Intervention against hypertension in the next generation programmed by developmental hypoxia.

Evidence derived from human clinical studies and experimental animal models shows a causal relationship between adverse pregnancy and increased cardiovascular disease in the adult offspring. However, translational studies isolating mechanisms to design intervention are lacking. Sheep and humans share similar precocial developmental milestones in cardiovascular anatomy and physiology. We tested the hypothesis in sheep that maternal treatment with antioxidants protects against fetal growth restriction and programmed hypertension in adulthood in gestation complicated by chronic fetal hypoxia, the most common adverse consequence in human pregnancy. Using bespoke isobaric chambers, chronically catheterized sheep carrying singletons underwent normoxia or hypoxia (10% oxygen [O2]) ± vitamin C treatment (maternal 200 mg.kg-1 IV daily) for the last third of gestation. In one cohort, the maternal arterial blood gas status, the value at which 50% of the maternal hemoglobin is saturated with oxygen (P50), nitric oxide (NO) bioavailability, oxidative stress, and antioxidant capacity were determined. In another, naturally delivered offspring were raised under normoxia until early adulthood (9 months). Lambs were chronically instrumented and cardiovascular function tested in vivo. Following euthanasia, femoral arterial segments were isolated and endothelial function determined by wire myography. Hypoxic pregnancy induced fetal growth restriction and fetal oxidative stress. At adulthood, it programmed hypertension by enhancing vasoconstrictor reactivity and impairing NO-independent endothelial function. Maternal vitamin C in hypoxic pregnancy improved transplacental oxygenation and enhanced fetal antioxidant capacity while increasing NO bioavailability, offsetting constrictor hyper-reactivity and replenishing endothelial function in the adult offspring. These discoveries provide novel insight into mechanisms and interventions against fetal growth restriction and adult-onset programmed hypertension in an animal model of complicated pregnancy in a species of similar temporal developmental milestones to humans.

Molecular regulation of lung maturation in near-term fetal sheep by maternal daily vitamin C treatment in late gestation.

BACKGROUND: In the fetus, the appropriate balance of prooxidants and antioxidants is essential to negate the detrimental effects of oxidative stress on lung maturation. Antioxidants improve respiratory function in postnatal life and adulthood. However, the outcomes and biological mechanisms of antioxidant action in the fetal lung are unknown. METHODS: We investigated the effect of maternal daily vitamin C treatment (200 mg/kg, intravenously) for a month in late gestation (105-138 days gestation, term ~145 days) on molecular regulation of fetal lung maturation in sheep. Expression of genes and proteins regulating lung development was quantified in fetal lung tissue. The number of surfactant-producing cells was determined by immunohistochemistry. RESULTS: Maternal vitamin C treatment increased fetal lung gene expression of the antioxidant enzyme SOD-1, hypoxia signaling genes (HIF-2α, HIF-3α, ADM, and EGLN-3), genes regulating sodium movement (SCNN1-A, SCNN1-B, ATP1-A1, and ATP1-B1), surfactant maturation (SFTP-B and ABCA3), and airway remodeling (ELN). There was no effect of maternal vitamin C treatment on the expression of protein markers evaluated or on the number of surfactant protein-producing cells in fetal lung tissue. CONCLUSIONS: Maternal vitamin C treatment in the last third of pregnancy in sheep acts at the molecular level to increase the expression of genes that are important for fetal lung maturation in a healthy pregnancy. IMPACT: Maternal daily vitamin C treatment for a month in late gestation in sheep increases the expression of gene-regulating pathways that are essential for normal fetal lung development. Following late gestation vitamin C exposure in a healthy pregnancy, an increase in lung gene but not protein expression may act as a mechanism to aid in the preparation for exposure to the air-breathing environment after birth. In the future, the availability/development of compounds with greater antioxidant properties than vitamin C or more specific targets at the site of oxidative stress in vivo may translate clinically to improve respiratory outcomes in complicated pregnancies at birth.

Maternal melatonin: Effective intervention against developmental programming of cardiovascular dysfunction in adult offspring of complicated pregnancy.

Adopting an integrative approach, by combining studies of cardiovascular function with those at cellular and molecular levels, this study investigated whether maternal treatment with melatonin protects against programmed cardiovascular dysfunction in the offspring using an established rodent model of hypoxic pregnancy. Wistar rats were divided into normoxic (N) or hypoxic (H, 10% O2 ) pregnancy ± melatonin (M) treatment (5 µg·ml-1 .day-1 ) in the maternal drinking water. Hypoxia ± melatonin treatment was from day 15-20 of gestation (term is ca. 22 days). To control for possible effects of maternal hypoxia-induced reductions in maternal food intake, additional dams underwent pregnancy under normoxic conditions but were pair-fed (PF) to the daily amount consumed by hypoxic dams from day 15 of gestation. In one cohort of animals from each experimental group (N, NM, H, HM, PF, PFM), measurements were made at the end of gestation. In another, following delivery of the offspring, investigations were made at adulthood. In both fetal and adult offspring, fixed aorta and hearts were studied stereologically and frozen hearts were processed for molecular studies. In adult offspring, mesenteric vessels were isolated and vascular reactivity determined by in-vitro wire myography. Melatonin treatment during normoxic, hypoxic or pair-fed pregnancy elevated circulating plasma melatonin in the pregnant dam and fetus. Relative to normoxic pregnancy, hypoxic pregnancy increased fetal haematocrit, promoted asymmetric fetal growth restriction and resulted in accelerated postnatal catch-up growth. Whilst fetal offspring of hypoxic pregnancy showed aortic wall thickening, adult offspring of hypoxic pregnancy showed dilated cardiomyopathy. Similarly, whilst cardiac protein expression of eNOS was downregulated in the fetal heart, eNOS protein expression was elevated in the heart of adult offspring of hypoxic pregnancy. Adult offspring of hypoxic pregnancy further showed enhanced mesenteric vasoconstrictor reactivity to phenylephrine and the thromboxane mimetic U46619. The effects of hypoxic pregnancy on cardiovascular remodelling and function in the fetal and adult offspring were independent of hypoxia-induced reductions in maternal food intake. Conversely, the effects of hypoxic pregnancy on fetal and postanal growth were similar in pair-fed pregnancies. Whilst maternal treatment of normoxic or pair-fed pregnancies with melatonin on the offspring cardiovascular system was unremarkable, treatment of hypoxic pregnancies with melatonin in doses lower than those recommended for overcoming jet lag in humans enhanced fetal cardiac eNOS expression and prevented all alterations in cardiovascular structure and function in fetal and adult offspring. Therefore, the data support that melatonin is a potential therapeutic target for clinical intervention against developmental origins of cardiovascular dysfunction in pregnancy complicated by chronic fetal hypoxia.

Blood-brain barrier dysfunction in hemorrhagic transformation: a therapeutic opportunity for nanoparticles and melatonin.

Stroke is the second leading cause of death worldwide, estimated that one-sixth of the world population will suffer it once in their life. The most common type of this medical condition is the ischemic stroke (IS), produced by a thrombotic or embolic occlusion of a major cerebral artery or its branches, leading to the formation of a complex infarct region caused by oxidative stress, excitotoxicity, and endothelial dysfunction. Nowadays, the immediate treatment for IS involves thrombolytic agents or mechanical thrombectomy, depending on the integrity of the blood-brain barrier (BBB). A common stroke complication is the hemorrhagic transformation (HT), which consists of bleeding into the ischemic brain area. Currently, better treatments for IS are urgently needed. As such, the neurohormone melatonin has been proposed as a good candidate due to its antioxidant, anti-inflammatory, and neuroprotective effects, particularly against lipid peroxidation and oxidative stress during brain ischemia. Here, we proposed to develop intravenous or intranasal melatonin nanoformulation to specifically target the brain in patients with stroke. Nowadays, the challenge is to find a formulation able to cross the barriers and reach the target organ in an effective dose to generate the pharmacological effect. In this review, we discuss the current literature about stroke pathophysiology, melatonin properties, and its potential use in nanoformulations as a novel therapeutic approach for ischemic stroke.

Melatonin long-lasting beneficial effects on pulmonary vascular reactivity and redox balance in chronic hypoxic ovine neonates.

Pulmonary arterial hypertension of the neonate (PAHN) is a pathophysiological condition characterized by maladaptive pulmonary vascular remodeling and abnormal contractile reactivity. This is a multifactorial syndrome with chronic hypoxia and oxidative stress as main etiological drivers, and with limited effectiveness in therapeutic approaches. Melatonin is a neurohormone with antioxidant and vasodilator properties at the pulmonary level. Therefore, this study aims to test whether a postnatal treatment with melatonin during the neonatal period improves in a long-lasting manner the clinical condition of PAHN. Ten newborn lambs gestated and born at 3600 m were used in this study, five received vehicle and five received melatonin in daily doses of 1 mg kg-1 for the first 3 weeks of life. After 1 week of treatment completion, lung tissue and small pulmonary arteries (SPA) were collected for wire myography, molecular biology, and morphostructural analyses. Melatonin decreased pulmonary arterial pressure the first 4 days of treatment. At 1 month old, melatonin decreased the contractile response to the vasoconstrictors K+ , TX2 , and ET-1. Further, melatonin increased the endothelium-dependent and muscle-dependent vasodilation of SPA. Finally, the treatment decreased pulmonary oxidative stress by inducing antioxidant enzymes and diminishing pro-oxidant sources. In conclusion, melatonin improved vascular reactivity and oxidative stress at the pulmonary level in PAHN lambs gestated and born in chronic hypoxia.

The role of nitric oxide signaling in pulmonary circulation of high- and low-altitude newborn sheep under basal and acute hypoxic conditions.

Nitric oxide (NO) is the main vasodilator agent that drives the rapid decrease of pulmonary vascular resistance for the respiratory onset during the fetal to neonatal transition. Nevertheless, the enhanced NO generation by the neonatal pulmonary arterial endothelium does not prevent development of hypoxic pulmonary hypertension in species without an evolutionary story at high altitude. Therefore, this study aims to describe the limits of the NO function at high-altitude during neonatal life in the sheep as an animal model without tolerance to perinatal hypoxia. We studied the effect of blockade of NO synthesis with l-NAME in the cardiopulmonary response of lowland (580 m) and highland (3600 m) newborn lambs basally and under an episode of acute hypoxia. We also determined the pulmonary expression of proteins that mediate the actions of the NO vasodilator pathway in the pulmonary vasoactive tone and remodeling. We observed an enhanced nitrergic function in highland lambs under basal conditions, evidenced as a markedly greater increase in basal mean pulmonary arterial pressure (mPAP) and resistance (PVR) under blockade of NO synthesis. Further, acute hypoxic challenge in lowland lambs infused with l-NAME markedly increased their mPAP and PVR to values greater than baseline, whilst in highland animals under NO synthesis blockade, these variables did not show additional increase in response to low PO2. Highland animals showed increased pulmonary RhoA expression, decreased PSer188-RhoA fraction, increased PSer311-p65-NFÒ›ß fraction and up-regulated smooth muscle α-actin, relative to lowland controls. Taken together our data suggest that NO-mediated vasodilation is important to keep a low pulmonary vascular resistance under basal conditions and acute hypoxia at low-altitude. At high-altitude, the enhanced nitrergic signaling partially prevents excessive pulmonary hypertension but does not protect against acute hypoxia. The decreased vasodilator efficacy of nitrergic tone in high altitude lambs could be in part due to increased RhoA signaling that opposes to NO action in the hypoxic pulmonary circulation.

Premature Vascular Aging in Guinea Pigs Affected by Fetal Growth Restriction.

Cardiovascular risk associated with fetal growth restriction (FGR) could result from an early impaired vascular function. However, whether this effect results in premature vascular aging has not been addressed. We studied the ex vivo reactivity of carotid and femoral arteries in fetal (near term), adults (eight months-old) and aged (16 months-old) guinea pigs in normal (control) and FGR offspring. Additionally, an epigenetic marker of vascular aging (i.e., LINE-1 DNA methylation) was evaluated in human umbilical artery endothelial cells (HUAEC) from control and FGR subjects. Control guinea pig arteries showed an increased contractile response (KCl-induced) and a progressive impairment of NO-mediated relaxing responses as animals get older. FGR was associated with an initial preserved carotid artery reactivity as well as a later significant impairment in NO-mediated responses. Femoral arteries from FGR fetuses showed an increased contractility but a decreased relaxing response compared with control fetuses, and both responses were impaired in FGR-adults. Finally, FGR-HUAEC showed decreased LINE-1 DNA methylation compared with control-HUAEC. These data suggest that the aging of vascular function occurs by changes in NO-mediated responses, with limited alterations in contractile capacity. Further, these effects are accelerated and imposed at early stages of development in subjects exposed to a suboptimal intrauterine environment.

[Melatonin modulates the expression of pulmonary prostanoids]. / El tratamiento postnatal con melatonina modula la expresión de agentes prostanoides en pulmón de neonatos de oveja con hipertensión pulmonar.

BACKGROUND: Living above 2,500 meters in hypobaric conditions induces pulmonary arterial hypertension of the neonate (PAHN), a syndrome whose main features are: pathological remodeling of the pulmonary vessels, abnormal vascular reactivity and increased oxidative stress. Melatonin could have pulmonary antioxidant, anti-remodeling and vasodilating properties for this condition. AIM: To determine the effect of melatonin at the transcript level of prostanoid pathways in the lung of neonatal lambs gestated and born under hypobaric hypoxia. MATERIAL AND METHODS: Vehicle (1.4% of ethanol, n = 6) or melatonin (1 mg * kg1, n = 5) were administered from the postnatal day 4 to 21 to lambs gestated and born at 3,600 meters above sea level. After one week of treatment completion, lung tissue was obtained, the transcript and protein levels of prostanoid synthases and receptors were assessed by RT-PCR and Western Blot. RESULTS: Melatonin induced the expression of prostacyclin synthase transcript and increased protein expression of the prostacyclin receptor. In addition, the treatment decreased the expression of transcript and protein of cyclooxygenase-2, without changes in the expression of the prostanoid vasoconstrictor (thromboxane) pathway. CONCLUSIONS: Postnatal treatment with melatonin increases the expression of the prostacyclin-vasodilator pathway without changing the vasoconstrictor thromboxane pathway. Further, the decreased COX-2 induced by melatonin could be an index of lesser oxidative stress and inflammation in the lung.

Digestive physiology of captive capybara (Hydrochoerus hydrochaeris).

The capybara (Hydrochoerus hydrochaeris), the largest living rodent, probably has a "mucus-trap" colonic separation mechanism. To test this hypothesis, we measured the mean retention time of a solute marker (MRTSolute ), 2 mm (MRT2 mm ), 10 mm (MRT10 mm ), and 20 mm (MRT20 mm ) particle markers and nutrient digestibility in adult captive capybaras (27-52 kg body mass (BM), 2-11 yr). In addition, total gut fill and the selectivity factor (MRTSolute /MRT2 mm ) were calculated, and mean faecal particle size and metabolic fecal nitrogen of captive capybaras were compared to those of free-ranging specimens. Finally, we also measured methane production in one animal. The MRT2 mm (29.2 ± 8.2 hr) was different (p < 0.01) from MRTSolute (37.0 ± 13.1 hr), MRT10 mm (36.5 ± 8.2 hr), and MRT20 mm (35.1 ± 9.6 hr). The selectivity factor (1.26 ± 0.30) was in the range considered typical for a "mucus-trap" colonic separation mechanism. The estimated total gut fill was 1.50 ± 0.37% and 1.73 ± 0.25% of BM calculated from the results of the 2-mm and 10-mm particle markers, respectively. The CH4 emission was 13.7 L/day. Captive capybaras had greater mean fecal particle size (0.44 ± 0.06 vs. 0.29 ± 0.05 mm, p < 0.001) and metabolic fecal nitrogen (65.5 ± 3.91 vs. 46.8 ± 10.5% of fecal nitrogen, p < 0.001) than free-ranging capybaras. Organic matter digestibility decreased less steeply with increasing dietary crude fiber content in capybaras as compared to published data from rabbits or guinea pigs. Accordingly, the digestive physiology of the capybara is characterized by a comparatively high fiber digestibility, with a "mucus-trap" colonic separation mechanism, allowing capybaras to thrive on forage-only diets.

The role of nitric oxide in the cardiopulmonary response to hypoxia in highland and lowland newborn llamas.

KEY POINTS: Perinatal hypoxia causes pulmonary hypertension in neonates, including humans. However, in species adapted to hypoxia, such as the llama, there is protection against pulmonary hypertension. Nitric oxide (NO) is a vasodilatator with an established role in the cardiopulmonary system of many species, but its function in the hypoxic pulmonary vasoconstrictor response in the newborn llama is unknown. Therefore, we studied the role of NO in the cardiopulmonary responses to acute hypoxia in high- and lowland newborn llamas. We show that high- compared to lowland newborn llamas have a reduced pulmonary vasoconstrictor response to acute hypoxia. Protection against excessive pulmonary vasoconstriction in the highland llama is mediated via enhancement of NO pathways, including increased MYPT1 and reduced ROCK expression as well as Ca2+ desensitization. Blunting of pulmonary hypertensive responses to hypoxia through enhanced NO pathways may be an adaptive mechanism to withstand life at high altitude in the newborn llama. ABSTRACT: Llamas are born in the Alto Andino with protection against pulmonary hypertension. The physiology underlying protection against pulmonary vasoconstrictor responses to acute hypoxia in highland species is unknown. We determined the role of nitric oxide (NO) in the cardiopulmonary responses to acute hypoxia in high- and lowland newborn llamas. The cardiopulmonary function of newborn llamas born at low (580 m) or high altitude (3600 m) was studied under acute hypoxia, with and without NO blockade. In pulmonary arteries, we measured the reactivity to potassium and sodium nitroprusside (SNP), and in lung we determined the content of cGMP and the expression of the NO-related proteins: BKCa, PDE5, PSer92-PDE5, PKG-1, ROCK1 and 2, MYPT1, PSer695-MYPT1, PThr696-MYPT1, MLC20 and PSer19-MLC20. Pulmonary vascular remodelling was evaluated by morphometry and based on α-actin expression. High- compared to lowland newborn llamas showed lower in vivo pulmonary arterial pressor responses to acute hypoxia. This protection involved enhanced NO function, as NO blockade reverted the effect and the pulmonary arterial dilatator response to SNP was significantly enhanced in highland neonates. The pulmonary expression of ROCK2 and the phosphorylation of MLC20 were lower in high-altitude llamas. Conversely, MYPT1 was up-regulated whilst PSer695-MYPT1 and PThr695-MYPT1 did not change. Enhanced NO-dependent mechanisms were insufficient to prevent pulmonary arterial remodelling. Combined, the data strongly support that in the highland newborn llama reduced ROCK, increased MYPT1 expression and Ca2+ desensitization in pulmonary tissue allow an enhanced NO biology to limit hypoxic pulmonary constrictor responses. Blunting of hypoxic pulmonary hypertensive responses may be an adaptive mechanism to life at high altitude.

Guinea pig models for translation of the developmental origins of health and disease hypothesis into the clinic.

Over 30 years ago Professor David Barker first proposed the theory that events in early life could explain an individual's risk of non-communicable disease in later life: the developmental origins of health and disease (DOHaD) hypothesis. During the 1990s the validity of the DOHaD hypothesis was extensively tested in a number of human populations and the mechanisms underpinning it characterised in a range of experimental animal models. Over the past decade, researchers have sought to use this mechanistic understanding of DOHaD to develop therapeutic interventions during pregnancy and early life to improve adult health. A variety of animal models have been used to develop and evaluate interventions, each with strengths and limitations. It is becoming apparent that effective translational research requires that the animal paradigm selected mirrors the tempo of human fetal growth and development as closely as possible so that the effect of a perinatal insult and/or therapeutic intervention can be fully assessed. The guinea pig is one such animal model that over the past two decades has demonstrated itself to be a very useful platform for these important reproductive studies. This review highlights similarities in the in utero development between humans and guinea pigs, the strengths and limitations of the guinea pig as an experimental model of DOHaD and the guinea pig's potential to enhance clinical therapeutic innovation to improve human health.

Improving pregnancy outcomes in humans through studies in sheep.

Experimental studies that are relevant to human pregnancy rely on the selection of appropriate animal models as an important element in experimental design. Consideration of the strengths and weaknesses of any animal model of human disease is fundamental to effective and meaningful translation of preclinical research. Studies in sheep have made significant contributions to our understanding of the normal and abnormal development of the fetus. As a model of human pregnancy, studies in sheep have enabled scientists and clinicians to answer questions about the etiology and treatment of poor maternal, placental, and fetal health and to provide an evidence base for translation of interventions to the clinic. The aim of this review is to highlight the advances in perinatal human medicine that have been achieved following translation of research using the pregnant sheep and fetus.

Mechanisms of Cardiovascular Protection Associated with Intermittent Hypobaric Hypoxia Exposure in a Rat Model: Role of Oxidative Stress.

More than 140 million people live and works (in a chronic or intermittent form) above 2500 m worldwide and 35 million live in the Andean Mountains. Furthermore, in Chile, it is estimated that 55,000 persons work in high altitude shifts, where stays at lowlands and interspersed with working stays at highlands. Acute exposure to high altitude has been shown to induce oxidative stress in healthy human lowlanders, due to an increase in free radical formation and a decrease in antioxidant capacity. However, in animal models, intermittent hypoxia (IH) induce preconditioning, like responses and cardioprotection. Here, we aimed to describe in a rat model the responses on cardiac and vascular function to 4 cycles of intermittent hypobaric hypoxia (IHH). Twelve adult Wistar rats were randomly divided into two equal groups, a four-cycle of IHH, and a normobaric hypoxic control. Intermittent hypoxia was induced in a hypobaric chamber in four continuous cycles (1 cycle = 4 days hypoxia + 4 days normoxia), reaching a barometric pressure equivalent to 4600 m of altitude (428 Torr). At the end of the first and fourth cycle, cardiac structural, and functional variables were determined by echocardiography. Thereafter, ex vivo vascular function and biomechanical properties were determined in femoral arteries by wire myography. We further measured cardiac oxidative stress biomarkers (4-Hydroxy-nonenal, HNE; nytrotirosine, NT), reactive oxygen species (ROS) sources (NADPH and mitochondrial), and antioxidant enzymes activity (catalase, CAT; glutathione peroxidase, GPx, and superoxide dismutase, SOD). Our results show a higher ejection and shortening fraction of the left ventricle function by the end of the 4th cycle. Further, femoral vessels showed an improvement of vasodilator capacity and diminished stiffening. Cardiac tissue presented a higher expression of antioxidant enzymes and mitochondrial ROS formation in IHH, as compared with normobaric hypoxic controls. IHH exposure determines a preconditioning effect on the heart and femoral artery, both at structural and functional levels, associated with the induction of antioxidant defence mechanisms. However, mitochondrial ROS generation was increased in cardiac tissue. These findings suggest that initial states of IHH are beneficial for cardiovascular function and protection.

N-Acetylcysteine, a glutathione precursor, reverts vascular dysfunction and endothelial epigenetic programming in intrauterine growth restricted guinea pigs.

KEY POINTS: Intrauterine growth restriction (IUGR) is associated with vascular dysfunction, oxidative stress and signs of endothelial epigenetic programming of the umbilical vessels. There is no evidence that this epigenetic programming is occurring on systemic fetal arteries. In IUGR guinea pigs we studied the functional and epigenetic programming of endothelial nitric oxide synthase (eNOS) (Nos3 gene) in umbilical and systemic fetal arteries, addressing the role of oxidative stress in this process by maternal treatment with N-acetylcysteine (NAC) during the second half of gestation. The present study suggests that IUGR endothelial cells have common molecular markers of programming in umbilical and systemic arteries. Notably, maternal treatment with NAC restores fetal growth by increasing placental efficiency and reverting the functional and epigenetic programming of eNOS in arterial endothelium in IUGR guinea pigs. ABSTRACT: In humans, intrauterine growth restriction (IUGR) is associated with vascular dysfunction, oxidative stress and signs of endothelial programming in umbilical vessels. We aimed to determine the effects of maternal antioxidant treatment with N-acetylcysteine (NAC) on fetal endothelial function and endothelial nitric oxide synthase (eNOS) programming in IUGR guinea pigs. IUGR was induced by implanting ameroid constrictors on uterine arteries of pregnant guinea pigs at mid gestation, half of the sows receiving NAC in the drinking water (from day 34 until term). Fetal biometry and placental vascular resistance were followed by ultrasound throughout gestation. At term, umbilical arteries and fetal aortae were isolated to assess endothelial function by wire-myography. Primary cultures of endothelial cells (ECs) from fetal aorta, femoral and umbilical arteries were used to determine eNOS mRNA levels by quantitative PCR and analyse DNA methylation in the Nos3 promoter by pyrosequencing. Doppler ultrasound measurements showed that NAC reduced placental vascular resistance in IUGR (P < 0.05) and recovered fetal weight (P < 0.05), increasing fetal-to-placental ratio at term (∼40%) (P < 0.001). In IUGR, NAC treatment restored eNOS-dependent relaxation in aorta and umbilical arteries (P < 0.05), normalizing eNOS mRNA levels in EC fetal and umbilical arteries (P < 0.05). IUGR-derived ECs had a decreased DNA methylation (∼30%) at CpG -170 (from the transcription start site) and this epigenetic signature was absent in NAC-treated fetuses (P < 0.001). These data show that IUGR-ECs have common molecular markers of eNOS programming in umbilical and systemic arteries and this effect is prevented by maternal treatment with antioxidants.

Maternal chronic hypoxia increases expression of genes regulating lung liquid movement and surfactant maturation in male fetuses in late gestation.

KEY POINTS: Chronic fetal hypoxaemia is a common pregnancy complication associated with intrauterine growth restriction that may influence respiratory outcome at birth. We investigated the effect of maternal chronic hypoxia for a month in late gestation on signalling pathways regulating fetal lung maturation and the transition to air-breathing at birth using isobaric hypoxic chambers without alterations to maternal food intake. Maternal chronic hypoxia in late gestation increases fetal lung expression of genes regulating hypoxia signalling, lung liquid reabsorption and surfactant maturation, which may be an adaptive response in preparation for the successful transition to air-breathing at birth. In contrast to other models of chronic fetal hypoxaemia, late gestation onset fetal hypoxaemia promotes molecular regulation of fetal lung maturation. This suggests a differential effect of timing and duration of fetal chronic hypoxaemia on fetal lung maturation, which supports the heterogeneity observed in respiratory outcomes in newborns following exposure to chronic hypoxaemia in utero. ABSTRACT: Chronic fetal hypoxaemia is a common pregnancy complication that may arise from maternal, placental and/or fetal factors. Respiratory outcome of the infant at birth likely depends on the duration, timing and severity of the hypoxaemic insult. We have isolated the effect of maternal chronic hypoxia (MCH) for a month in late gestation on fetal lung development. Pregnant ewes were exposed to normoxia (21% O2 ) or hypoxia (10% O2 ) from 105 to 138 days of gestation (term ∼145 days). At 138 days, gene expression in fetal lung tissue was determined by quantitative RT-PCR. Cortisol concentrations were determined in fetal plasma and lung tissue. Numerical density of surfactant protein positive cells was determined by immunohistochemistry. MCH reduced maternal PaO2 (106 ± 2.9 vs. 47 ± 2.8 mmHg) and fetal body weight (4.0 ± 0.4 vs. 3.2 ± 0.9 kg). MCH increased fetal lung expression of the anti-oxidant marker CAT and decreased expression of the pro-oxidant marker NOX-4. MCH increased expression of genes regulating hypoxia signalling and feedback (HIF-3α, KDM3A, SLC2A1, EGLN-3). There was no effect of MCH on fetal plasma/lung tissue cortisol concentrations, nor genes regulating glucocorticoid signalling (HSD11B-1, HSD11B-2, NR3C1, NR3C2). MCH increased expression of genes regulating sodium (SCNN1-B, ATP1-A1, ATP1-B1) and water (AQP-4) movement in the fetal lung. MCH promoted surfactant maturation (SFTP-B, SFTP-D, ABCA3) at the molecular level, but did not alter the numerical density of surfactant positive cells in lung tissue. MCH in late gestation promotes molecular maturation of the fetal lung, which may be an adaptive response in preparation for the successful transition to air-breathing at birth.

Trypanosoma evansi (Kinetoplastida: Trypanosomatidae) in capybaras (Hydrochoerus hydrochaeris, Rodentia: Hydrochoeridae): prevalence, effect and sexual selection.

Parasites play a crucial role in the ecology of animals. They also appear to be important in mechanisms underlying sexual selection processes. In this article we study the prevalence, effect and potential role in sexual selection of the protozoon Trypanosoma evansi in capybaras, Hydrochoerus hydrochaeris. We collected our samples from the annual capybara cull of a ranch in Venezuela, using the volume of the snout scent gland as an indicator of dominance; the residuals of body weight as indicators of condition; and the residuals of the spleen mass as indicators of immune function. Overall prevalence was 30.9% (N=97) with no difference between males and females and no relation between infection with T. evansi and condition. However, we found that infected animals had larger spleens (residuals), indicating an immunological cost of the infection. Further, males with larger snout scent glands (more dominant) were less likely to be infected than males with smaller glands (less dominant) suggesting that by choosing males with a large gland, females may be using the gland as an indicator of health, which is consistent with the "good genes" view of sexual selection.

Cardiovascular function in term fetal sheep conceived, gestated and studied in the hypobaric hypoxia of the Andean altiplano.

High-altitude hypoxia causes intrauterine growth restriction and cardiovascular programming. However, adult humans and animals that have evolved at altitude show certain protection against the effects of chronic hypoxia. Whether the highland fetus shows similar protection against high altitude gestation is unclear. We tested the hypothesis that high-altitude fetal sheep have evolved cardiovascular compensatory mechanisms to withstand chronic hypoxia that are different from lowland sheep. We studied seven high-altitude (HA; 3600 m) and eight low-altitude (LA; 520 m) pregnant sheep at ∼90% gestation. Pregnant ewes and fetuses were instrumented for cardiovascular investigation. A three-period experimental protocol was performed in vivo: 30 min of basal, 1 h of acute superimposed hypoxia (∼10% O2) and 30 min of recovery. Further, we determined ex vivo fetal cerebral and femoral arterial function. HA pregnancy led to chronic fetal hypoxia, growth restriction and altered cardiovascular function. During acute superimposed hypoxia, LA fetuses redistributed blood flow favouring the brain, heart and adrenals, whereas HA fetuses showed a blunted cardiovascular response. Importantly, HA fetuses have a marked reduction in umbilical blood flow versus LA. Isolated cerebral arteries from HA fetuses showed a higher contractile capacity but a diminished response to catecholamines. In contrast, femoral arteries from HA fetuses showed decreased contractile capacity and increased adrenergic contractility. The blunting of the cardiovascular responses to hypoxia in fetuses raised in the Alto Andino may indicate a change in control strategy triggered by chronic hypoxia, switching towards compensatory mechanisms that are more cost-effective in terms of oxygen uptake.

Assessment of in vivo fetal growth and placental vascular function in a novel intrauterine growth restriction model of progressive uterine artery occlusion in guinea pigs.

Intra-uterine growth restriction (IUGR) is associated with short and long-term metabolic and cardiovascular alterations. Mice and rats have been extensively used to study the effects of IUGR, but there are notable differences in fetal and placental physiology relative to those of humans that argue for alternative animal models. This study proposes that gradual occlusion of uterine arteries from mid-gestation in pregnant guinea pigs produces a novel model to better assess human IUGR. Fetal biometry and in vivo placental vascular function were followed by sonography and Doppler of control pregnant guinea pigs and sows submitted to surgical placement of ameroid constrictors in both uterine arteries (IUGR) at mid-gestation (35 days). The ameroid constrictors induced a reduction in the fetal abdominal circumference growth rate (0.205 cm day(-1) ) compared to control (0.241 cm day(-1) , P < 0.001) without affecting biparietal diameter growth. Umbilical artery pulsatility and resistance indexes at 10 and 20 days after surgery were significantly higher in IUGR animals than controls (P < 0.01). These effects were associated with a decrease in the relative luminal area of placental chorionic arteries (21.3 ± 2.2% vs. 33.2 ± 2.7%, P < 0.01) in IUGR sows at near term. Uterine artery intervention reduced fetal (∼30%), placental (∼20%) and liver (∼50%) weights (P < 0.05), with an increased brain to liver ratio (P < 0.001) relative to the control group. These data demonstrate that the ameroid constrictor implantations in uterine arteries in pregnant guinea pigs lead to placental vascular dysfunction and altered fetal growth that induces asymmetric IUGR.

2-AMINOETHYLDIPHENYLBORINATE MODIFIES THE PULMONARY CIRCULATION IN PULMONARY HYPERTENSIVE NEWBORN LAMBS WITH PARTIAL GESTATION AT HIGH ALTITUDE.

Calcium signaling through store operated channels (SOC) is involved in hypoxic pulmonary hypertension. We determined whether a treatment with 2-aminoethyldiphenylborinate (2-APB), a compound with SOC blocker activity, reduces pulmonary hypertension and vascular remodeling. Twelve newborn lambs exposed to perinatal chronic hypoxia were studied, 6 of them received a 2-APB treatment and the other 6 received vehicle treatment, for 10 days in both cases. Throughout this period, we recorded cardiopulmonary variables and on day 11 we evaluated the response to an acute hypoxic challenge. Additionally, we assessed the vasoconstrictor and vasodilator function in isolated pulmonary arteries as well as their remodeling in lung slices. 2-APB reduced pulmonary arterial pressure at the third and tenth days, cardiac output between the fourth and eighth days, and pulmonary vascular resistance at the tenth day of treatment. The pulmonary vasoconstrictor response to acute hypoxia was reduced by the end of treatment. 2-APB also decreased maximal vasoconstrictor response to the thromboxane mimetic U46619 and endothelin-1 and increased maximal relaxation to 8-Br-cGMP. The maximal relaxation and potency to phosphodiesterase-5 and Rho-kinase inhibition with sildenafil and fasudil respectively, were also increased. Finally, 2-APB reduced the medial and adventitial layers' thickness, the expression of α-actin and the percentage of Ki67+ nuclei of small pulmonary arteries. Taken together, our results indicate that 2-APB reduces pulmonary hypertension, vasoconstrictor responses and pathological remodeling in pulmonary hypertensive lambs. We conclude that SOC targeting may be a useful strategy for the treatment of neonatal pulmonary hypertension, however, further testing of specific blockers is needed.

Role of the RhoA/ROCK pathway in high-altitude associated neonatal pulmonary hypertension in lambs.

Exposure to high-altitude chronic hypoxia during pregnancy may cause pulmonary hypertension in neonates, as a result of vasoconstriction and vascular remodeling. We hypothesized that susceptibility to pulmonary hypertension, due to an augmented expression and activity of the RhoA/Rho-kinase (ROCK) pathway in these neonates, can be reduced by daily administration of fasudil, a ROCK inhibitor. We studied 10 highland newborn lambs with conception, gestation, and birth at 3,600 m in Putre, Chile. Five highland controls (HLC) were compared with 5 highland lambs treated with fasudil (HL-FAS; 3 mg·kg(-1)·day(-1) iv for 10 days). Ten lowland controls were studied in Lluta (50 m; LLC). During the 10 days of fasudil daily administration, the drug decreased pulmonary arterial pressure (PAP) and resistance (PVR), basally and during a superimposed episode of acute hypoxia. HL-FAS small pulmonary arteries showed diminished muscular area and a reduced contractile response to the thromboxane analog U46619 compared with HLC. Hypoxia, but not fasudil, changed the protein expression pattern of the RhoA/ROCKII pathway. Moreover, HL-FAS lungs expressed less pMYPT1(T850) and pMYPT1T(696) than HLC, with a potential increase of the myosin light chain phosphatase activity. Finally, hypoxia induced RhoA, ROCKII, and PKG mRNA expression in PASMCs of HLC, but fasudil reduced them (HL-FAS) similarly to LLC. We conclude that fasudil decreases the function of the RhoA/ROCK pathway, reducing the PAP and PVR in chronically hypoxic highland neonatal lambs. The inhibition of ROCKs by fasudil may offer a possible therapeutic tool for the pulmonary hypertension of the neonates.