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

Am J Physiol Regul Integr Comp Physiol 299: R1676 R1684, 2010. First published September 29, 2010; doi:10.1152/ajpregu.00123.2010. Long-term exposure to high-altitude chronic hypoxia during gestation induces neonatal pulmonary hypertension at sea level Emilio A. Herrera, 1,2,4 Raquel A. Riquelme, 3 Germán Ebensperger, 1 Roberto V. Reyes, 1 César E. Ulloa, 1 Gertrudis Cabello, 6 Bernardo J. Krause, 1 Julian T. Parer, 5 Dino A. Giussani, 4 and Aníbal J. Llanos 1,2,6 1 Laboratorio de Fisiología y Fisiopatología del Desarrollo, Programa de Fisiopatología, Facultad de Medicina, Instituto de Ciencias Biomédicas, 2 International Center for Andean Studies, 3 Facultad de Ciencias Químicas y Farmacéuticas, Departamento de Bioquímica y Biología Molecular, Universidad de Chile, Santiago, and 6 Facultad de Ciencias, Departamento de Biología, Universidad de Tarapacá, Arica, Chile; 4 Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, United Kingdom; 5 Department of Obstetrics, Gynecology and Reproductive Sciences, University of California San Francisco, San Francisco, California Submitted 19 February 2010; accepted in final form 24 September 2010 Herrera EA, Riquelme RA, Ebensperger G, Reyes RV, Ulloa CE, Cabello G, Krause BJ, Parer JT, Giussani DA, Llanos AJ. Long-term exposure to high-altitude chronic hypoxia during gestation induces neonatal pulmonary hypertension at sea level. Am J Physiol Regul Integr Comp Physiol 299: R1676 R1684, 2010. First published September 29, 2010; doi:10.1152/ajpregu.00123.2010. 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, ) and conception at low altitude, pregnancy at HA (3,600 m) from 30% of gestation until delivery, and return to lowland (). 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. lambs had higher basal PAP and a greater increment in PAP after N G -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 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 relative to. 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 Ca 2 -activated K channel (0.76 0.16 vs. 0.30 0.01) were greater in compared with lambs. In contrast, 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. pulmonary hypoxic vasoconstriction; pulmonary vascular reactivity; nitric oxide; pulmonary vasodilators; pulmonary vasoconstrictors THE ETIOLOGY OF PULMONARY hypertension in the postnatal period is complex and not completely understood (2, 44, 48). Proposed mechanisms underlying the physiology mediating elevations in postnatal pulmonary arterial pressure include impaired endothelial function promoting an increase in pulmonary vascular resistance (1, 2, 44). Pulmonary hypertension in Address for reprint requests and other correspondence: A. J. Llanos, Programa de Fisiopatología, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile. Avda. Salvador 486, Providencia, CP 6640871, Santiago, Chile (e-mail: allanos@med.uchile.cl). the postnatal period is associated with high mortality, and children who survive may have decreased postnatal growth and devastating neurological, respiratory, and cardiac complications that often persist into childhood (9, 44). One condition that may lead to elevations in pulmonary arterial pressure in the postnatal period is sustained fetal hypoxia (1, 44). In humans and animals, a common form of sustained fetal hypoxia is pregnancy at high altitude (16, 23). Pulmonary hypertension in the postnatal period due to this condition is an important problem, since currently nearly 140 million people reside at over 2,500 meters above sea level, being permanently exposed to chronic hypoxic conditions (34, 41). Sustained or partial exposure to high altitude of pregnant women, either permanently resident at high altitude or native to low altitude, is therefore a current problem. Several mediators act upon the pulmonary vasculature, triggering alterations in vascular tone and structure. A potent vasoconstrictor is endothelin-1 (ET-1), which acts via the ET A receptor to stimulate both contraction and remodeling of the pulmonary vascular bed. ET-1 therefore plays an important role in the regulation of pulmonary vascular resistance (3, 4, 26). Interestingly, it has been reported that ET-1 function is increased in neonatal pulmonary hypertension (3). Nitric oxide (NO) is also another important modulator of the pulmonary circulation, and its vasodilator actions are mediated via several mechanisms, including the activation and opening of Ca 2 - activated K channels (BK Ca ) and the balance between the synthesis of cgmp through the activation of soluble guanylate cyclase (sgc) and its degradation by the isoenzyme phosphodiesterase 5 (PDE5) (2, 42). The impairment of NO-dependent dilatation has also been closely related to pulmonary hypertension in the postnatal period (1, 44). In addition, the endogenous gas carbon monoxide (CO) is a dilator in the pulmonary vascular bed, and it protects against pulmonary vascular remodeling (31, 37, 48). In newborn llamas, augmented pulmonary CO, rather than pulmonary NO, helps to prevent pulmonary hypertension in the newborn period at high altitude (25). Using ovine pregnancy at high altitude as an experimental model, we have previously reported that pregnancy and delivery at high altitude yields offspring with pulmonary hypertension, coupled with increased constrictor reactivity of isolated pulmonary vessels despite enhanced pulmonary NO function (23, 24, 25). In those studies, the in vivo and in vitro measurements were performed at high altitude. It remains unknown whether long-term exposure of the pregnancy to high altitude R1676 0363-6119/10 Copyright 2010 the American Physiological Society http://www.ajpregu.org

results in altered pulmonary vascular function and anatomy in offspring, even following return to sea level. Therefore, this study tested the hypothesis that long-term exposure of the pregnancy to high altitude results in postnatal pulmonary hypertension even following return to sea level and that this is associated with cardiopulmonary remodeling and alterations in the pulmonary vascular function. We used an integrative approach at the whole animal, isolated organ, and molecular level to determine the effects of 70% of gestation at high altitude on: 1) in vivo pulmonary arterial pressure under basal and acute hypoxic conditions, both before and after NO blockade; 2) the reactivity of isolated small pulmonary arteries to KCl, ET-1, and to sodium nitroprusside (SNP) before and after treatment with the sgc inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin- 1-one (ODQ); 3) the mrna and protein expression of endothelial NO synthase (enos), and protein expression of sgc, BK Ca, PDE5, heme oxygenase-1 (HO-1), and CO production in the postnatal lung; and 4) the morphology of small pulmonary arteries and weight ratios of the heart and lungs. All studies were performed at sea level in lambs within the first two weeks of postnatal life. MATERIALS AND METHODS The Faculty of Medicine Ethics Committee of the University of Chile approved all experimental procedures (Protocol CBA No. 097, FMUCH). The studies on animals were performed according with the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH Publication No. 85-23, revised 1996) and adheres to the American Physiological Society s Guiding Principles in the Care and Use of Animals. Animals Twenty-eight pregnant ewes (Ovis aries) were divided into the following two groups: conception, pregnancy, and delivery at lowland (Santiago, 580 m,, n 14) and conception at lowland, pregnancy at high altitude (Putre, 3,600 m) from 30% of gestation until delivery, and return to lowland (, n 14). Mothers and newborns were housed in an open yard with access to food and water ad libitum. Surgical Preparation and In Vivo Experiments A subgroup of the lambs was surgically prepared between 3 and 8 days of age for in vivo experimentation (, n 8;, n 4). In brief, the animals were anesthetized with ketamine (10 mg/kg im) and diazepam (0.1 0.5 mg/kg im) with additional local infiltration of 2% lidocaine. Polyvinyl catheters were placed in the descending aorta and inferior vena cava, and a Swan-Ganz catheter was placed in the pulmonary artery, as previously described in detail (23). Following 3 4 days of postsurgical recovery, the animals were subjected to a 3-h experimental protocol, consisting of 1 h of normoxia, 1 h of hypoxia, and 1 h of recovery. Acute isocapnic hypoxia was induced via a transparent, loosely tied polyethylene bag placed over the animal s head into which a known mixture of air, N 2, and CO 2 ( 10% O 2 and 2 3% CO 2 in N 2) was passed at a rate of 20 l/min. Acute hypoxia was induced on separate days during vehicle infusion (0.9% NaCl) and during NO blockade [N G -nitro-l-arginine methyl ester (L-NAME), 20 mg/kg bolus plus 0.5 mg kg 1 min 1 in 0.9% NaCl infusion] in random order. Infusions started 15 min before hypoxia and ran continuously until the end of the hypoxemic challenge. Arterial blood samples were taken during each experimental protocol to determine arterial ph, PO 2,PCO 2, hemoglobin concentration ([Hb]), and percentage saturation of hemoglobin (Sa O2 ) [IL-Synthesis 25 (Instrumentation Laboratories, Lexington, MA); measurements corrected to 39 C]. Pulmonary and systemic arterial pressures and heart rate were recorded continually via a data acquisition system (Powerlab/8SP System and Chart v4.1.2 Software; ADInstruments, New South Wales, Australia) connected to a computer. Cardiac output was determined at set intervals by the thermodilution method by the injection of 3 ml of chilled (0 C) 0.9% NaCl in the pulmonary artery through the Swan- Ganz catheter connected to a cardiac output computer (COM-2 model; Baxter, Irvine, CA). Pulmonary vascular resistance was calculated as described previously (23). The production of CO by the pulmonary circulation was calculated as follows: cardiac output multiplied by the difference in the concentration of CO between the aorta and the pulmonary artery (percent of carboxyhemoglobin; IL-Synthesis 25) (25). Ex Vivo and In Vitro Experiments R1677 The remaining uninstrumented lambs (, n 6;, n 5) underwent euthanasia with an overdose of sodium thiopentone (100 mg/kg iv) and were studied ex vivo. Wire myography. The left lung was removed by dissection and immediately immersed in cold saline. Fourth-generation pulmonary arteries (counting from the pulmonary artery trunk, id : 410 20 m and : 365 22 m) were dissected from the caudal lobule of the left lung. Isolated arteries were mounted in a wire myograph, maintained at 37 C, and aerated with 95% O 2-5% CO 2. Concentration-response curves (CRCs) were analyzed in terms of sensitivity and maximal or minimal responses by fitting experimental data to the Boltzmann equation (Prism 5.0; GraphPad Software, La Jolla, CA). Contractile responses were expressed in terms of tension (N/m) and contraction or relaxation responses as a percentage of increase or reduction of 125 mm K -induced contraction or tension (N/m). Sensitivity was calculated as pd 2, where pd 2 log[ec 50], with EC 50 being the concentration at which 50% of the maximal response was obtained (22). CRCs were constructed for KCl, ET-1, and for the NO donor SNP following precontraction with 125 mm K. CRCs to SNP were repeated following blockade of sgc with 50 M ofodq (10 5 M) incubation for 10 min. RT-PCR and Western blot. Total RNA purification from lung tissue, cdna synthesis, and PCR amplification was performed as described previously (11). Primers for amplification of partial DNA sequences from enos (forward 5=-AGCTTGAGACCCTCAGT- CAGGA-3= and reverse 5=-GTCTCCAGTCTTGAGCTGGC-3=, accession no. DQ015701) and 18S rrna (forward 5=-TCAAGAAC- GAAAGTCGGAGG-3= and reverse 5=-GGACATCTAAGGGCAT- CACA-3=, the housekeeping gene, accession no. BK00096) were derived from the corresponding sheep and mouse genes, respectively. All of the PCR products were sequenced to verify their identity. The PCR products were visualized under ultraviolet light and quantified by densitometry. Protein expression of enos, HO-1, sgc, BK Ca, PDE5, and -actin was determined in total lung lysates by immunoblot with specific anti-enos monoclonal antibody (Transduction Laboratories), anti-ho-1 monoclonal antibody (Research Diagnostic), anti-sgc polyclonal antibody (Cayman Laboratories), anti-bk Ca polyclonal antibody (Alomone laboratories), anti-pde5 monoclonal antibody (BD Transduction Laboratories), and anti- -actin monoclonal antibody (Sigma) as described elsewhere (25). The signals obtained on immunoblot or RT-PCR determinations were quantified by densitometric analysis using the Scion Image Software (Scion Image Beta 4.02 Win; Scion). Heart and lung biometry. The neonatal heart was obtained, and the free wall of the right ventricle, the left ventricle, and the septum were dissected. The ratio of the weights of the right ventricle to the left ventricle and septum was calculated (20). In addition, the lungs were removed and weighed. Lung weight-to-body weight ratio was calculated. Histology. We isolated and perfused the right lung with 4% paraformaldehyde. Excised lungs were fixed in 4% paraformaldehyde for 24 h at 4 C and embedded in paraffin; van Giesson staining was

R1678 performed on 10- m slides. At least four arteries (100 200 m diameter) per lung were chosen, and an average of four measurements from each artery was recorded. Images of parenchymal arterioles were acquired using a workstation (Olympus trinocular microscope-bx51 plus digital camera QimaginGO3) linked to Image Pro software 6.3, and vascular areas were calculated using an image analysis program. The wall-to-vessel area ratio was calculated and expressed as a percentage, as previously described (24, 33). Briefly, the percent wall thickness was calculated as follows: wall thickness (%) external area internal area/external area 100, where external area and internal area are the area bounded by external and internal elastic laminae, respectively. In addition, the area of vascular smooth muscle was calculated as follows: muscle area (%) external muscle area internal area/external muscle area 100, where the external muscle area and the internal area are the external and internal boundaries of the tunica media, respectively. Statistical Analysis Data are expressed as means SE. Groups were compared by two-way ANOVA and the post hoc Newman-Keuls test, or the Student s t-test for unpaired data, as appropriate. We used the Fisher Exact Test to compare survival between groups. For all comparisons, differences were considered statistically significant when P 0.05 (18). RESULTS Survival and Weight In marked contrast to sea level pregnancies () with 100% survival, pregnancies after 70% exposure to high altitude () had increased mortality, with 21% abortions and 14% stillbirths (P 0.05). No lambs died after birth in either group. Surviving lambs were much lighter than lambs (3.8 0.3 kg, n 9 vs. 7.0 0.4 kg, n 14, P 0.001, weights at the time of experimentation between 6 and 11 days of age). In Vivo Experiments Because of differences in survival, eight and four lambs were studied in vivo. Values for basal ph a,pa O2,Pa CO2, Sa O2, and [Hb] were similar in both groups of lambs (Table 1). During acute hypoxia on a background of saline infusion, a similar fall in Pa O2 and Sa O2 occurred in both groups of lambs, without any alteration to Pa CO2 from baseline (Table 1). During recovery, all variables returned toward basal values in both groups. However, values for Pa CO2 were significantly depressed from baseline in lambs (Table 1). Treatment with L-NAME had no significant effect on arterial blood gas and acid base status either during basal or acute hypoxic conditions (Table 1). Basal values for pulmonary arterial pressure, pulmonary vascular resistance, and cardiac output were significantly greater in than lambs (Fig. 1 and Table 1). Basal values for heart rate were similar between the groups (Table 1). Basal systemic arterial pressure was similar in and lambs (87 3 vs. 82 1 mmhg, respectively). During acute hypoxia on a background of saline infusion, pulmonary arterial pressure, pulmonary vascular resistance, cardiac output, and heart rate increased significantly in both groups of lambs. However, in relative to lambs, values for pulmonary arterial pressure and cardiac output reached significantly greater values during the acute hypoxic challenge (Fig. 1 and Table 1). No changes in systemic arterial pressure were seen in either of the experimental groups during hypoxia. During recovery, pulmonary arterial pressure, and cardiac output remained significantly elevated from baseline, but heart rate and pulmonary vascular resistance returned toward basal values in lambs. In contrast, all variables returned toward basal values in lambs (Fig. 1 and Table 1). Treatment of the lambs with L-NAME during the basal period led to an increase in pulmonary arterial pressure and pulmonary vascular resistance and a decrease in heart rate and cardiac output in both groups of lambs. Although the fall in heart rate and cardiac output was similar between the groups, the increment in pulmonary arterial pressure and in pulmonary vascular resistance was significantly greater in than in lambs (Fig. 1 and Table 1). Treatment with L-NAME did not affect the magnitude of the pulmonary hemodynamic and blood gas responses to acute hypoxia in either group of lambs, with the exception that values for pulmonary arterial pressure reached greater values in than in lambs (Fig. 1 and Table 1). At the time of surgery up until the time of study, there was no evidence of a patent ductus arteriosus. At the time of dissection, after the last study, the ductus arteriosus was examined, and no lumen was visible in any of the studied animals. Additional evidence for the closure of the ductus arteriosus is provided by the similarities of oxygen saturation and PO 2 in samples obtained from the ascending and descending aorta (data not shown). This is further supported by the fact that systemic arterial pressure was always higher than the pulmonary arterial pressure in all animals. The presence of a left-to-right shunt is unlikely because there were no differences in pulmonary arterial pulse pressure, between and lambs, either during basal conditions or during hypoxia. Ex Vivo Experiments Isolated small pulmonary arteries from relative to lambs showed a greater maximal contraction to KCl (K max : 1.34 0.05 vs. 1.16 0.05 N/m, P 0.05) with similar sensitivity (EC 50 : 28.54 2.48 vs. 31.53 4.44; Fig. 2A). In contrast, the maximal contraction with ET-1 was similar in the two groups, although the sensitivity to the contraction elicited by ET-1 was significantly greater in than in lambs (PD 2 : 8.08 0.13 vs. 7.22 0.28, P 0.05; Fig. 2B). The NO donor SNP evoked a similar maximal relaxation in pulmonary vessels from and lambs (%K max : 98.1 3.0 vs. 100.0 2.3%; Fig. 3A). However, the relaxant sensitivity of the pulmonary vessels to SNP was significantly greater in than in lambs (PD 2 : 7.31 0.12 vs. 5.77 0.07, P 0.05; Fig. 3A). This SNP-induced vasorelaxation in the pulmonary vasculature was completely abolished by blocking sgc with ODQ in lambs (Fig. 3B). In marked contrast, SNP-induced vasorelaxation in vessels isolated from lambs persisted following treatment with ODQ, but the maximal relaxation and sensitivity were significantly diminished (before ODQ, %K max : 98.1 3.0%, PD 2 : 7.31 0.12; after ODQ, %K max : 37.7 3.2%, PD 2 : 4.97 0.13, P 0.05; Fig. 3B). Western Blot The expression of enos mrna and protein in lung tissue was significantly greater in lambs than in lambs (Figs. 4 and 5). This was associated with a significantly

Table 1. Cardiorespiratory variables in and lambs R1679 Basal Basal I Hypoxia I Recovery ph a NaCl (0.9%) 7.412 0.014 7.407 0.013 7.391 0.019 7.423 0.018 L-NAME 7.459 0.007 7.441 0.010 7.393 0.027 7.409 0.033 NaCl (0.9%) 7.475 0.024 7.444 0.009 7.462 0.022 7.459 0.023 L-NAME 7.464 0.005 7.450 0.011 7.417 0.032 7.432 0.014 Pa O2, mmhg NaCl (0.9%) 79.5 1.9 77.7 3.4 30.9 0.6* 82.4 3.8 L-NAME 78.3 2.8 82.6 5.5 32.0 0.7* 77.8 6.1 NaCl (0.9%) 85.0 4.4 83.7 3.3 29.2 0.1* 79.6 6.0 L-NAME 86.8 1.8 81.5 5.3 30.2 0.8* 81.0 5.5 Pa CO2, mmhg NaCl (0.9%) 36.8 1.1 36.9 1.3 35.8 1.2 32.4 1.4* L-NAME 33.3 0.8 33.4 0.9 32.5 0.8 28.9 1.2* NaCl (0.9%) 36.9 1.8 37.4 3.0 37.8 0.8 36.1 1.4 L-NAME 38.7 1.4 35.8 1.8 37.2 2.2 32.8 0.8* Sa O2,% NaCl (0.9%) 94.7 0.7 93.5 1.2 52.7 3.1* 96.0 0.7 L-NAME 94.9 1.1 93.9 1.6 55.9 5.6* 90.9 4.6 NaCl (0.9%) 95.1 1.6 93.1 1.7 55.4 5.7* 93.6 2.3 L-NAME 93.8 1.2 91.3 1.1 47.0 2.3* 90.7 1.5 [Hb], g/dl NaCl (0.9%) 10.9 0.5 11.0 0.5 11.3 0.4 10.3 0.4 L-NAME 9.7 0.7 9.5 0.8 10.8 0.5 10.1 0.4 NaCl (0.9%) 11.9 0.7 12.6 1.3 12.3 1.1 10.8 0.5 L-NAME 12.2 1.3 12.6 1.2 13.2 1.4 12.4 1.4 Heart rate, min 1 NaCl (0.9%) 198.2 8.0 192.8 7.6 267.9 12.32* 207.6 6.7 L-NAME 173.4 11.6 132.1 8.0* 203.8 12.2 146.6 9.0 NaCl (0.9%) 188.5 7.4 188.9 4.9 265.1 13.4* 216.3 5.7 L-NAME 167.3 7.4 148.0 8.3* 196.1 19.3 155.1 8.6 Cardiac output, ml min 1 kg 1 NaCl (0.9%) 281.0 13.3 276.0 13.0 394.0 15.3* 304.9 10.8 L-NAME 298.8 14.6 194.1 9.1* 261.2 12.6* 192.4 9.9* NaCl (0.9%) 373.2 13.0 326.8 19.1 474.5 7.9* 456.9 32.6* L-NAME 373.5 41.8 216.2 16.8* 308.4 49.8 267.5 41.0* PVR, mmhg min 1 kg 1 NaCl (0.9%) 0.049 0.003 0.054 0.004 0.067 0.004* 0.054 0.003 L-NAME 0.040 0.002 0.091 0.006* 0.149 0.009* 0.090 0.013* NaCl (0.9%) 0.056 0.004 0.064 0.007 0.066 0.005* 0.056 0.004 L-NAME 0.052 0.005 0.186 0.025* 0.180 0.050* 0.132 0.025* Values are the means SE, in units, for arterial ph (ph a), partial pressure of oxygen (Pa O2 ), partial pressure of carbon dioxide (Pa CO2 ), saturation of hemoglobin with oxygen (Sa O2 ), hemoglobin concentration ([Hb]), heart rate, cardiac output, and pulmonary vascular resistance (PVR)., conception, pregnancy, and delivery at low altitude (580 m);, pregnancy at high altitude (3,600 m) from 30% of gestation until delivery, and return to lowland; N G -nitro-l-arginine methyl ester, L-NAME. Blood samples and cardiovascular variables were taken, measured, and calculated during preinfusion baseline (Basal), during infusion with saline or L-NAME (Basal I), during acute hypoxia (Hypoxia I), and during recovery. Significant differences (P 0.05) are as follows: vs. basal (*), vs. ( ), and L-NAME vs. 0.9% NaCl ( ). greater protein expression of pulmonary BK Ca and PDE5 but not sgc in than in lambs (Fig. 5). Furthermore, protein expression of pulmonary HO-1 and the production of CO by the pulmonary circulation were both diminished in compared with lambs (Fig. 6). Heart and Lung Biometry The ratio of the weight of the right ventricle to the left ventricle plus septum was augmented in the compared with the group (0.370 0.017 vs. 0.328 0.009, P

R1680 Fig. 1. Pulmonary arterial pressure (PAP) during the in vivo acute hypoxia protocol in lambs in the following 2 groups: conception, pregnancy, and delivery at low altitude (580 m) (, Œ) and conception at low altitude, pregnancy at high altitude (3,600 m) from 30% of gestation until delivery, and return to lowland (, ). Acute hypoxia was induced following a background infusion (gray bar) with 0.9% NaCl (A) or with the nitric oxide synthase (NOS) blockade inducer N G -nitro-l-arginine methyl ester (L-NAME, B). Values are means SE, calculated every minute during the experimental protocol. Significant differences (P 0.05) are as follows: vs. preinfusion baseline (*), vs. ( ), and L-NAME vs. 0.9% NaCl ( ). A striking difference between the groups of lambs in the present study was the much greater mortality and pronounced growth restriction in lambs born from pregnancies after prolonged exposure to high altitude. Pregnancy at high altitude induces maternofetal hypobaric hypoxia, and we have previously reported lower maternal and fetal arterial PO 2 in a separate cohort of animals exposed to the same altitude during the whole pregnancy (12). A similar effect on fetal growth restriction and mortality during development at high altitude has been reported in highland human populations (16, 30, 34, 38) and in chick embryos following highland incubation (17, 43). Malnutrition during early gestation in high-altitude cattle also resulted in a higher incidence of elevated pulmonary arterial pressure and right ventricular hypertrophy compared with controls when measured in the offspring at 15 mo. This was associated with differential gene expression in the right ventricle, but the resulting interaction between undernutrition and high-altitude hypoxia is unclear (21). In our study, both groups of lambs received the same nutrition, so the changes observed in pulmonary arterial pressure and growth restriction appear to be independent of nutrition. Accordingly, the effects on fetal growth restriction and mortality of developmental hypoxia at high altitude have been shown to be independent of the maternal nutritional status and of highland hypobaria in other species, since fetal growth restriction persists in ewes undergoing pregnancy at high altitude with food intake values similar to those as sea level pregnancies (23, 39). These effects have also been shown in the chick embryo, where incubation at high altitude of sea level eggs with oxygen supplementation completely prevented the high altitude-induced fetal growth restriction and mortality (17). The present study extends these 0.01). The ratio of the lung weight to the body weight was similar in compared with lambs (0.0200 0.0009 vs. 0.0180 0.0005, not significant). Histology Morphometric analysis of the pulmonary vasculature revealed no significant difference in vascular wall thickness between and lambs (48.79 3.59 vs. 55.03 4.35%, respectively, P 0.31, n 5 for each group). However, there was a significant increase in the area of vascular smooth muscle in compared with lambs (29.28 2.96 vs. 21.09 1.73%; P 0.05; Fig. 7). DISCUSSION These studies show that 70% exposure to high-altitude chronic hypoxia during gestation yields postnatal lambs with basal pulmonary hypertension and an increased pulmonary vascular response to an episode of acute hypoxia even following return to sea level. These findings persist despite evidence of enhanced pulmonary NO function obtained through in vivo, isolated organ and molecular approaches. Furthermore, the results show a decrease in pulmonary CO function and an increase in the vascular reactivity of constrictors associated with cardiopulmonary remodeling processes. Combined, the data support the hypothesis tested and provide a mechanistic explanation for the persistence of neonatal pulmonary hypertension at sea level induced by high-altitude pregnancy. Fig. 2. Vasoconstrictor function of small pulmonary vessels isolated from (Œ) and ( ) lambs. Values are the means SE for the vascular response to KCL (A) and to endothelin-1 (ET-1, B). Maximal response (E max) and sensitivity (EC 50 or pd 2) were calculated (see text). Significant differences (P 0.05) are as follows: vs. for E max ( ) and vs. for sensitivity ( ). Brackets denote concentration.

R1681 Fig. 3. Vasodilator function of small pulmonary vessels isolated from (Œ) and ( ) lambs. Values are the means SE for the vascular response to sodium nitroprusside (SNP, A) and to SNP in the presence of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 5 M, B). E max and sensitivity (EC 50 or pd 2) were calculated (see text). Significant differences (P 0.05) are as follows: vs. for E max ( ) and vs. for sensitivity ( ). findings and reports that 70% rather than 100% exposure to high altitude during fetal development can also have dramatic effects on the maintenance of pregnancy, on fetal growth, and on fetal mortality (abortion and stillbirth). In contrast, we did not have neonatal mortality. Fig. 5. Components of the NO signaling pathway in (open bars) and (filled bars) lambs. Values are the means SE for the protein expression of enos (A), soluble guanylate cyclase (sgc, B), phosphodiesterase 5 (PDE5, C), and Ca 2 -activated K channels (BK Ca, D). E: individual immunoblots. Significant differences (P 0.05) are vs. ( ). Fig. 4. mrna expression of endothelial nitric oxide synthase (enos) in (open bar) and (filled bar) lambs. Values are the means SE for the mrna expression of enos. Individual immunoblots are shown. Significant differences (P 0.05) are vs. ( ). Lambs born from pregnancies after 70% exposure to high altitude had a greater basal cardiac output, pulmonary vascular resistance, and pulmonary arterial pressure, even when their Pa O2 had recovered to normoxic levels, and also showed a greater pulmonary pressor response to L-NAME and to acute hypoxia. The greater basal cardiac output in the highland group is independent of differences in basal heart rate, suggesting a greater resting stroke volume in lambs from pregnancies after long exposure to high altitude. The differences in basal pulmonary arterial pressure and vascular resistance between the groups may be explained, in part, by the larger area of vascular smooth muscle, the greater pulmonary vessel maximal constrictor response to KCl, and the increased sensitivity to ET-1 in the lambs. ET-1 is induced by chronic hypoxia and is a potent pulmonary vasoconstrictor and a mitogen, leading to smooth muscle cell proliferation (3). Previous studies have correlated an increased vascular response with greater smooth muscle cell remodeling (29, 45), conditions that were both

R1682 Fig. 6. Components of the heme oxygenase (HO)-CO pathway in (open bars) and (filled bars) lambs. Values are means SE for the protein expression of HO-1 (A) and CO production by the pulmonary circulation (B). Individual immunoblots are shown (A). Significant differences (P 0.05) are: vs. ( ). observed in lambs in our study. Moreover, it has been suggested that the longer the exposure to high altitude, the greater the vascular smooth muscle remodeling (29, 40, 45). Dissociation between changes in vascular wall area and in wall thickness is a common finding with established explanations. Elegant studies by Baumbach and Heistad (5, 6) and by Mulvany (35, 36) have made it clear that an increase in the ratio of the vascular wall to lumen may be achieved by at least two very different situations. For instance, the ratio may be increased by a reduction in lumenal diameter without a change in medial volume. There is thus rearrangement of the same volume of vessel wall around a smaller-diameter lumen, what is now termed inward eutrophic vascular remodeling. Conversely, an increase in the vascular wall-to-lumen ratio may be achieved by an increase in wall material with or without a change in lumen diameter, what has been termed outward hypertrophic vascular growth. An increase in wall material with an increase in lumen diameter is what is occurring in the vessels. Interestingly, the main driving forces that promote this type of vascular remodeling are increased flow and pressure (36), both of which are present in the pulmonary bed of lambs. The present study also reports an increase in right ventricular mass in neonates. This is a common finding in humans and animals that have suffered arterial pulmonary hypertension (1, 15, 46, 47). Other components contributing to basal pulmonary hypertension in lambs from pregnancies after prolonged exposure to high altitude may include alterations in the tonic balance between dilator and constrictor influences on the pulmonary vascular bed. For instance, we have previously reported in highland lambs reduced synthesis of dilators, such as CO (25). In this set of experiments, it was also found that lambs had an important decrease in the production of CO by the pulmonary circulation concordant with the reduced HO-1 protein expression. Interestingly, a study in fetal lambs showed them to be unresponsive to CO (19). However, this was performed in ventilated (hypoxic, 10% FI O2 ) fetuses rather than normally oxygenated postnatal lambs. Lambs native to high altitude do not increase HO as do llamas, which suggests that they are insensitive to endogenous CO, although they may be responsive to induced CO production (19). CO is a dilatator via activation of sgc (13, 27, 32) and via hyperpolarizing the vascular smooth muscle secondary to activation of BK Ca channels (7, 10, 50). CO can also diminish the vasoconstrictor responses to phenylephrine and 20-hydroxyeicosatetraenoic acid while reducing the synthesis and release of ET (28, 51). The diminished production of CO by the pulmonary circulation determined in this study may play a putative role in the maintenance of persistent pulmonary hypertension of the newborn at sea level. In addition, chronic developmental hypoxia is known to result in lung hypoplasia and immaturity, pulmonary edema, and altered endothelial function (2, 20, 39, 46). Alterations in the synthesis and function of vasoconstrictors such as ET-1, as reported in this paper, thromboxane, IGF, Fig. 7. Representative micrograph of parenchymal pulmonary small arteries from (A) and (B) lams. van Giesson staining. PA, pulmonary artery. Bar: 100 m. Magnification: 40.

serotonin, and leukotriene C 4 /D 4 have also been implicated in the pulmonary hypertensive phenotype during chronic hypoxia (29, 45). In the present study, the greater pulmonary hypertension under basal and stimulated conditions in lambs from pregnancies after 70% exposure to high altitude occurred despite evidence of enhanced NO-dependent dilator function in the pulmonary vascular bed. The greater pressor response to treatment with L-NAME, the increased expression of enos mrna and protein, and the enhanced isolated vessel dilator response to SNP all strongly support enhanced NO function in the pulmonary vasculature of lambs from pregnancies after longterm exposure to high altitude. PDE is an enzyme that breaks down cgmp and thus halts the NO vasodilator cascade (42). In this study, also showed greater pulmonary protein expression of PDE5, findings similar to those reported in hypertensive lambs and lambs native to high altitude (22, 24). Although a greater protein expression of pulmonary PDE5 may itself favor constriction in the pulmonary vascular bed, it is likely that the increased expression of PDE5 occurs to match all other components of the enhanced NO cascade, and it does not underlie a cause but it is likely a consequence of the pulmonary hypertension in lambs from pregnancies exposed to high altitude. In the present study, blockade of sgc with ODQ completely prevented the pulmonary dilator response to the NO donor SNP in control lambs but not in lambs from pregnancies after prolonged exposure to high altitude. In the latter group, the dilator response to SNP persisted, albeit at a reduced level. This suggests that long-term exposure to high altitude during pregnancy may trigger an enhancement of NO dilatation pathways in addition to the activation of sgc in vascular smooth muscle. One possibility is the direct action of NO on the activation of K channels, as has already been described for the BK Ca channel (8). Accordingly, in the present study, lambs showed a significantly greater pulmonary BK Ca protein expression. What is important to highlight is that, despite evidence of enhanced pulmonary NO function via at least two different signaling cascades, this adaptive response is insufficient to offset pulmonary hypertension and vascular remodeling in lambs even following return to sea level. In conclusion, postnatal pulmonary hypertension induced by long-term exposure of the pregnancy to high altitude persists at sea level, despite enhanced pulmonary NO function. This condition is associated with a decrease in the production of pulmonary CO coupled with an increase in the vascular reactivity of constrictors associated with cardiopulmonary remodeling processes. Perspectives and Significance During acute episodes of hypoxia, the pulmonary vascular bed undergoes constriction to match the reduced oxygenation with reduced perfusion. During sustained hypoxia, this initial homeostatic response becomes maladaptive, triggering sustained increases in pulmonary vascular resistance, leading to the establishment of pulmonary hypertension. Our studies show that this maladaptive pulmonary constrictor response to hypoxia can be triggered in the newborn lamb following pregnancy at high altitude, when the measurements are performed at high altitude (23, 24, 25) and, even, following return to sea level. Sustained pulmonary hypertension and remodeling of the pulmonary vasculature suggest possible persistence of this maladaptive response until adulthood. The implications of these findings are not only relevant to women of reproductive age native to sea level countries, considering trips or work at high altitude, but also to the developmental programming of pulmonary hypertension in adulthood by prenatal hypoxia (14, 34). ACKNOWLEDGMENTS We are grateful to Carlos López, Jaime Figueroa, and Dr. Patricio Alvarez from Escuela Agrícola Francisco Napolitano, Lluta, Chile, for their collaboration on the studies and to Carlos Brito, Gabino Llusco, and Enrique Pérez for excellent technical assistance. GRANTS This work was supported by National Fund for Scientific and Technological Development (FONDECYT) Grant Nos. 1050479, 1080663, and 1090355; The Wellcome Trust Collaborative Research Initiative Grant No. 072256; ALFA programme Project No. II-0379-FCD; and Convenio de Desempeño Universidad de Tarapacá-MECESUP 2. E. Herrera is a Fellow of Beca Presidente de la República, Gobierno de Chile. D. A Giussani is a Wolfson Research Merit Award Holder of The Royal Society. DISCLOSURES No conflicts of interest are declared by the authors. REFERENCES R1683 1. Abman SH, Shanley PF, Accurso FJ. Failure of postnatal adaptation of the pulmonary circulation after chronic intrauterine pulmonary hypertension in fetal lambs. J Clin Invest 83: 1849 1858, 1989. 2. Abman SH. Recent advances in the pathogenesis and treatment of persistent pulmonary hypertension of the newborn. Neonatology 91: 283 290, 2007. 3. Abman SH. Role of endothelin receptor antagonists in the treatment of pulmonary arterial hypertension. Annu Rev Med 60: 13 23, 2009. 4. Ambalavanan N, Bulger A, Murphy-Ullrich J, Oparil S, Chen YF. Endothelin-A receptor blockade prevents and partially reverses neonatal hypoxic pulmonary vascular remodelling. Pediatr Res 57: 631 636, 2005. 5. Baumbach GL, Heistad DD. Remodeling of cerebral arterioles in chronic hypertension. Hypertension 13: 968 972, 1989. 6. Baumbach GL, Heistad DD. Adaptive changes in cerebral blood vessels during chronic hypertension. J Hypertens 9: 987 991, 1991. 7. Bolognesi M, Sacerdoti D, Piva A, Di Pascoli M, Zampieri F, Quarta S, Motterlini R, Angeli P, Merkel C, Gatta A. Carbon monoxidemediated activation of large-conductance calcium-activated potassium channels contributes to mesenteric vasodilatation in cirrhotic rats. J Pharmacol Exp Ther 321: 187 194, 2007. 8. Bolotina VM, Najibi S, Palacino JJ, Pagano PH, Cohen RA. Nitric oxide directly activates calcium-dependent potassium channels in vascular smooth muscle. Nature 368: 850 853, 1994. 9. de Meer K, Heymans HS, Zijlstra WG. Physical adaptation of children to life at high altitude. Eur J Pediatr 154: 263 272, 1995. 10. Dubuis E, Potier M, Wang R, Vandier C. Continuous inhalation of carbon monoxide attenuates hypoxic pulmonary hypertension development presumably through activation of BKCa channels. Cardiovasc Res 65: 751 761, 2005. 11. Ebensperger G, Ebensperger R, Herrera EA, Riquelme RA, Sanhueza EM, Lesage F, Marengo JJ, Tejo RI, Llanos AJ, Reyes RV. Fetal brain hypometabolism during prolonged hypoxaemia in the llama. J Physiol 567: 963 975, 2005. 12. Ebensperger G, Herrera EA, Riquelme RA, Parer JT, Llanos AJ. The fetal chronic hypoxia during partial gestation at highlands modifies the cardiovascular response to acute hypoxia in fetal sheep (Abstract). Physiol Mini Rev 2: 176, 2006. 13. Foresti R, Hammad J, Clark JE, Johnson TR, Mann BE, Friebe A, Green CJ, Motterlini R. Vasoactive properties of CORM-3, a novel water-soluble carbon monoxide-releasing molecule. Br J Pharmacol 142: 453 460, 2004. 14. Fowden AL, Giussani DA, Forhead AJ. Intrauterine programming of physiological systems: causes and consequences. Physiology (Bethesda) 21: 29 37, 2006.

R1684 15. Fulton RM, Hutchinson EC, Jones AM. Ventricular weight in cardiac hypertrophy. Br Heart J 14: 413 420, 1952. 16. Giussani DA, Phillips PS, Anstee S, Barker DJ. Effects of altitude versus economic status on birth weight and body shape at birth. Pediatr Res 49: 490 494, 2001. 17. Giussani DA, Salinas CE, Villena M, Blanco CE. The role of oxygen in prenatal growth: studies in the chick embryo. J Physiol 585: 911 917, 2007. 18. Glantz SA, Slinker BK. Primer of Applied Regression and Analysis of Variance (2nd ed.). New York, NY: McGraw-Hill, 2001, p. 418 507. 19. Grover TR, Rairigh RL, Zenge JP, Abman SH, Kinsella JP. Inhaled carbon monoxide does not cause pulmonary vasodilation in the lategestation fetal lamb. Am J Physiol Lung Cell Mol Physiol 278: L779 L784, 2000. 20. Grover TR, Parker TA, Balasubramaniam V, Markham NE, Abman SH. Pulmonary hypertension impairs alveolarization and reduces lung growth in the ovine fetus. Am J Physiol Lung Cell Mol Physiol 288: L648 L654, 2005. 21. Han H, Hansen TR, Berg B, Hess BW, Ford SP. Maternal undernutrition induces differential cardiac gene expression in pulmonary hypertensive steers at high elevation. Am J Physiol Heart Circ Physiol 295: H382 H389, 2008. 22. Hanson KA, Ziegler JW, Rybalkin SD, Miller JW, Abman SH, Clarke WR. Chronic pulmonary hypertension increases fetal lung cgmp phosphodiesterase activity. Am J Physiol Lung Cell Mol Physiol 275: L931 L941, 1998. 23. Herrera EA, Pulgar VM, Riquelme RA, Sanhueza EM, Reyes VR, Ebensperger G, Parer JT, Valdez EA, Giussani DA, Blanco CE, Hanson MA, Llanos AJ. High altitude chronic hypoxia during gestation and after birth modifies cardiovascular responses in newborn sheep. Am J Physiol Regul Integr Comp Physiol 292: R2234 R2240, 2007. 24. Herrera EA, Ebensperger G, Krause BJ, Riquelme RA, Reyes VR, Capetillo M, González S, Parer JT, Llanos AJ. Sildenafil reverses hypoxic pulmonary hypertension in lowland and highland newborn sheep. Pediatr Res 63: 169 175, 2008. 25. Herrera EA, Reyes RV, Giussani DA, Riquelme RA, Sanhueza EM, Ebensperger G, Casanello P, Méndez N, Ebensperger R, Sepúlveda- Kattan E, Pulgar VM, Cabello G, Blanco CE, Hanson MA, Parer JT, Llanos AJ. Carbon monoxide: a novel pulmonary artery vasodilator in neonatal llamas of the Andean altiplano. Cardiovasc Res 1: 197 201, 2008. 26. Ivy DD, le Cras TD, Parker TA, Zenge JP, Jakkula M, Markham NE, Kinsella JP, Abman SH. Developmental changes in endothelin expression and activity in the ovine fetal lung. Am J Physiol Lung Cell Mol Physiol 278: L785 L793, 2000. 27. Jackson EB Jr, Mukhopadhyay S, Tulis DA. Pharmacologic modulators of soluble guanylate cyclase/cyclic guanosine monophosphate in the vascular system-from bench top to bedside. Curr Vasc Pharmacol 5: 1 14, 2007. 28. Kaide J, Zhang F, Wei Y, Wang W, Gopal VR, Falck JR, Laniado- Schwartzman M, Nasjletti A. Vascular CO counterbalances the sensitizing influence of 20-HETE on agonist-induced vasoconstriction. Hypertension 44: 210 216, 2004. 29. Kelly DA, Hislop AA, Hall SM, Haworth SG. Relationship between structural remodeling and reactivity in pulmonary resistance arteries from hypertensive piglets. Pediatr Res 58: 525 530, 2005. 30. Keyes LE, Armaza JF, Niermeyer S, Vargas E, Young DA, Moore LG. Intrauterine growth restriction, preeclampsia, and intrauterine mortality at high altitude in Bolivia. Pediatr Res 54: 20 25, 2003. 31. Kourembanas S. Hypoxia and carbon monoxide in the vasculature. Antioxid Redox Signal 4: 291 299, 2002. 32. Mandarim-de-Lacerda CA, Fernandes-Santos C, Aguila MB. Image analysis and quantitative morphology. Methods Mol Biol 611: 211 225, 2010. 33. McGrath JJ, Smith DL. Response of rat coronary circulation to carbon monoxide and nitrogen hypoxia. Proc Soc Exp Biol Med 177: 132 136, 1984. 34. Moore LG, Niermeyer S, Vargas E. Does chronic mountain sickness (CMS) have perinatal origins? Respir Physiol Neurobiol 158: 180 189, 2007. 35. Mulvany MJ. Vascular remodelling in hypertension. Eur Heart J 14, Suppl C: 2 4, 1993. 36. Mulvany MJ. Small artery remodeling and significance in the development of hypertension. News Physiol Sci 17: 105 109, 2002. 37. Ndisang JF, Wang R. Alterations in heme oxygenase/carbon monoxide system in pulmonary arteries in hypertension. Exp Biol Med 228: 557 563, 2003. 38. Niermeyer S, Yang P, Shanmina D, Zhuang J, Moore LG. Arterial oxygen saturation in Tibetan and Han infants born in Lhasa, Tibet. N Engl J Med 333: 1248 1252, 1995. 39. Parraguez VH, Atlagich M, Diaz R, Bruzzone ME, Behn C, Raggi LA. Effect of hypobaric hypoxia on lamb intrauterine growth: comparison between high- and low-altitude native ewes. Reprod Fertil Dev 17: 497 505, 2005. 40. Pearce WJ, Hull AD, Long DM, Longo LD. Developmental changes in ovine cerebral artery composition and reactivity. Am J Physiol Regul Integr Comp Physiol 261: R458 R465, 1991. 41. Penaloza D, Arias-Stella J. The heart and pulmonary circulation at high altitudes: healthy highlanders and chronic mountain sickness. Circulation 115: 1132 1146, 2007. 42. Rybalkin SD, Yan C, Bornfeldt KE, Beavo JA. Cyclic GMP phosphodiesterases and regulation of smooth muscle function. Circ Res 93: 280 291, 2003. 43. Salinas CE, Blanco CE, Villena M, Camm EJ, Tuckett JD, Weerakkody RA, Kane AD, Shelley AM, Wooding FBP, Quy M, Giussani DA. Cardiac and vascular disease prior to hatching in chick embryos incubated at high altitude. J DOHaD 00: 1 7, 2009. 44. Steinhorn RH, Farrow KN. Pulmonary hypertension in the neonate. NeoReviews 8: e14 e21, 2007. 45. Stenmark KR, Fagan KA, Frid MG. Hypoxia-induced pulmonary vascular remodeling: cellular and molecular mechanisms. Circ Res 99: 675 691, 2006. 46. Sui GJ, Li YH, Cheng XS, Anand IS, Harris E, Harris P, Heath D. Subacute infantile mountain sickness. J Pathol 155: 161 170, 1988. 47. Tucker A, McMurtry IF, Reeves JT, Alexander AF, Will DH, Grover RF. Lung vascular smooth muscle as a determinant of pulmonary hypertension at high altitude. Am J Physiol 228: 762 767, 1975. 48. Vitali SH, Mitsialis SA, Christou H, Fernandez-Gonzalez A, Liu X, Kourembanas S. Mechanisms of heme oxygenase-1-mediated cardiac and pulmonary vascular protection in chronic hypoxia: roles of carbon monoxide and bilirubin. Chest 128: 578S 579S, 2005. 49. Widlitz A, Barst RJ. Pulmonary arterial hypertension in children. Eur Respir J 21: 155 176, 2003. 50. Williams SE, Wootton P, Mason HS, Bould J, Iles DE, Riccardi D, Peers C, Kemp PJ. Hemoxygenase-2 is an oxygen sensor for a calciumsensitive potassium channel. Science 306: 2093 2097, 2004. 51. Zhang F, Kaide JI, Yang L, Jiang H, Quan S, Kemp R, Gong W, Balazy M, Abraham NG, Nasjletti A. CO modulates pulmonary vascular response to acute hypoxia: relation to endothelin. Am J Physiol Heart Circ Physiol 286: H137 H344, 2004.