Perinatal hypoxia increases susceptibility to high-altitude polycythemia and attendant pulmonary vascular dysfunction.

Perinatal exposures exert a profound influence on physiological function, including developmental processes vital for efficient pulmonary gas transfer throughout the lifespan. We extend the concept of developmental programming to chronic mountain sickness (CMS), a debilitating syndrome marked by polycythemia, ventilatory impairment, and pulmonary hypertension that affects ∼10% of male high-altitude residents. We hypothesized that adverse perinatal oxygenation caused abnormalities of ventilatory and/or pulmonary vascular function that increased susceptibility to CMS in adulthood. Subjects were 67 male high-altitude (3,600-4,100 m) residents aged 18-25 yr with excessive erythrocytosis (EE, Hb concentration ≥18.3 g/dl), a preclinical form of CMS, and 66 controls identified from a community-based survey (n = 981). EE subjects not only had higher Hb concentrations and erythrocyte counts, but also lower alveolar ventilation, impaired pulmonary diffusion capacity, higher systolic pulmonary artery pressure, lower pulmonary artery acceleration time, and more frequent right ventricular hypertrophy, than controls. Compared with controls, EE subjects were more often born to mothers experiencing hypertensive complications of pregnancy and hypoxia during the perinatal period, with each increasing the risk of developing EE (odds ratio = 5.25, P = 0.05 and odds ratio = 6.44, P = 0.04, respectively) after other factors known to influence EE status were taken into account. Adverse perinatal oxygenation is associated with increased susceptibility to EE accompanied by modest abnormalities of the pulmonary circulation that are independent of increased blood viscosity. The association between perinatal hypoxia and EE may be due to disrupted alveolarization and microvascular development, leading to impaired gas exchange and/or pulmonary hypertension.

Divergent physiological responses in laboratory rats and mice raised at high altitude.

Ecological studies show that mice can be found at high altitude (HA - up to 4000 m) while rats are absent at these altitudes, and there are no data to explain this discrepancy. We used adult laboratory rats and mice that have been raised for more than 30 generations in La Paz, Bolivia (3600 m), and compared their hematocrit levels, right ventricular hypertrophy (index of pulmonary hypertension) and alveolar surface area in the lungs. We also used whole-body plethysmography, indirect calorimetry and pulse oxymetry to measure ventilation, metabolic rate (O2 consumption and CO2 production), heart rate and pulse oxymetry oxygen saturation (pO2 ,sat) under ambient conditions, and in response to exposure to sea level PO2 (32% O2=160 mmHg for 10 min) and hypoxia (18% and 15% O2=90 and 75 mmHg for 10 min each). The variables used for comparisons between species were corrected for body mass using standard allometric equations, and are termed mass-corrected variables. Under baseline, compared with rats, adult mice had similar levels of pO2 ,sat, but lower hematocrit and hemoglobin levels, reduced right ventricular hypertrophy and higher mass-corrected alveolar surface area, tidal volume and metabolic rate. In response to sea level PO2 and hypoxia, mice and rats had similar changes of ventilation, but metabolic rate decreased much more in hypoxia in mice, while pO2 ,sat remained higher in mice. We conclude that laboratory mice and rats that have been raised at HA for >30 generations have different physiological responses to altitude. These differences might explain the different altitude distribution observed in wild rats and mice.

Erythropoietin and caffeine exert similar protective impact against neonatal intermittent hypoxia: Apnea of prematurity and sex dimorphism.

Apnea of prematurity (AoP) is associated with severe and repeated episodes of arterial oxygen desaturation (intermittent hypoxia - IH), which in turn increases the number of apneas. So far, there is no data addressing whether IH leads to sex-specific respiratory consequences, neither if drugs targeting AoP are more effective in males or females. We used rat pups for investigating whether IH-mediated increase of apneas is sex-specific. We also tested whether caffeine (treatment of choice of AoP), erythropoietin (Epo - a neuroprotective factor and potent respiratory stimulant), and combination of both (caffeine+Epo) prevent the IH-mediated formation of apneas in a sex-dependent manner. Newborn rats exposed to IH (21% - 10% FIO2-8 h a day - 10 cycles per hour) during postnatal days (P) 3-10 were used in this work. Animals were administered drug vehicle, Epo, caffeine and Epo + caffeine (daily from P3 to P10) gavage. At P10 the frequency of apneas at rest (as an index of respiratory dysfunction induced by IH), and respiratory parameters were measured by plethysmography. Our results showed that IH significantly increases the number of apneas in male but not in female rat pups. Moreover, caffeine and Epo in males similarly prevented the increase of apneas induced by IH, and the administration of both drugs together did not provide a cumulative beneficial effect. No impact of drugs was evidenced in females. Apart from apneas, IH increased the normoxic basal ventilation (ventilation at rest) of male animals, and treatments did not prevent such alteration. Besides, no IH- nor treatment-mediated modulation of basal ventilation was found in the basal ventilation of female animals. Analysis of the activity of pro- and antioxidative molecules revealed that IH induces oxidative stress in the brainstem of male and female animals and that all tested treatments similarly prevented such oxidative imbalance in pups of both sexes. We concluded that neonatal IH and the treatments tested to prevent its respiratory consequences are sex-specific. The mechanics associated with such prevention are directly linked with the prevention of oxidative stress and the maturation of the brain. These findings are relevant to understanding better the AoP disorder and for proposing Epo as a new therapeutical tool.

Divergent Mitochondrial Antioxidant Activities and Lung Alveolar Architecture in the Lungs of Rats and Mice at High Altitude.

Compared with mice, adult rats living at 3,600 m above sea level (SL-La Paz, Bolivia) have high hematocrit, signs of pulmonary hypertension, and low lung volume with reduced alveolar surface area. This phenotype is associated with chronic mountain sickness in humans living at high altitude (HA). We tested the hypothesis that this phenotype is associated with impaired gas exchange and oxidative stress in the lungs. We used rats and mice (3 months old) living at HA (La Paz) and SL (Quebec City, Canada) to measure arterial oxygen saturation under graded levels of hypoxia (by pulse oximetry), the alveolar surface area in lung slices and the activity of pro- (NADPH and xanthine oxidases-NOX and XO) and anti- (superoxide dismutase, and glutathione peroxidase-SOD and GPx) oxidant enzymes in cytosolic and mitochondrial lung protein extracts. HA rats have a lower arterial oxygen saturation and reduced alveolar surface area compared to HA mice and SL rats. Enzymatic activities (NOX, XO, SOD, and GPx) in the cytosol were similar between HA and SL animals, but SOD and GPx activities in the mitochondria were 2-3 times higher in HA vs. SL rats, and only marginally higher in HA mice vs. SL mice. Furthermore, the maximum activity of cytochrome oxidase-c (COX) measured in mitochondrial lung extracts was also 2 times higher in HA rats compared with SL rats, while there was only a small increase in HA mice vs. SL mice. Interestingly, compared with SL controls, alterations in lung morphology are not observed for young rats at HA (15 days after birth), and enzymatic activities are only slightly altered. These results suggest that rats living at HA have a gradual reduction of their alveolar surface area beyond the postnatal period. We can speculate that the elevation of SOD, GPx, and COX activities in the lung mitochondria are not sufficient to compensate for oxidative stress, leading to damage of the lung tissue in rats.

Unique DNA Methylation Patterns in Offspring of Hypertensive Pregnancy.

Epigenomic processes are believed to play a pivotal role for the effect of environmental exposures in early life to modify disease risk throughout the lifespan. Offspring of women with hypertensive complications of pregnancy (HTNPREG ) have an increased risk of developing systemic and pulmonary vascular dysfunction in adulthood. In this preliminary report, we sought to determine whether epigenetic modifications of genes involved in the regulation of vascular function were present in HTNPREG offspring. We contrasted DNA methylation and gene expression patterns of peripheral blood mononuclear cells obtained from young male offspring of HTNPREG (n = 5) to those of normotensive controls (n = 19). In HTNPREG offspring we identified six differentially methylated regions (DMRs) including three genes (SMOC2, ARID1B and CTRHC1) relevant to vascular function. The transcriptional activity of ARID1B and CTRCH1 was inversely related to methylation status. HTNPREG offspring had higher systolic pulmonary artery pressure (sPPA ) versus controls. Our findings demonstrate that epigenetic marks are altered in offspring of HTNPREG with a modest elevation of sPPA and introduce novel epigenomic targets for further study. On the basis of these findings we speculate that epigenomic mechanisms may be involved in mediating the effect of HTNPREG to raise the risk of vascular disease later in life.

Sleep-disordered breathing and oxidative stress in preclinical chronic mountain sickness (excessive erythrocytosis).

Chronic mountain sickness (CMS) is considered to be a loss of ventilatory acclimatization to high altitude (>2500m) resulting in marked arterial hypoxemia and polycythemia. This case-control study explores the possibility that sleep-disordered breathing (SDB) and associated oxidative stress contribute to the etiology of CMS. Nocturnal respiratory and [Formula: see text] patterns were measured using standard polysomnography techniques and compared between male high-altitude residents (aged 18-25) with preclinical CMS (excessive erythrocytosis (EE), n=20) and controls (n=19). Measures of oxidative stress and antioxidant status included isoprostanes (8-iso-PGF2alpha), superoxide dismutase and ascorbic acid. EE cases had a greater apnea-hypopnea index, a higher frequency of apneas (central and obstructive) and hypopneas during REM sleep, and lower nocturnal [Formula: see text] compared to controls. 8-iso-PGF2alpha was greater in EE than controls, negatively associated with nocturnal [Formula: see text] , and positively associated with hemoglobin concentration. Mild sleep-disordered breathing and oxidative stress are evident in preclinical CMS, suggesting that the resolution of nocturnal hypoxemia or antioxidant treatment may prevent disease progression.

Life-long consequences of postnatal normoxia exposure in rats raised at high altitude.

We tested the hypothesis that exposure of high-altitude (HA) rats to a period of postnatal normoxia has long-term consequences on the ventilatory and hematological acclimatization in adults. Male and female HA rats (3,600 m, Po(2) ≃ 100 Torr; La Paz, Bolivia) were exposed to normal room air [HA control (HACont)] or enriched oxygen (32% O(2); Po(2) ≃ 160 Torr) from 1 day before to 15 days after birth [HA postnatal normoxia (HApNorm)]. Hematocrit and hemoglobin values were assessed at 2, 12, and 32 wk of age. Cardiac and lung morphology were assessed at 12 wk by measuring right ventricular hypertrophy (pulmonary hypertension index) and lung air space-to-tissue ratio (indicative of alveolarization). Respiratory parameters under baseline conditions and in response to 32% O(2) for 10 min (relieving the ambient hypoxic stimulus) were measured by whole body plethysmography at 12 wk. Finally, we performed a survival analysis up to 600 days of age. Compared with HACont, HApNorm rats had reduced hematocrit and hemoglobin levels at all ages (both sexes); reduced right ventricular hypertrophy (both sexes); lower air space-to-tissue ratio in the lungs (males only); reduced CO(2) production rate, but higher oxygen uptake (males only); and similar respiratory frequency, tidal volume, and minute ventilation. When breathing 32% O(2), HApNorm male rats had a stronger decrease of minute ventilation than HACont. HApNorm rats had a marked tendency toward longer survival throughout the study. We conclude that exposure to ambient hypoxia during postnatal development in HA rats has deleterious consequences on acclimatization to hypoxia as adults.

Enhanced erythropoietin expression in the brainstem of newborn rats at high altitude.

In addition to its role in elevating red blood cell number, erythropoietin (Epo) exerts protective functions against acute and delayed degenerative diseases of the brain. Moreover, we have recently demonstrated that endogenously synthesized Epo and soluble Epo receptor (a negative regulator of Epo binding to the Epo receptor) in the central nervous system play a crucial role in facilitating the ventilatory response and acclimatization to hypoxia. Here we hypothesized that cerebral Epo in the brainstem is implicated in the process that allows cardiorespiratory acclimatization to high altitude hypoxia during the postnatal period. Thus, we evaluated the postnatal ontogeny of cerebral Epo concentration of Sprague-Dawley rats living and reproducing at high altitude for longer than 19 years (3600 m in La Paz, Bolivia). Our results show that postnatal Epo concentration in high-altitude rats is higher in the brainstem than in the forebrain. Moreover, although Epo concentration in the forebrain of high-altitude rats is similar to sea-level controls, Epo level in the brainstem is surprisingly 2-fold higher in high-altitude rats than in sea-level controls. These findings strongly suggest that brainstem Epo plays an important role in tolerance to high altitude hypoxia after birth. From a clinical perspective, a better understanding of the role of Epo in the postnatal development of cardiorespiratory responses in neonates exposed to acute or chronic hypoxia might be useful.