subjected to intermittent hypoxia alone; which is in agreement with several other studies (Li et al., 2005; Li et al., 2007; Jun et al., 2008; Yang et al., 2011). Oxidative stress is also influenced by the severity of IH; a study reported that lipid peroxidation is higher in mice subjected to IH at an inspired oxygen nadir of 5% than 10% (Li et al., 2007). Other studies also show that IH causes oxidative stress in organs such as the brain, liver and carotid body (Jun et al., 2008). Each organ differs in its susceptibility to oxidative stress caused by IH. This suggests that IH-induced oxidative stress is a phenomenon that is species, organ, and time dependent. When high fat diet was added to hypoxia, MDA levels were significantly higher, showing that high fat diet contributes to exacerbation of oxidative stress caused by hypoxia. We used a mouse model of diet-induced obesity since this model resembles human obesity, which is a polygenic disease and strongly linked to high caloric intake. Diet induced obesity increases oxidative stress, although in my study there was no significant elevation in MDA levels in mice fed high fat diet alone, possibly because this study lasted only for six weeks. Other studies reported increased oxidative stress after 112 days (Kobayasi et al., 2010) or 100 days of feeding mice a high fat diet (Ketonen et al., 2010). It is likely that both IH and high fat diet increase oxidative stress in mice through different mechanisms, and that combining IH and a high fat diet further augments oxidative stress. 50!!Many studies in mice and rats show that IH causes systemic inflammation by upregulating transcription factors such as NF-?B, which is important in inflammatory and innate immune responses.  Activated NF-?B controls the expression of several genes including those encoding inflammatory cytokines such as tumour necrosis factor alpha (TNF-?) and interleukin 6 (IL-6), chemokines such as IL-8, adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1), and upregulation of these inflammatory molecules will eventually lead to endothelial dysfunction and atherosclerosis. Diet induced obesity also causes inflammation in mice fed a high fat diet for 12 weeks, where there was a 3.5-fold increased whole body NF-?B activity (Carlsen et al., 2009). My study demonstrates that intermittent hypoxia alone increased CRP levels, an important marker of inflammation and a potent inducer of NF-?B (Hattori et al., 2003). CRP levels are recognized as potent, independent predictors of future cardiovascular events (Liuzzo et al., 1994; Haverkate et al., 1997; Ridker and Haughie, 1998), but adding high fat diet led to further increase in CRP levels, indicating a more inflammatory status.  Recent large-scale studies suggest that the elevated CRP levels are attributable to the presence of cardiovascular risk factors, in particular obesity (Miller et al., 2005; Khera et al., 2006; Cao et al., 2007). Also numerous case-control studies have identified increased levels of CRP in OSAS patients (Yokoe et al., 2003; Can et al., 2006; Saletu et al., 2006). A recent study  (The Icelandic Sleep Apnea Cohort) found that BMI has a significantly high correlation with CRP levels in OSA patients (Arnardottir et al., 2012), which is consistent with my data. Thus, both obesity and OSAS are pro-inflammatory 51!!conditions that can mutually enhance progression and severity of cardiovascular diseases.  Antioxidants play an important role in reducing the formation, and increase the scavenging of free radicals and other potentially toxic species. Measuring antioxidant capacity of biological fluids and other samples provides an indication of the overall capability to counteract ROS, resist oxidative damage and combat oxidative stress related diseases. In my study, antioxidant capacity was not significantly changed in the four experimental groups. My experiment only lasted 6 week , perhaps it?s not chronic enough to deplete all cellular reductants since I?m measuring the total antioxidant capacity and not specific antioxidants.  The effect of intermittent hypoxia on antioxidant capacity is controversial. Our results are consistent with some studies on OSA patients were there was no difference in antioxidant capacity between OSA patients and controls (Wali et al., 1998; Alzoghaibi and Bahammam, 2005). OSA severity not only affects antioxidant capacity in OSA patients, but also other factors could such as obesity, smoking, age and sex and other characteristics exacerbates this effect. This might explain the divergent results in different studies (Simiakakis et al., 2012). Studies that recorded decreased antioxidant capacity suggested that hypoxia induced reduction of antioxidant capacity may be associated with dysregulation of genes involved in the modulation of ROS or enzymes involved in the production of the antioxidant barriers (Simiakakis et al., 2012) or hypoxic conditions may also result in transient depletion of cellular reductants (such as 52!!glutathione and vitamins such as Vit E and C), which constitute a main line of antioxidant defense (Christou et al., 2003).   4-4 Expression of eNOS in intermittent hypoxia and diet induced obesity  Expression and activity of eNOS, a main source of basal and stimulated NO release, have been reported to be upregulated (Arnet et al., 1996; Le Cras et al., 1998; Shirai et al., 2003) downregulated (Toporsian et al., 2000; Takemoto et al., 2002), or unchanged (Murata et al., 2001; Tahawi et al., 2001) in various experimental models of hypoxia and repetitive hypoxia/reoxygenation. My results show that neither intermittent hypoxia nor diet induced obesity or their combination affected eNOS expression in the aorta. One study shows that high-fat diet-fed wild-type C57BL/6J mice showed decreased eNOS protein in adipose tissue without significant changes in eNOS levels in skeletal muscle or aorta which is consistent with my results (Sansbury et al., 2012) Contradictory reports of eNOS expression and activity appear to be due to temporal variations in experimental hypoxemic conditions, and differences in the species and vascular bed from which endothelial cells were derived. Several mechanisms of hypoxia-induced eNOS down-regulation have been proposed (Coulet et al., 2003; Tai et al., 2004). On a transcriptional level, hypoxia-induced activation of hypoxia inducible factor-2 initially upregulates eNOS mRNA followed by a prolonged decrease in eNOS mRNA levels (Coulet et al., 2003). On a post-transcriptional level, hypoxia destabilizes eNOS mRNA, in part via the Rho kinase pathway in human venous and pulmonary artery endothelial cells (Tabrizi-Fard et al., 1999; Takemoto et al., 2002).  53!!5- Summary Many studies report that intermittent hypoxia/high fat diet causes oxidative stress and inflammation leading to endothelial dysfunction and atherosclerosis. My study indicates that the combination of intermittent hypoxia and high fat diet results in impairment of endothelium function (ACH-induced vasodilation and basal NO production) after 6 weeks. High fat diet did not cause inflammation or oxidative stress but intermittent hypoxia did; adding high fat diet to intermittent hypoxia exacerbated oxidative stress and inflammation although antioxidant capacity and aortic eNOS gene expression remained unchanged.     54!! Figure 5-1: Intermittent hypoxia and diet-induced obesity lead to endothelial dysfunction    55!!6- Limitations of the animal model and study 1) My model does not incorporate airway obstruction as the perturbing factor altering arterial blood-gas levels during sleep. 2) The absence of airway obstruction in my model eliminates the large negative intrathoracic pressure swings that characterize obstructive sleep apnea. 3) My study lasted only 6 weeks, but it is likely that if longer, I might show endothelial dysfunction in intermittent hypoxia and diet induced obesity alone.         56!!7- Future directions I am planning to study the effects of intermittent hypoxia and high fat diet on the vascular endothelium on a longer period than this study. i will also not only look at the aorta, but other arteries and arterioles such as the femoral artery and the carotid artery to assess the endothelial function. 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