Aidan Bradford

Intravascular thrombosis after hypoxia-induced pulmonary hypertension: regulation by cyclooxygenase-2.

Arachidonic acid metabolism leads to the generation of biologically active metabolites that regulate cell growth and proliferation, as well as survival and apoptosis. We have demonstrated previously that platelet-type 12-lipoxygenase (LOX) regulates the growth and survival of a number of cancer cells. In this study, we show that overexpression of platelet-type 12-LOX in prostate cancer PC3 cells or epithelial cancer A431 cells significantly extended their survival and delayed apoptosis when cultured under serum-free conditions. These effects were shown to be a result of enhanced surface integrin expression, resulting in a more spread morphology of the cells in culture. PC3 cells transfected with 12-LOX displayed increased alpha(v)beta(3) and alpha(v)beta(5) integrin expression, whereas other integrins were unaltered. Transfected A431 cells did not express alpha(v)beta(3); however, alpha(v)beta(5) integrin expression was increased. Treatment of both transfected cell lines with monoclonal antibody to alpha(v)beta(5) (and in the case of PC3 cells, anti-alpha(v)beta(3)) resulted in significant apoptosis. In addition, treatment with 100 nM 12(S)-hydroxy-eicosatetraenoic acid, the end product of platelet-type 12-LOX, but not other hydroxy-eicosatetraenoic acids, enhanced the survival of wild-type PC3 and A431 cells and resulted in increased expression of alpha(v)beta(5). Furthermore, Baicalein or N-benzyl-N-hydroxy-5-phenylpentamide, specific 12-LOX inhibitors, significantly decreased alpha(v)beta(5)-mediated adhesion and survival in 12-LOX-overexpressing cells. The results show that 12-LOX regulates cell survival and apoptosis by affecting the expression and localization of the vitronectin receptors, alpha(v)beta(3) and alpha(v)beta(5), in two cancer cell lines.Background—Pulmonary hypertension induced by chronic hypoxia is characterized by thickening of pulmonary artery walls, elevated pulmonary vascular resistance, and right-heart failure. Prostacyclin analogues reduce pulmonary pressures in this condition; raising the possibility that cycloxygenase-2 (COX-2) modulates the response of the pulmonary vasculature to hypoxia. Methods and Results—Sprague-Dawley rats in which pulmonary hypertension was induced by hypobaric hypoxia for 14 days were treated concurrently with the selective COX-2 inhibitor SC236 or vehicle. Mean pulmonary arterial pressure (mPAP) was elevated after hypoxia (28.1±3.2 versus 17.2±3.1 mm Hg; n=8, P<0.01), with thickening of small pulmonary arteries and increased COX-2 expression and prostacyclin formation. Selective inhibition of COX-2 aggravated the increase in mPAP (42.8±5.9 mm Hg; n=8, P<0.05), an effect that was attenuated by the thromboxane (TX) A2/prostaglandin endoperoxide receptor antagonist ifetroban. Urinary TXB2 increased during hypoxia (5.9±0.9 versus 1.2±0.2 ng/mg creatinine; n=6, P<0.01) and was further increased by COX-2 inhibition (8.5±0.7 ng/mg creatinine; n=6, P<0.05). In contrast, urinary excretion of the prostacyclin metabolite 6-ketoprostaglandin F1&agr; decreased with COX-2 inhibition (8.6±3.0 versus 27.0±4.8 ng/mg creatinine; n=6, P<0.05). Platelet activation was enhanced after chronic hypoxia. COX-2 inhibition further reduced the PFA-100 closure time and enhanced platelet deposition in the smaller pulmonary arteries, effects that were attenuated by ifetroban. Mice with targeted disruption of the COX-2 gene exposed to chronic hypoxia had exacerbated right ventricular end-systolic pressure, whereas targeted disruption of COX-1 had no effect. Conclusions—COX-2 expression is increased and regulates platelet activity and intravascular thrombosis in hypoxia-induced pulmonary hypertension.

Chronic intermittent hypoxia increases haematocrit and causes right ventricular hypertrophy in the rat.

Chronic continuous hypoxia increases haematocrit and causes right ventricular hypertrophy and pulmonary hypertension. In obstructive sleep apnoea, the exposure to hypoxia is intermittent rather than continuous but the effects of chronic intermittent hypoxia on haematocrit and right ventricular mass are unclear. Wistar rats were exposed to alternating periods of hypoxia and normoxia twice per min for 8 h per day for 5 weeks in order to mimic the intermittent hypoxia of obstructive sleep apnoea in humans. Haematocrit was significantly raised at day 7, 14, 21, 28 and 35 of the treatment period. At the end of the treatment, there was a significant increase in right ventricular mass. Therefore, chronic intermittent hypoxia increases haematocrit and right heart mass. These results suggest that the raised haematocrit and pulmonary arterial pressure observed in some cases of obstructive sleep apnoea in humans may be caused by intermittent nocturnal hypoxaemia.

Chronic intermittent hypercapnic hypoxia increases pulmonary arterial pressure and haematocrit in rats

Sleep-disordered breathing is associated with pulmonary hypertension and raised haematocrit. The multiple episodes of apnoea in this condition cause chronic intermittent hypoxia and hypercapnia but the effects of such blood gas changes on pulmonary pressure or haematocrit are unknown. The present investigation tests the hypothesis that chronic intermittent hypercapnic hypoxia causes increased pulmonary arterial pressure and erythropoiesis. Rats were treated with alternating periods of normoxia and hypercapnic hypoxia every 30 s for 8 h per day for 5 days per week for 5 weeks, as a model of the intermittent blood gas changes which occur in sleep-disordered breathing in humans. Haematocrit, red blood cell count and haemoglobin concentration were measured each week and systemic and pulmonary arterial blood pressure and heart weight were measured after 5 weeks. In relation to control, chronic intermittent hypercapnic hypoxia caused a significant increase in systemic (104.3+/-4.7 mmHg versus 121.0+/-10.4 mmHg) and pulmonary arterial pressure (20.7+/-6.8 mmHg versus 31.3+/-7.2 mmHg), right ventricular weight (expressed as ratios) and haematocrit (45.2+/-1.0% versus 51.5+/-1.5%). It is concluded that the pulmonary hypertension and elevated haematocrit associated with sleep-disordered breathing is caused by chronic intermittent hypercapnic hypoxia.

Does episodic hypoxia affect upper airway dilator muscle function? Implications for the pathophysiology of obstructive sleep apnoea.

Obstructive sleep apnoea (OSA) is characterised by repetitive collapse of the upper airway during sleep owing to a sleep-related decrement in upper airway muscle activity with consequent failure of the pharyngeal dilator muscles to oppose the collapsing pressure that is generated by the diaphragm and accessory muscles during inspiration. The causes of upper airway obstruction during sleep are multi-factorial but there is evidence implicating intrinsic upper airway muscle function and impaired central regulation of the upper airway muscles in the pathophysiology of OSA. The condition is associated with episodic hypoxia due to recurrent apnoea. However, despite its obvious importance very little is known about the effects of episodic hypoxia on upper airway muscle function. In this review, we examine the evidence that chronic intermittent hypoxia can affect upper airway muscle structure and function and impair CNS control of the pharyngeal dilator muscles. We review the literature and discuss results from our laboratory showing that episodic hypoxia/asphyxia reduces upper airway muscle endurance and selectively impairs pharyngeal dilator EMG responses to physiological stimulation. Our observations lead us to speculate that episodic hypoxia--a consequence of periodic airway occlusion--is responsible for progression of OSA through impairment of the neural control systems that regulate upper airway patency and through altered respiratory muscle contractile function, leading to the establishment of a vicious cycle of further airway obstruction and hypoxic insult that chronically exacerbates and perpetuates the condition. We conclude that chronic intermittent hypoxia/asphyxia contributes to the pathophysiology of sleep-disordered breathing.

Tempol Ameliorates Pharyngeal Dilator Muscle Dysfunction in a Rodent Model of Chronic Intermittent Hypoxia

Respiratory muscle dysfunction is implicated in the pathophysiology of obstructive sleep apnea syndrome (OSAS), an oxidative stress disorder prevalent in men. Pharmacotherapy for OSAS is an attractive option, and antioxidant treatments may prove beneficial. We examined the effects of chronic intermittent hypoxia (CIH) on breathing and pharyngeal dilator muscle structure and function in male and female rats. Additionally, we tested the efficacy of antioxidant treatment in preventing (chronic administration) or reversing (acute administration) CIH-induced effects in male rats. Adult male and female Wistar rats were exposed to alternating cycles of normoxia and hypoxia (90 s each; Fi(O(2)) = 5% O(2) at nadir; Sa(O(2)) ∼ 80%) or sham treatment for 8 h/d for 9 days. Tempol (1 mM, superoxide dismutase mimetic) was administered to subgroups of sham- and CIH-treated animals. Breathing was assessed by whole-body plethysmography. Sternohyoid muscle contractile and endurance properties were examined in vitro. Muscle fiber type and cross-sectional area and the activity of key metabolic enzymes were determined. CIH decreased sternohyoid muscle force in male rats only. This was not attributable to fiber transitions or alterations in oxidative or glycolytic enzyme activity. Muscle weakness after CIH was prevented by chronic Tempol supplementation and was reversed by acute antioxidant treatment in vitro. CIH increased normoxic ventilation in male rats only. Sex differences exist in the effects of CIH on the respiratory system, which may contribute to the higher prevalence of OSAS in male subjects. Antioxidant treatment may be beneficial as an adjunct OSAS therapy.

Oxidative stress impairs upper airway muscle endurance in an animal model of sleep-disordered breathing.

Obstructive sleep apnoea is characterised by intermittent hypoxia due to recurrent obstructions of the pharyngeal airway during sleep. We have shown that chronic intermittent hypoxia impairs respiratory muscle function and CNS control of upper airway patency. In this study, we tested the hypothesis that disruption of an endogenous antioxidant defence system exacerbates the effects of intermittent hypoxia on upper airway muscle contractile function. Thirty-two male Wistar rats were placed in restrainers with their heads in hoods in which the ambient oxygen concentration could be modified by controlling the gas supply to the hoods. Sixteen rats were exposed to alternating equal periods of hypoxia and normoxia, twice per minute, 8 hours per day for 1 week. The remaining 16 animals were exposed to normoxia continuously under identical experimental conditions. In both groups, half the animals received daily injections of buthionine sulfoxamine (BSO), an inhibitor of the rate-limiting enzyme in glutathione synthesis. The other half received daily vehicle injections. At the end of the 1-week treatment period, the sternohyoid muscles were removed and fatigue characteristics were determined in vitro. Intermittent hypoxia was associated with a decrease in sternohyoid muscle endurance, an effect that was exacerbated by treatment with BSO. In separate experiments, daily treatment with the antioxidant N-acetyl cysteine blocked the deleterious effects of intermittent hypoxia on respiratory muscle function. We suggest that oxidative stress contributes to impaired upper airway muscle endurance in our animal model and that endogenous glutathione may be especially important in limiting free radical-induced muscle dysfunction. Our results may have particular relevance to respiratory disorders associated with recurrent hypoxia, such as the sleep apnoea/hypopnoea syndrome.

Chronic hypoxia increases rat diaphragm muscle endurance and sodium–potassium ATPase pump content

The effects of chronic hypoxia (CH) on respiratory muscle are poorly understood. The aim of the present study was to examine the effects of CH on respiratory muscle structure and function, and to determine whether nitric oxide is implicated in respiratory muscle adaptation to CH. Male Wistar rats were exposed to CH for 1–6 weeks. Sternohyoid and diaphragm muscle contractile properties, muscle fibre type and size, the density of fibres expressing sarco/endoplasmic reticulum calcium-ATPase (SERCA) 2 and sodium–potassium ATPase (Na+,K+-ATPase) pump content were determined. Muscle succinate dehydrogenase (SDH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) dehydrogenase activities were also assessed. Acute and chronic blockade of nitric oxide synthase (NOS) was employed to determine whether or not NO is critically involved in functional remodelling in CH muscles. CH improved diaphragm, but not sternohyoid, fatigue tolerance in a time-dependent fashion. This adaptation was not attributable to increased SDH or NADPH dehydrogenase activities. The areal density of muscle fibres and relative area of fibres expressing SERCA2 were unchanged. Na+,K+-ATPase pump content was significantly increased in CH diaphragm. Chronic NOS inhibition decreased diaphragm Na+,K+-ATPase pump content and prevented CH-induced increase in muscle endurance. This study provides novel insight into the mechanisms involved in CH-induced muscle plasticity. The results may be of relevance to respiratory disorders characterised by CH, such as chronic obstructive pulmonary disease.

The effects of changes in laryngeal airway CO2 concentration on genioglossus muscle activity in the anaesthetized cat

In the anaesthetized cat the larynx was isolated in situ, artificially ventilated and used to assess reflex effects exerted by respiration‐related laryngeal stimuli on genioglossus electromyographic activity (Gg EMG) and respiratory frequency (RF). Phasic Gg EMG was not observed when the larynx was unventilated but was evoked, with a concurrent decrease in RF, when negative pressures or oscillatory pressures similar to those of normal ventilation were applied to the larynx. Increases in laryngeal airway CO2 concentration also enhanced Gg EMG and reduced RF. All reflex effects were abolished by bilateral section of the superior laryngeal nerves. We propose that negative intralaryngeal pressure and CO2 may act together to restore pharyngeal patency during obstructive apnoea.

Reactive oxygen species mediated diaphragm fatigue in a rat model of chronic intermittent hypoxia.

What is the central question of this study? The effects of chronic intermittent hypoxia (CIH) on respiratory muscles are relatively underexplored. It is speculated that muscle dysfunction and other key morbidities associated with sleep apnoea are the result of CIH‐induced oxidative stress. We sought to investigate the putative role of CIH‐induced reactive oxygen species in the development of respiratory muscle dysfunction. What is the main finding and its importance? The CIH‐induced diaphragm muscle fatigue is time and intensity dependent and is associated with a modest oxidative stress. Supplementation with N‐acetyl cysteine prevents CIH‐induced diaphragm muscle dysfunction, suggesting that antioxidant supplementation may have therapeutic value in respiratory muscle disorders characterized by CIH, such as obstructive sleep apnoea.

Chronic intermittent asphyxia increases platelet reactivity in rats

Sleep‐disordered breathing is associated with chronic intermittent asphyxia and with a variety of cardiovascular abnormalities. Cardiovascular morbidity and mortality are linked to altered platelet function, and platelet function is affected in sleep‐disordered breathing. As there is evidence that chronic continuous hypoxia may alter platelet number and function, the aim of the present study was to test the hypothesis that chronic intermittent asphyxia affects platelet count, activation and aggregation. Rats were treated with a hypercapnic hypoxic gas mixture (minimum of 6–8% O2, maximum of 10–14% CO2) for 15 s, twice per minute for 8 h per day for 3 weeks. Blood was analysed for platelet count, platelet activation (CD62p expression using flow cytometry), response to low dose ADP, haematocrit, red cell count and haemoglobin concentration. A platelet function analyser measured the closure time of an aperture, dependent on platelet aggregation. Compared to controls (n= 16), chronic intermittent asphyxia (n= 13) reduced body weight and increased right ventricular weight but had no significant effect on platelet count (control, 880.4 ± 20.1; treated: 914.1 ± 35.2 × 103μl−1; mean ±s.e.m.), on the reduction in platelet count in response to ADP (control, reduced to 206.7 ± 49.0; treated, reduced to 193.8 ± 35.9 × 103μl−1), or on the percentage of platelets positive for CD62p (control, 5.2 ± 0.7; treated, 6.0 ± 0.8%). Chronic intermittent asphyxia significantly (P= 0.037) reduced the closure time (control, 90.9 ± 7.7; treated, 77.7 ± 3.8 s), indicating greater adhesion and aggregation. There was no significant difference in haematocrit, red cell count and haemoglobin concentration. In conclusion, chronic intermittent asphyxia has no effect on platelet count but does increase platelet aggegation in rats. These data support the idea that chronic intermittent asphyxia alters platelet function in sleep‐disordered breathing.