Neonatal lambs, as other neonates, have physiologically a very low plasma melatonin concentration throughout 24 h. Previously, we found that melatonin given to neonates daily for 5 days decreased heart weight and changed plasma cortisol and gene expression in the adrenal and heart. Whether these changes could compromise the responses to life challenges is unknown. Therefore, firstly, we studied acute effects of melatonin on the defense mechanisms to acute hypoxia in the neonate. Eleven lambs, 2 weeks old, were instrumented and subjected to an episode of acute isocapnic hypoxia, consisting of four 30 min periods: normoxia (room air), normoxia after an i.v. bolus of melatonin (0.27 mg kg −1 , n = 6) or vehicle (ethanol 1:10 NaCl 0.9%, n = 5), hypoxia (PaO 2 : 30 ± 2 mmHg), and recovery (room air). Mean pulmonary and systemic blood pressures, heart rate, and cardiac output were measured, and systemic and pulmonary vascular resistance and stroke volume were calculated. Blood samples were taken every 30 min to measure plasma norepinephrine, cortisol, glucose, triglycerides, and redox markers (8-isoprostane and FRAP). Melatonin blunted the increase of pulmonary vascular resistance triggered by hypoxia, markedly exacerbated the heart rate response, decreased heart stroke volume, and lessened the magnitude of the increase of plasmatic norepinephrine and cortisol levels induced by hypoxia. No changes were observed in pulmonary blood pressure, systemic blood pressures and resistance, cardiac output, glucose, triglyceride plasma concentrations, or redox markers. Melatonin had no effect on cardiovascular, endocrine, or metabolic variables, under normoxia. Secondly, we examined whether acute melatonin administration under normoxia could have an effect in gene expression on the adrenal, lung, and heart. Lambs received a bolus of vehicle or melatonin and were euthanized 30 min later to collect tissues. We found that melatonin affected expression of the immediate early genes egr1 in adrenal, ctgf in lung, and nr3c1 , the glucocorticoid receptor, in adrenal and heart. We speculate that these early gene responses may contribute to the observed alterations of the newborn defense mechanisms to hypoxia. This could be particularly important since the use of melatonin is proposed for several diseases in the neonatal period in humans.

Neonatal pulmonary hypertension (NPHT) is produced by sustained pulmonary vasoconstriction and increased vascular remodeling. Soluble guanylyl cyclase (sGC) participates in signaling pathways that induce vascular vasodilation and reduce vascular remodeling. However, when sGC is oxidized and/or loses its heme group, it does not respond to nitric oxide (NO), losing its vasodilating effects. sGC protein expression and function is reduced in hypertensive neonatal lambs. Currently, NPHT is treated with NO inhalation therapy; however, new treatments are needed for improved outcomes. We used Cinaciguat (BAY-582667), which activates oxidized and/or without heme group sGC in pulmonary hypertensive lambs studied at 3,600 m. Our study included 6 Cinaciguat-treated (35 ug kg−1 day−1x 7 days) and 6 Control neonates. We measured acute and chronic basal cardiovascular variables in pulmonary and systemic circulation, cardiovascular variables during a superimposed episode of acute hypoxia, remodeling of pulmonary arteries and changes in the right ventricle weight, vasoactive functions in small pulmonary arteries, and expression of NO-sGC-cGMP signaling pathway proteins involved in vasodilation. We observed a decrease in pulmonary arterial pressure and vascular resistance during the acute treatment. In contrast, the pulmonary pressure did not change in the chronic study due to increased cardiac output, resulting in lower pulmonary vascular resistance in the last 2 days of chronic study. The latter may have had a role in decreasing right ventricular hypertrophy, although the direct effect of Cinaciguat on the heart should also be considered. During acute hypoxia, the pulmonary vascular resistance remained low compared to the Control lambs. We observed a higher lung artery density, accompanied by reduced smooth muscle and adventitia layers in the pulmonary arteries. Additionally, vasodilator function was increased, and vasoconstrictor function was decreased, with modifications in the expression of proteins linked to pulmonary vasodilation, consistent with low pulmonary vascular resistance. In summary, Cinaciguat, an activator of sGC, induces cardiopulmonary modifications in chronically hypoxic and pulmonary hypertensive newborn lambs. Therefore, Cinaciguat is a potential therapeutic tool for reducing pulmonary vascular remodeling and/or right ventricular hypertrophy in pulmonary arterial hypertension syndrome.

Newborn lambs at high altitudes (3,600 m) develop pulmonary hypertension due to a sustained hypoxic vasoconstriction followed by a pathological remodeling of small pulmonary arteries (SPA). The latter is produced by several agents, including the transcription factor Hypoxia Inducible Factor‐1α (HIF‐1α). HIF‐1α increases the pulmonary vascular remodeling in chronic hypoxia and the enzyme PHD‐2 is a key factor participating in the degradation of HIF‐1α. Cinaciguat an activator of sGC is able to decrease HIF‐1α, in adults, and increase HO‐1 in neonatal lambs. This enzyme reduces reactive oxygen species (ROS) and cytokines, whereas no information in PHD‐2 and cinaciguat is available. Therefore, we use two groups of 6 newborn lambs each, that were catheterized under general anesthesia and a Swan Ganz catheter was placed into the pulmonary artery, to measure pulmonary arterial pressure (mPAP), cardiac output (CO) and calculate pulmonary vascular resistance (PVR). The groups received, either the activator of sGC (cinaciguat 35 μg kg−1) or vehicle (DMSO : NaCl 0,9%, 1:10), for seven days. Additionally, we took lung samples to perform histology (muscle & adventitia layers in small pulmonary arteries, SPA) and western blots (HIF‐1α and PHD‐2). Forty‐eight hour after the end of treatment, euthanasia was accomplished. All procedures were approved by the local Bioethical Committee (CBA#0643 FMUCH). Cinaciguat did not change the mPAP but did vary the PVR. Mean PAP was not modified because CO increased. Concordant with the lower PVR there was a reduction of the muscular and adventitia layers in SPA. Further, cinaciguat did not modify the HIF‐1α protein expression but showed a decrease in PHD‐2 in pulmonary hypertensive newborn lambs. If ROS and cytokines are diminished (unmeasured) by cinaciguat, both factors could generate a reduction in HIF‐1α, that could lessen PHD‐2 (feedforward). The latter should produce a decrease in HIF‐1α hydroxylation and therefore a reduced destruction in the proteasome, which would lead to an increase in HIF‐1α availability, and eventually to its initial state (feedback). HIF‐1α did not decrease because has a short half‐life in contrast with PHD‐2 that has a half‐life over 48h. In conclusion, the decrease in PHD‐2 could be the consequence of an initial HIF‐1α reduction, producing a diminution in muscle and adventitia layers in small pulmonary arteries consistent with a lesser PVR. The reduction of HIF‐1α could be considered a useful tool to treat the pulmonary hypertension of the neonate. Support or Funding Information Conicyt 21191677, Fondecyt 1140647, Chile.

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