—Exposure to high-altitude chronic hypoxia during pregnancy may cause pulmonary hypertension in n... more —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. Rho kinase; hypoxia; pulmonary hypertension; newborn; high altitude EXPOSURE to chronic hypoxia causes pulmonary arterial hyper-tension (PAH), characterized by a progressive elevation of pulmonary artery pressure and resistance, associated with va-soconstriction and vascular remodeling (14, 43). Although PAH can develop at any stage during an individual's lifetime, neonates are especially vulnerable to this syndrome due to the marked changes that take place in the pulmonary circulation at this period of life. At birth, there is a rapid transition from a high resistance-low flow pulmonary circulation in the fetus to a very low resistance condition in the newborn capable of accommodating the total cardiac output (1, 2). Exposure to chronic hypoxia in utero, as seen in some complicated pregnancies at low altitude and in high altitude populations, may lead to persistent pulmonary hypertension of the newborn (37). This is a life-threatening syndrome with a prevalence from 0.43 to 6.82 per 1,000 live births in lowlands (52), and some authors suggested that at high altitude this prevalence is higher than in lowlands (37, 40). Despite the available therapeutic strategies, a high percentage of patients are refractory to these treatments and the mortality rate ranges from 4 to 33%, accompanied by significant morbidity (45, 52). The mechanisms of chronic hypoxia inducing PAH are complex and not completely understood, but the involvement of the RhoA/Rho-kinase (ROCK) pathway has been demonstrated in the rat and mouse, contributing to vascular remod-eling and vasoconstriction through Ca 2 sensitization (38, 54). Thus, ROCK-specific pharmacological inhibitors, fasudil and Y-27632, have been shown to inhibit adult and fetal hypoxic pulmonary artery myogenic responses (9, 51), reversing hy-poxic pulmonary vasoconstriction (12). Furthermore, treatment with ROCK inhibitors suppresses the development of hypoxic PAH in adult rodents (12, 35). ROCKs are cytoplasmic serine/threonine kinases translo-cated to the membrane after activation of the small GTPase RhoA (26). The binding of active RhoA to ROCK RBD (RhoA binding domain) stimulates the phosphotransferase activity of ROCKs (4, 30). Once activated in the smooth muscle, ROCK acts on the regulatory subunit of myosin light chain phospha-tase MYPT1 and phosphorylates MYPT1's threonine 850 and 696. This results in a decreased myosin light chain phosphatase (MLCP) activity and increased levels of phosphorylated myo-sin light chain (MLC) at a constant cytosolic Ca 2 concentration , a mechanism known as Ca 2 sensitization (48, 50). There are two ROCK isoforms, ROCKI and ROCKII, and both are expressed in vascular smooth muscle cells (VSMC) and phos-phorylate MLCP (36). Nevertheless, these proteins seem to
Please cite this article in press as: Llanos, A.J., et al., The heme oxygenase-carbon monoxide sy... more Please cite this article in press as: Llanos, A.J., et al., The heme oxygenase-carbon monoxide system in the regulation of cardiorespiratory function at high altitude. Respir. Physiol. Neurobiol. (2012), http://dx.
Lowland mammals at high altitude constrict the pulmonary vessels, augmenting vascular resistance ... more Lowland mammals at high altitude constrict the pulmonary vessels, augmenting vascular resistance and developing pulmonary arterial hypertension. In contrast, highland mammals, like the llama, do not present pulmonary arterial hypertension. Using wire myography, we studied the sensitivity to norepinephrine (NE) and NO of small pulmonary arteries of fetal llamas and sheep at high altitudes. The sensitivity of the contractile responses to NE was decreased whereas the relaxation sensitivity to NO was augmented in the llama fetus compared to the sheep fetus. Altogether these data show that the fetal llama has a lower sensitivity to a vasoconstrictor (NE) and a higher sensitivity to a vasodilator (NO), than the fetal sheep, consistent with a lower pulmonary arterial pressure found in the neonatal llama in the Andean altiplano. Additionally, we investigated carbon monoxide (CO) in the pulmonary circulation in lowland and highland newborn sheep and llamas. Pulmonary arterial pressure was augmented in neonatal sheep but not in llamas. These sheep had reduced soluble guanylate cyclase and heme oxygenase expression and CO production than at lowland. In contrast, neonatal llamas increased markedly pulmonary CO production and HO expression at high altitude. Thus, enhanced pulmonary CO protects against pulmonary hypertension in the highland neonate. Further, we compared pulmonary vascular responses to acute hypoxia in the adult llama versus the adult sheep. The rise in pulmonary arterial pressure was more marked in the sheep than in the llama. The llama pulmonary dilator strategy may provide insights into new treatments for pulmonary arterial hypertension of the neonate and adult.
Chronic hypoxia during gestation and delivery results in oxidative stress and cerebrovascular dys... more Chronic hypoxia during gestation and delivery results in oxidative stress and cerebrovascular dysfunction in the neonate. We assessed whether melatonin, a potent antioxidant and potential vasodilator, improves the cerebral vascular function in chronically hypoxic neonatal lambs gestated and born in the highlands (3600 m). Six lambs received melatonin (1 mg/kg per day oral) and six received vehicle, once a day for 8 days. During treatment, biometry and hemodynamic variables were recorded. After treatment, lambs were submitted to a graded FiO 2 protocol to assess cardiovascular responses to oxygenation changes. At 12 days old, middle cerebral arteries (MCA) were collected for vascular reactivity, morphostructural, and immunostaining evaluation. Melatonin increased fractional growth at the beginning and improved carotid blood flow at all arterial PO 2 levels by the end of the treatment (P < 0.05). Further, melatonin treatment improved vascular responses to potassium, serotonin, methacholine, and melatonin itself (P < 0.05). In addition, melatonin enhanced the endothelial response via nitric oxide-independent mechanisms in isolated arteries (162 AE 26 versus 266 AE 34 AUC, P < 0.05). Finally, nitrotyrosine staining as an oxidative stress marker decreased in the MCA media layer of melatonin-treated animals (0.01357 AE 0.00089 versus 0.00837 AE 0.00164 pixels/lm 2 , P < 0.05). All the melatonin-induced changes were associated with no systemic cardiovascular alterations in vivo. In conclusion, oral treatment with melatonin modulates cerebral vascular function, resulting in a better cerebral perfusion and reduced oxidative stress in the neonatal period in chronically hypoxic lambs. Melatonin is a potential therapeutic agent for treating cerebrovascular dysfunction associated with oxidative stress and developmental hypoxia in neonates.
La exposición a la hipoxia crónica en el altiplano andino durante el período perinatal induce hip... more La exposición a la hipoxia crónica en el altiplano andino durante el período perinatal induce hipertensión pulmonar y cambios estructurales y funcionales en la circulación pulmonar y sistémica. El objetivo de este estudio fue analizar los efectos de la gestación y vida perinatal temprana a diferentes altitudes, en el sistema cardiovascular sistémico y pulmonar en recién nacidos de oveja. Corderos de tierras bajas (200m) y tierras altas (dos grupos: 3.600m y 4.600m) fueron cateterizados bajo anestesia general y sometidos a episodios de hipoxia/hiperoxia. Gasometría arterial y venosa, frecuencia cardiaca, gasto cardiaco, presión arterial sistémica y pulmonar, y resistencia vascular sistémica y pulmonar fueron analizadas a diferentes niveles de oxigenación en corderos de 1-2 semanas de edad. En condiciones basales, los corderos de altura (3,600m y 4,600m) presentaron un menor peso, PO 2 y SaO 2 . Ambos grupos de altura tuvieron un gasto cardiaco mayor y una menor resistencia vascular sistémica. La altura determinó un aumento significativo de la presión arterial pulmonar con cambios en la sensibilidad al oxígeno de la circulación pulmonar. Interesantemente, estos efectos fueron mayores en corderos de 3,600m que en el grupo de 4,600m. La hipoxia graduada demostró que la presión pulmonar media fue mayor en corderos de 3,600m a cualquier nivel de PO 2 . Existe una relación importante entre el peso del cordero y los efectos cardiovasculares sistémicos con la altura. Las respuestas pulmonares fueron importantes en ambos grupos de altura pero más marcadas en los corderos de 3,600m. Corderos provenientes de alturas extremas (4,600m) han desarrollado una adaptación pulmonar particular, la cual les permite compensar la baja disponibilidad de oxígeno ambiental en el Altiplano Andino. La hipoxia crónica afecta la salud y producción del rebaño. Conocer los mecanismos involucrados en el desarrollo y establecimiento del sistema cardiovascular podría dar información para un mejor entendimiento de la fisiología y fisiopatología perinatal en tierras altas y en enfermedades que evolucionan con hipoxia crónica fetal y neonatal en tierras bajas. Esto podría dar claves para mejorar la producción animal local en poblaciones de tierras altas y entender la fisiopatología de la hipoxia crónica.
Pulmonary arterial hypertension is one of the most serious pathologies that could affect the 140 ... more Pulmonary arterial hypertension is one of the most serious pathologies that could affect the 140 million people living at altitudes over 2,500m. The primary emphasis of the review is pulmonary artery hypertension in mammals (sheep and llamas) at high altitude, with specific focus on the heme oxygenase and carbon monoxide (HO-CO) system. We highlight the fact that the neonatal llama has neither pulmonary artery hypertension nor pulmonary vascular remodeling in the Andean altiplano. These neonates have an enhanced HO-CO system function, increasing the HO-1 protein expression and CO production by the pulmonary vessels when compared to low altitude raised llamas, or highland raised neonatal sheep. The neonatal sheep has high altitude pulmonary artery hypertension in spite of enhancement of the NO system, with high eNOS protein expression and NO production by the lung. It appears that the HO-CO pathway has been selected to participate in the control of the pulmonary circulation in the llama, a native dweller of the altiplano.
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