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.
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 Po2
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
) and in chick embryos following highland incubation (17
). 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
). 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 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.
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 PaO2
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 LHL 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
), conditions that were both observed in LHL 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
). 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
) and by Mulvany (35
) 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 LHL 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 LHL lambs.
The present study also reports an increase in right ventricular mass in LHL neonates. This is a common finding in humans and animals that have suffered arterial pulmonary hypertension (1
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 LHL 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% FiO2
) 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
) and via hyperpolarizing the vascular smooth muscle secondary to activation of BKCa
). CO can also diminish the vasoconstrictor responses to phenylephrine and 20-hydroxyeicosatetraenoic acid while reducing the synthesis and release of ET (28
). 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
). Alterations in the synthesis and function of vasoconstrictors such as ET-1, as reported in this paper, thromboxane, IGF, serotonin, and leukotriene C4
have also been implicated in the pulmonary hypertensive phenotype during chronic hypoxia (29
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 long-term exposure to high altitude. PDE is an enzyme that breaks down cGMP and thus halts the NO vasodilator cascade (42
). In this study, LHL also showed greater pulmonary protein expression of PDE5, findings similar to those reported in hypertensive lambs and lambs native to high altitude (22
). 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 BKCa
). Accordingly, in the present study, LHL lambs showed a significantly greater pulmonary BKCa
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
) 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