Aníbal J. Llanos

Hypoxia-related lipid peroxidation: evidences, implications and approaches.

Hypoxia may be intensified by concurrent oxidative stress. Lack of oxygen in relation to aerobic ATP requirements, as hypoxia has been defined, goes along with an increased generation of reactive oxygen species (ROS). Polyunsaturated fatty acids (PUFAs) range among the molecules most susceptible to ROS. Oxidative breakdown of n-3 PUFAs may compromise not only membrane lipid matrix dynamics, and hence structure and function of membrane-associated proteins like enzymes, receptors, and transporters, but also gene expression. Eicosapentaenoic acid depletion, products of lipid peroxidation (LP), as well as, lack of oxygen may combine in exacerbating activity of nuclear factor kappa B (NFkappaB), an ubiquitous pro-inflammatory and anti-apoptotic transcription factor. Field studies at high altitude show malondialdehyde (MDA) content in exhaled breath condensate (EBC) of mountaineers to correlate with Lake Louis score of acute mountain sickness. A pathogenic role of LP in hypoxia can therefore be expected. By control of LP, some species seem to cope more efficiently than others with naturally occurring hypoxia. Limitation of potential pro-inflammatory effects of hypoxia-related LP by an adequate provision of n-3 PUFAs and antioxidants may contribute to increase survival under conditions where oxygen is lacking in relation to aerobic ATP requirements. A need for antioxidant intervention, however, should be weighed against the ROS requirement for triggering adaptive processes in response to an increased demand of oxygen.

Adrenergic and vasopressinergic contributions to the cardiovascular response to acute hypoxaemia in the llama fetus

1 The effects of fetal intravenous treatment with phentolamine or a vasopressinergic V1‐receptor antagonist on the fetal cardiovascular responses to acute hypoxaemia in the llama were investigated. 2 Six llama fetuses were surgically prepared between 60 and 70% of gestation under general halothane anaesthesia with vascular catheters and transit‐time ultrasonic flow probes around a carotid artery and a femoral artery. At least 4 days after surgery all fetuses were subjected to a 3 h experiment: 1 h of normoxia, 1 h of hypoxaemia and 1 h of recovery while on slow i.v. infusion with saline. On separate days this experiment was repeated with fetal i.v. treatment with either phentolamine or a V1‐receptor antagonist dissolved in saline. 3 During saline infusion all llama fetuses responded to acute hypoxaemia with intense femoral vasoconstriction. Phentolamine during normoxia produced hypotension, tachycardia and vasodilatation in both the carotid and the femoral circulations. During hypoxaemia, fetuses treated with phentolamine did not elicit the pronounced femoral vasoconstriction and all died within 20 min of the onset of hypoxaemia. A V1‐receptor antagonist produced a femoral vasodilatation during normoxia but did not affect the fetal cardiovascular responses to acute hypoxaemia. 4 In conclusion, α‐adrenergic and V1‐vasopressinergic mechanisms contribute to a basal vasoconstrictor tone in the femoral circulation in the llama fetus. The enhanced femoral vasoconstriction during acute hypoxaemia in the llama fetus is not mediated by stimulation of V1‐vasopressin receptors, but is dependent on α‐adrenergic receptor stimulation. Such α‐adrenergic efferent mechanisms are indispensable to fetal survival during hypoxaemia in the llama since their abolition leads to cardiovascular collapse and death.

The fetal llama versus the fetal sheep: different strategies to withstand hypoxia

The pregnant llama (Lama glama) has walked for millions of years through the thin oxygen trail of the Andean altiplano. We hypothesize that a pool of genes has been selected in the llama that express efficient mechanisms to withstand this low-oxygen milieu. The llama fetus responds to acute hypoxia with an intense peripheral vasoconstriction that is not affected by bilateral section of the carotid sinus nerves. Moreover, the increase in fetal plasma concentrations of vasoconstrictor hormones, such as catecholamines, neuropeptide Y, and vasopressin, is much greater in the llama than in the sheep fetus. Furthermore, treatment of fetal llamas with an alpha-adrenergic antagonist abolished the peripheral vasoconstriction and resulted in fetal cardiovascular collapse and death during acute hypoxia, suggesting an indispensable upregulation of alpha-adrenergic mechanisms in this high altitude species. Local endothelial factors such as nitric oxide (NO) also play a key role in the regulation of fetal adrenal blood flow and in the adrenal secretion of catecholamines and cortisol. Interestingly, in contrast to the human or sheep fetus, the llama fetus showed a small increase in brain blood flow during acute hypoxia, with no increase in oxygen extraction across the brain, and thereby a decrease in brain oxygen consumption. These results suggest that the llama fetus responds to acute hypoxia with hypometabolism. How this reduction in metabolism is produced and how the cells are preserved during this condition remain to be elucidated.

Long-term exposure to high-altitude chronic hypoxia during gestation induces neonatal pulmonary hypertension at sea level

We determined whether postnatal pulmonary hypertension induced by 70% of pregnancy at high altitude (HA) persists once the offspring return to sea level and investigated pulmonary vascular mechanisms operating under these circumstances. Pregnant ewes were divided into two groups: conception, pregnancy, and delivery at low altitude (580 m, LLL) and conception at low altitude, pregnancy at HA (3,600 m) from 30% of gestation until delivery, and return to lowland (LHL). Pulmonary arterial pressure (PAP) was measured in vivo. Vascular reactivity and morphometry were assessed in small pulmonary arteries (SPA). Protein expression of vascular mediators was determined. LHL lambs had higher basal PAP and a greater increment in PAP after NG-nitro-l-arginine methyl ester (20.9 ± 1.1 vs. 13.7 ± 0.5 mmHg; 39.9 ± 5.0 vs. 18.3 ± 1.3 mmHg, respectively). SPA from LHL had a greater maximal contraction to K+ (1.34 ± 0.05 vs. 1.16 ± 0.05 N/m), higher sensitivity to endothelin-1 and nitroprusside, and persistence of dilatation following blockade of soluble guanylate cyclase. The heart ratio of the right ventricle-to-left ventricle plus septum was higher in the LHL relative to LLL. The muscle area of SPA (29.3 ± 2.9 vs. 21.1 ± 1.7%) and the protein expression of endothelial nitric oxide synthase (1.7 ± 0.1 vs. 1.1 ± 0.2), phosphodiesterase (1.4 ± 0.1 vs. 0.7 ± 0.1), and Ca2+-activated K+ channel (0.76 ± 0.16 vs. 0.30 ± 0.01) were greater in LHL compared with LLL lambs. In contrast, LHL had decreased heme oxygenase-1 expression (0.82 ± 0.26 vs. 2.22 ± 0.44) and carbon monoxide production (all P < 0.05). Postnatal pulmonary hypertension induced by 70% of pregnancy at HA promotes cardiopulmonary remodeling that persists at sea level.

Evidence of a role for melatonin in fetal sheep physiology: direct actions of melatonin on fetal cerebral artery, brown adipose tissue and adrenal gland

Although the fetal pineal gland does not secrete melatonin, the fetus is exposed to melatonin of maternal origin. In the non‐human primate fetus, melatonin acts as a trophic hormone for the adrenal gland, stimulating growth while restraining cortisol production. This latter physiological activity led us to hypothesize that melatonin may influence some fetal functions critical for neonatal adaptation to extrauterine life. To test this hypothesis we explored (i) the presence of G‐protein‐coupled melatonin binding sites and (ii) the direct modulatory effects of melatonin on noradrenaline (norepinephrine)‐induced middle cerebral artery (MCA) contraction, brown adipose tissue (BAT) lypolysis and ACTH‐induced adrenal cortisol production in fetal sheep. We found that melatonin directly inhibits the response to noradrenaline in the MCA and BAT, and also inhibits the response to ACTH in the adrenal gland. Melatonin inhibition was reversed by the melatonin antagonist luzindole only in the fetal adrenal. MCA, BAT and adrenal tissue displayed specific high‐affinity melatonin binding sites coupled to G‐protein (Kd values: MCA 64 ± 1 pm, BAT 98.44 ± 2.12 pm and adrenal 4.123 ± 3.22 pm). Melatonin binding was displaced by luzindole only in the adrenal gland, supporting the idea that action in the MCA and BAT is mediated by different melatonin receptors. These direct inhibitory responses to melatonin support a role for melatonin in fetal physiology, which we propose prevents major contraction of cerebral vessels, restrains cortisol release and restricts BAT lypolysis during fetal life.

Fetal and maternal blood oxygen affinity: A comparative study in llamas and sheep☆

We compared blood oxygen affinity (P50) and hemoglobin concentration among fetal and maternal llamas and sheep, as respective examples of species native to high and low altitudes. P50, hemoglobin concentration and blood oxygen content were determined at sea level in 16 pregnant llamas, 6 pregnant sheep and their respective fetuses. P50 was similar in fetal llamas and sheep, but maternal llamas had higher blood oxygen affinity than maternal sheep. As a consequence, the P50 difference between mother and fetus was less in llamas than in sheep. Fetal llamas had higher hemoglobin concentrations than fetal sheep. In contrast, the maternal hemoglobin concentrations were similar. The blood oxygen content was higher in fetal and maternal llamas than in fetal and maternal sheep. We conclude that the llama, a species native to the altiplano, has a higher blood oxygen content than the sheep, as determined in the fetus by a high hemoglobin concentration and in the mother by a low P50.

The placental pursuit for an adequate oxidant balance between the mother and the fetus

The placenta is the exchange organ that regulates metabolic processes between the mother and her developing fetus. The adequate function of this organ is clearly vital for a physiologic gestational process and a healthy baby as final outcome. The umbilico-placental vasculature has the capacity to respond to variations in the materno-fetal milieu. Depending on the intensity and the extensity of the insult, these responses may be immediate-, mediate-, and long-lasting, deriving in potential morphostructural and functional changes later in life. These adjustments usually compensate the initial insults, but occasionally may switch to long-lasting remodeling and dysfunctional processes, arising maladaptation. One of the most challenging conditions in modern perinatology is hypoxia and oxidative stress during development, both disorders occurring in high-altitude and in low-altitude placental insufficiency. Hypoxia and oxidative stress may induce endothelial dysfunction and thus, reduction in the perfusion of the placenta and restriction in the fetal growth and development. This Review will focus on placental responses to hypoxic conditions, usually related with high-altitude and placental insufficiency, deriving in oxidative stress and vascular disorders, altering fetal and maternal health. Although day-to-day clinical practice, basic and clinical research are clearly providing evidence of the severe impact of oxygen deficiency and oxidative stress establishment during pregnancy, further research on umbilical and placental vascular function under these conditions is badly needed to clarify the myriad of questions still unsettled.

Melatonin reduces oxidative stress and improves vascular function in pulmonary hypertensive newborn sheep

Pulmonary hypertension of the newborn (PHN) constitutes a critical condition with severe cardiovascular and neurological consequences. One of its main causes is hypoxia during gestation, and thus, it is a public health concern in populations living above 2500 m. Although some mechanisms are recognized, the pathophysiological facts that lead to PHN are not fully understood, which explains the lack of an effective treatment. Oxidative stress is one of the proposed mechanisms inducing pulmonary vascular dysfunction and PHN. Therefore, we assessed whether melatonin, a potent antioxidant, improves pulmonary vascular function. Twelve newborn sheep were gestated, born, and raised at 3600 meters. At 3 days old, lambs were catheterized and daily cardiovascular measurements were recorded. Lambs were divided into two groups, one received daily vehicle as control and another received daily melatonin (1 mg/kg/d), for 8 days. At 11 days old, lung tissue and small pulmonary arteries (SPA) were collected. Melatonin decreased pulmonary pressure and resistance for the first 3 days of treatment. Further, melatonin significantly improved the vasodilator function of SPA, enhancing the endothelial‐ and muscular‐dependent pathways. This was associated with an enhanced nitric oxide‐dependent and nitric oxide independent vasodilator components and with increased nitric oxide bioavailability in lung tissue. Further, melatonin reduced the pulmonary oxidative stress markers and increased enzymatic and nonenzymatic antioxidant capacity. Finally, these effects were associated with an increase of lumen diameter and a mild decrease in the wall of the pulmonary arteries. These outcomes support the use of melatonin as an adjuvant in the treatment for PHN.

Fetal brain hypometabolism during prolonged hypoxaemia in the llama

In this study we looked for additional evidence to support the hypothesis that fetal llama reacts to hypoxaemia with adaptive brain hypometabolism. We determined fetal llama brain temperature, Na+ and K+ channel density and Na+–K+‐ATPase activity. Additionally, we looked to see whether there were signs of cell death in the brain cortex of llama fetuses submitted to prolonged hypoxaemia. Ten fetal llamas were instrumented under general anaesthesia to measure pH, arterial blood gases, mean arterial pressure, heart rate, and brain and core temperatures. Measurements were made 1 h before and every hour during 24 h of hypoxaemia (n= 5), which was imposed by reducing maternal inspired oxygen fraction to reach a fetal arterial partial pressure of oxygen (P  a,O 2 ) of about 12 mmHg. A normoxaemic group was the control (n= 5). After 24 h of hypoxaemia, we determined brain cortex Na+–K+‐ATPase activity, ouabain binding, and the expression of NaV1.1, NaV1.2, NaV1.3, NaV1.6, TREK1, TRAAK and KATP channels. The lack of brain cortex damage was assessed as poly ADP‐ribose polymerase (PARP) proteolysis. We found a mean decrease of 0.56°C in brain cortex temperature during prolonged hypoxaemia, which was accompanied by a 51% decrease in brain cortex Na+–K+‐ATPase activity, and by a 44% decrease in protein content of NaV1.1, a voltage‐gated Na+ channel. These changes occurred in absence of changes in PARP protein degradation, suggesting that the cell death of the brain was not enhanced in the fetal llama during hypoxaemia. Taken together, these results provide further evidence to support the hypothesis that the fetal llama responds to prolonged hypoxaemia with adaptive brain hypometabolism, partly mediated by decreases in Na+–K+‐ATPase activity and expression of NaV channels.

Sildenafil reverses hypoxic pulmonary hypertension in highland and lowland newborn sheep.

Perinatal exposure to chronic hypoxia induces sustained hypertension and structural and functional changes in the pulmonary vascular bed. We hypothesized that highland newborn lambs (HLNB, 3600 m) have a higher pulmonary arterial pressure (PAP) due in part to a higher activity/expression of phosphodiesterase 5 (PDE5). We administered sildenafil, a PDE5 inhibitor, during basal and hypoxic conditions in the pulmonary hypertensive HLNB and compared them to lowland newborn lambs (LLNB, 580 m). Additionally, we compared the vasodilator responses to sildenafil in isolated small pulmonary arteries and the PDE5 mRNA expression and evaluated the vascular remodeling by histomorphometric analysis in these newborn lambs. Under basal conditions, HLNB had a higher PAP and cardiac output compared with LLNB. Sildenafil decreased the PAP during basal conditions and completely prevented the PAP increase during hypoxia in both groups. HLNB showed a greater contractile capacity and a higher maximal dilation to sildenafil. PDE5 mRNA expression did not show significant differences between HLNB and LLNB. The distal pulmonary arteries showed an increased wall thickness in HLNB. Our results showed that HLNB are more sensitive to sildenafil and therefore could be useful for treatment of pulmonary hypertension in high-altitude neonates.