Deletion of the eNOS gene has a greater impact on the pulmonary circulation of male than female mice

Alyson A. Miller,1 Alison A. Hislop,1 Patrick J. Vallance,2 and Sheila G. Haworth1

1Institute of Child Health, University College London; and 2Centre for Clinical Pharmacology, The Rayne Institute, University College London, London, United Kingdom

Submitted 12 January 2005 ; accepted in final form 6 April 2005


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Nitric oxide is involved in development and postnatal adaptation of the pulmonary circulation. This study aimed to determine whether genetic deletion of nitric oxide synthase (NOS) would lead to maldevelopment of the pulmonary arteries in fetal life, compromise adaptation to extrauterine life, and be associated with a pulmonary hypertensive phenotype in adult life and if any abnormalities were detected, were they sex dependent. Morphometric analyses were made on lung tissue from male and female fetal, newborn, 14-day-old, and adult endothelial NOS-deficient (eNOS–/–) or inducible NOS-deficient (iNOS–/–) and wild-type mice. Hemodynamic studies were carried out on adult mice with deletion of either eNOS or iNOS genes. We found that in eNOS–/– mice, lung development was normal in fetal, newborn, and adult lungs. Pulmonary arterial muscularity was greater than normal in both male and female eNOS–/– during fetal life and at birth, but the abnormality persisted only in male mice. Right ventricular hypertrophy was present in 14-day-old and adult male eNOS–/– but not in female mice. Adult male eNOS–/– mice had higher mean right ventricular and systemic pressures than female eNOS–/– mice (P < 0.05). Thus deletion of the eNOS gene was associated with structural evidence of pulmonary hypertension in both sexes during fetal life, but pulmonary hypertension persisted only in the male. In neither sex did iNOS or neuronal NOS appear to compensate for the eNOS deletion. Adult iNOS–/– mice did not have structural or hemodynamic evidence of pulmonary hypertension. Possible compensatory mechanisms are discussed.

pulmonary hypertension; vasculature; development; endothelial nitric oxide synthase


IN THE NORMAL ADULT LUNG, endothelium-derived nitric oxide (NO) is thought to be important in maintaining pulmonary vasodilatation and a low pulmonary vascular resistance, and it contributes to the postnatal decrease in pulmonary vascular resistance. During fetal life, endothelial nitric oxide synthase (eNOS) protein levels are high (12) and decrease within days of birth. Activity is high immediately after birth during adaptation (2, 11). NO also influences vascular remodeling; it has angiogenic effects and can inhibit smooth muscle migration and proliferation (22). Abnormalities of the NO pathway are a feature of persistent pulmonary hypertension of the newborn (PPHN) when the pulmonary vasculature fails to adapt to extrauterine life and when postnatal structural remodeling is abnormal (1, 30). In animal models of PPHN, basal release of endothelium-derived NO is impaired (6, 10), and eNOS protein is decreased (6, 10, 15, 27).

Mice with homozygous disruptions of the eNOS gene have been used to investigate the role of NO in the pathogenesis of cardiovascular and respiratory disease in the adult. (8, 31). These studies showed that eNOS-deficient (–/–) mice have mild pulmonary hypertension in normoxic conditions but surprisingly showed little structural evidence of hypertension in either the pulmonary arteries or the heart. However, the animals have an exaggerated response to hypoxic exposure, shown by an excessive increase in pulmonary artery smooth muscle. Whether this relatively mild phenotypic abnormality is present at birth or is acquired later is unknown. Recently it has been reported that neonatal mortality is higher in eNOS–/– mice and that their lungs display major defects in airway development that lead to respiratory distress and death (13). The aim of the present study was to test the hypothesis that genetic deletion of eNOS would alter pulmonary vascular growth in fetal life and give structural abnormalities consistent with PPHN and that it would deter normal postnatal remodeling and possibly be associated with pulmonary hypertension in adult life. Because studies on the systemic circulation in adult mice have suggested sex-dependent functional differences in response to eNOS deletion (17, 35), we compared the findings in male and female mice. Having found structural differences between male and female eNOS–/– mice, we investigated whether this sex difference was specific to eNOS deficiency by studying mice with a genetic deletion of inducible (i) NOS, which, like eNOS, is expressed in fetal lung (28).


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. All experiments were conducted according to the Animals (Scientific Procedures) Act 1986, United Kingdom, and under Home Office license. Mutant mice lacking eNOS or iNOS gene and the wild-type mice from which they were derived (SV129/C57BL/6J for eNOS and C57BL/6J for iNOS) were bred in-house as pure lines. Adult animals were killed at 3 mo of age, young mice at 14 days of age, and newborns at >15 h of age. For eNOS–/– mice and the respective wild-type mice, time-dated pregnant mothers were killed at embryonic day (E) 14.5 and E17.5, the fetuses were removed from the uterus, and sex was determined.

Hemodynamic measurements. Male and female adult (~25–30 g) eNOS–/–, iNOS–/–, and the respective wild-type mice (n = 5 for each group) were anesthetized with 5% isoflurane (Abbott Laboratories) and anesthesia was then maintained with 2–3% isoflurane inhalation through a small mask. The right carotid artery was isolated, and a heparinized saline cannula (0.28 cm internal diameter; Critchley Electrical Products Ply, Auburn, New South Wales, Australia) was introduced into the artery. After a stabilization period (~5 min), systemic arterial blood pressure was measured using a MacLab (version 3.4/e) data analysis system. To measure mean right ventricular (RV) pressures, the left jugular vein was isolated and a heparinized saline cannula (0.28 cm) introduced into the vein and advanced into the right ventricle. RV pressure was recorded using the MacLab.

Tissue collection and preparation. The thorax of the fetal mice was fixed intact in formol saline for 24 h and then processed and embedded in paraffin wax. Sections (4 µm) were cut through the central part of the lungs. Newborn, 14-day-old, and adult male and female, NOS-deficient, and wild-type mice were killed by an intraperitoneal injection of pentobarbital sodium (100 mg/kg). Heart and lungs were removed en bloc. Lungs were frozen in liquid nitrogen for use in Western blots (n > 5 for each age and sex). Others were perfused through the trachea with 10% neutral buffered formol saline (20 cmH2O for 5 min), and the trachea was tied. After being immersed in formol saline for 24 h, lungs were weighed, sliced, and prepared for histological study (n > 5 for each group).

To assess ventricular hypertrophy, the atria were removed, and the total ventricular weight was determined. In the 14-day-old and adult hearts, the RV free wall was dissected from the left ventricle and intraventricular septum (LV+S), both segments were weighed, and the RV/LV+S ratio was calculated (8).

Immunohistochemistry. Tissue sections (4 µm) were stained with antibodies to smooth muscle specific {alpha}-actin isoform (Sigma, Poole, UK), eNOS (Santa Cruz c-20), or iNOS (Santa Cruz M19). Heat antigen retrieval was performed, and endogenous peroxidase activity and nonspecific antibody binding were blocked. Sections were incubated with primary antibody for 1 h at room temperature for {alpha}-actin (1:3,000) or overnight at 4°C for eNOS and iNOS (1:600 and 1:300, respectively, with 4% normal swine serum). The secondary antibody was biotin-conjugated sheep anti-mouse ({alpha}-actin, Amersham) or swine anti-rabbit (NOS antibodies, DAKO). This was followed by streptavidin biotinylated horseradish peroxidase (HRP) complex (Amersham) incubated for 30 min at room temperature and 3,3'-diaminobenzidine (DAB, Sigma). The sections were lightly counterstained with hematoxylin. In control sections, incubation with the primary antibody was omitted. For iNOS, a section of aorta from a mouse exposed to LPS was used a positive control.

Measurement of muscle in peripheral arteries. Color images from {alpha}-actin-stained slides were obtained with a digital camera (Zeiss Axiocam; Imaging Associates, Thame, UK). Analysis was made using Openlab v3.15 software (Improvision, Coventry, UK). Muscular arteries accompanying specific airway levels were studied in each age group of eNOS and iNOS–/– and wild-type animals (n ≥ 5 male and 5 female animals in each group and at least 10 arteries at each level for each animal). The total area of the artery (including the lumen, endothelium, and media) and the area of the lumen and endothelium were measured. The external diameter and lumen plus endothelium diameter were also measured. The medial wall area was calculated and expressed as percentage of the total area. Twice the medial wall thickness was calculated and expressed as a percentage of external diameter. A mean value for each airway level in each group was calculated for external diameter, percentage medial area, and percentage medial wall thickness, since there was no statistical difference between animals in the same group. Arteries at the level of alveoli (or saccules in the case of the newborn mice) were scored for the presence of {alpha}-actin staining. The percentage of nonmuscular arteries was calculated for each group of animals. This study was not done on fetal lungs.

Western blots. Frozen lung tissue from newborn, 14-day-old, and adult mice was crushed and homogenized in a 1 g/5 ml ratio in cold extraction buffer (protease inhibitor cocktail, pH 7.4; Roche, Basel, Switzerland). The homogenate was then centrifuged at 15,000 g for 30 min at 4 °C. The supernatant was removed, and protein concentration was measured using a Bradford assay. Lung homogenates were loaded as 20 µg of protein on an SDS-PAGE gel (8%) and subsequently electrophoretically transferred onto a polyvinylidene difluoride membrane. The membrane was blocked in 5% nonfat milk overnight at 4°C and incubated for 2 h at room temperature with iNOS (1:2,000, Santa Cruz, sc-650), eNOS (1:1,000, Santa Cruz sc-654), or neuronal (n) NOS (1:3,000, Santa Cruz 5302) antibody. Membranes were incubated with secondary antibody for 1 h at room temperature (donkey anti-rabbit HRP, Santa Cruz, 1:3,000 for iNOS and eNOS and goat anti-mouse HRP, DAKO, 1:4,000 for nNOS). Labeling was visualized using ECL Plus (Amersham).

Statistical analysis. All results are expressed as means ± SE. Statistical comparisons were performed by two-tailed t-tests or one-way analysis of variance with Bonferroni's multiple-comparison post hoc test. P < 0.05 was considered statistically significant.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The litter size of the dated-pregnant eNOS–/– mice and in the wild-type mice from which the eNOS–/– mice were derived was the same at E14.5 and E17.5 (~8) and was similar to the litter size at term. This suggests that there was no loss of eNOS–/– animals during late fetal life or in the perinatal period. There was no preponderance of male or female animals in –/– or wild-type litters.

RV and systemic pressure measurements in adult mice. In male adult eNOS–/– mice, the mean RV pressure was significantly greater than in female eNOS–/– mice (P < 0.02, Table 1), and the systemic arterial pressure was greater than in both female eNOS–/– mice and male wild-type mice (P < 0.01). The RV and systemic pressures in the female eNOS–/– mice were not significantly different from the pressure in the wild-type mice. Male and female iNOS–/– mice had RV and systemic arterial pressures that were similar to those in the wild-type mice from which they were derived.


View this table:
[in this window]
[in a new window]
 
Table 1. Hemodynamic measurements in adult mice

 
Body and heart weights. Body weights (BW) of male and female, wild-type, and eNOS–/– mice increased from birth to adulthood (Table 2). At 14 days of age, both male and female eNOS–/– mice had a lower BW than wild-type mice (P < 0.05, Table 2), but only male adult eNOS–/– mice were significantly smaller than wild-type mice (P < 0.01, Table 2). The fixed lung weight did not vary between male and female mice, and lung weight and total ventricular weight in both male and female eNOS–/– mice were not significantly different from values in wild-type mice at any age. In the 14-day-old and adult male eNOS–/– mice, the RV/BW and RV/LV+ S ratios were greater than in male age-matched wild-type mice (P < 0.05). The female eNOS–/– mice did not show evidence of RV hypertrophy (Table 2).


View this table:
[in this window]
[in a new window]
 
Table 2. Weight of body, lung, and heart in NOS–/– and wild-type mice

 
In male adult iNOS–/– mice the BW and total ventricular weight were greater than in wild-type mice (P < 0.05), but the RV/BW and RV/LV+S ratios were normal (Table 2). Female iNOS–/– mice were not significantly different from wild-type animals.

Structure of the lungs in eNOS–/– and wild-type mice. The lungs of the fetal, newborn, 14-day-old, and adult eNOS–/– and iNOS–/– mice were not different in gross and microscopic appearance from those of age-matched wild-type mice. The lungs from both eNOS–/– and wild-type mice at E14.5 were in the glandular phase of development. Lung buds lined with columnar epithelium were surrounded by mesenchyme (Fig. 1). The lungs at E17.5 in –/– and wild-type animals were in the canalicular phase of development. Proximal airways were lined with columnar epithelium and peripheral airways with cuboidal epithelium. Peripheral airways had a larger airway lumen than proximal airways, and less mesenchyme surrounded the airways. The first peripheral airway generation of this type was called a terminal bronchiolus in these lungs. The lungs at birth had airways lined by columnar epithelium near the hilum, then cuboidal epithelium, and the epithelium became flattened in the peripheral airways. Terminal bronchioli were the last airway to have a complete cuboidal epithelium. Prospective respiratory bronchioli had part of the wall flattened and part cuboidal. These bronchioli led into and were surrounded by large thin-walled saccules, the prospective alveolar ducts (Fig. 1). There were no cup-shaped alveoli at this time. The appearance was the same in both eNOS–/–, iNOS–/–, and wild-type mice. Fourteen days after birth, alveoli were present, having developed from the saccules, and all had a thin wall with a single capillary network. The appearance was similar to the adult lung (Fig. 1). Pulmonary arteries were found alongside the airways. For the purpose of measuring the amount of muscle in the pulmonary arteries, we identified the arteries by the airway that they accompanied, that is from hilum to periphery, bronchioli, terminal bronchioli, respiratory bronchioli (airways with alveolar epithelium in part of their wall), and alveolar ducts (prospective alveolar ducts in the newborn group) (Fig. 2, A and B). Pulmonary veins could be identified by having a thin wall relative to lumen diameter and by their position, usually being located between rather than alongside the airways.



View larger version (82K):
[in this window]
[in a new window]
 
Fig. 1. Photomicrographs illustrating lung development in endothelial nitric oxide synthase (eNOS) wild-type and eNOS-deficient (–/–) mice. Embryonic day (E) 14.5 mice are in the glandular phase of development, E17.5 in the canalicular phase, newborn mice have saccules, and by 14 days alveoli are present. In the adult the alveoli are similar to those at 14 days. All sections are immunostained with {alpha}-actin and at the same magnification. Bar = 50 µm.

 


View larger version (153K):
[in this window]
[in a new window]
 
Fig. 2. Photomicrographs of lungs from 14-day-old eNOS–/– animals immunostained with smooth muscle {alpha}-actin. A and B: illustration of the peripheral airway structure with terminal bronchiolus (TB), respiratory bronchiolus (RB), and alveolar duct (AD) with accompanying pulmonary artery. C and D: peripheral region from male and female mice, fewer arteries stain for smooth muscle in the female mice. Bar = 50 µm.

 
External diameter and percentage medial area of pulmonary arteries. For wild-type animals the mean size of the arteries accompanying each type of airway decreased toward the periphery, and arteries increased in diameter with age (Table 3). There was no difference in external diameter between male and female animals at any age in either eNOS wild-type or –/–. The arteries were significantly larger in eNOS–/– animals than in the wild-type at 14 days but not in the other age groups (Table 3).


View this table:
[in this window]
[in a new window]
 
Table 3. External diameter of arteries accompanying peripheral airways in eNOS–/– and wt mice

 
In the fetal lungs the percentage medial area in arteries accompanying bronchioli was high, ranging from 45 to 58% and was significantly greater in eNOS–/– lungs than wild type (P < 0.01). There was no difference between E14.5 and E17.5 arteries, and no consistent difference with sex (Fig. 3). Alongside the terminal bronchioli, which were only identified at E17.5, the pulmonary arteries were small with a complete or incomplete muscle layer. The percentage medial area was greater in eNOS–/– animals than in the wild type for both male and female animals (Fig. 3).



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 3. Percentage medial area of arteries from E14.5 and E17.5 eNOS–/– and wild-type (wt) mice accompanying bronchioli (br) and terminal bronchioli (TB). *P < 0.01 compared with age- and sex-matched wt animals.

 
In the newborn animals aged <15 h, the percentage medial area at the level of the terminal bronchioli was less than in the E17.5 animals in arteries from wild-type and eNOS–/– mice (30.6–44.5%). The reduction in percentage medial area continued, and by 14 days muscularity at terminal bronchiolar (15.3–29.1%), respiratory bronchiolar (21.4–28.9%), and alveolar duct (18.7–28.1%) level was significantly less than in the newborns in both male and female eNOS–/– and wild-type animals (P < 0.01 for all). Muscularity decreased further between 14 days and adulthood in all arteries in the wild-type mice (terminal bronchiolar, 20.9–22.3%; respiratory bronchiolar, 18.7–19.5%; alveolar duct, 10.7–13%) and in those accompanying alveolar ducts in the –/– mice (10.6–22.9%, P < 0.01, Fig. 4).



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 4. Percentage medial area of arteries accompanying terminal bronchioli (TB), respiratory bronchioli (RB), and alveolar ducts (AD) in eNOS–/– and wt newborn, 14-day-old, and adult male and female mice. *P < 0.05 compared with age- and sex-matched wt animals.

 
In the wild-type mice at all ages and at all airway levels, the percentage medial area was similar in male and female animals. In the newborn, 14-day-old, and adult male eNOS–/– mice, the percentage medial area was significantly greater than in age-matched wild-type mice at all three airway levels (P < 0.05 for all) (Fig. 4). In female –/– mice, at birth the percentage medial area was greater than in wild-type mice in arteries accompanying terminal and respiratory bronchioli (P < 0.05) but not in the smallest arteries accompanying alveolar ducts. Here the percentage medial area in the female was significantly less than in the male eNOS–/– (P < 0.01). By 14 days all the values in female eNOS–/– mice were similar to those in the wild-type mice and remained normal in adults. The female eNOS–/– mice showed a 50% decrease in muscularity between birth and 14 days, which brought their values similar to normal for age. By contrast, the male eNOS–/– mice showed only a 33% reduction, as in the wild-type mice, leaving the eNOS–/– males with a higher percentage medial area than normal.

Results for percentage wall thickness showed a similar pattern of change with age and sex in eNOS–/– and wild-type animals as described for the percentage medial area (results not shown).

Muscle in the alveolar region. In the alveolar region of the newborn and adult male and female eNOS wild-type animals, the majority of arteries were nonmuscular (Fig. 2C). At 14 days ~30% were nonmuscular (Table 4). By contrast, in male eNOS–/– mice few arteries were nonmuscular (Fig. 2D) at any age (P < 0.01 for all age groups, Table 4), although the proportion did increase with age (P < 0.05, newborn to adult). In the female eNOS–/– mice the findings were similar to those in wild-type mice at all ages.


View this table:
[in this window]
[in a new window]
 
Table 4. Percentage of nonmuscular arteries in the alveolar region of eNOS–/– and wt mice

 
In the adult iNOS mice no difference in percentage medial wall area was found between male and female or between the –/– and wild-type mice (Fig. 5). The percentage medial wall thickness and the percentage of nonmuscular vessels in the alveolar region were also not different between wild-type and –/– mice (results not shown). No increase in smooth muscle was demonstrated in newborn or 14-day-old iNOS–/– mice compared with wild type (results not shown).



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 5. Percentage medial area of arteries accompanying TB, RB, and AD in inducible (i) NOS–/– and wt adult male and female mice.

 
Western blots for eNOS, iNOS, and nNOS. Immunoreactive eNOS and iNOS protein was detected in lung homogenates from all wild-type mice, and the amount was not influenced by age or sex. Figure 6 illustrates representative blots from three separate blots from different adult animals. No eNOS was detected in homogenates from any eNOS–/– mice, and no iNOS in the iNOS–/– mice (Fig. 6). eNOS was detected at all ages in the lung tissue of iNOS–/– mice, did not vary with age or sex, and was similar in amount to that seen in wild-type animals (Fig. 6). iNOS was detected in eNOS–/– mice and was similar in amount to that in wild-type mice and was not different in male and female animals or with age (Fig. 6). A small amount of nNOS was found in all animals studied, wild-type and eNOS and iNOS–/– mice. There was no consistent difference between male and female animals.



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 6. Representative Western blot of adult mouse lungs with eNOS and iNOS antibody. For each antibody all lanes are from the same gel. e-wtM/F, eNOS wild-type male/female; e-koM/F, eNOS–/– male/female; i-wtM/F, iNOS wild-type male/female; i-koM/F, iNOS–/– male/female. lps, mouse lung treated with lipopolysaccharide as a positive control. Std, rainbow standard.

 
Immunohistochemistry. Positive eNOS immunostaining was found on the pulmonary artery endothelial cells of male and female wild-type animals of both strains and in both male and female iNOS–/– animals. It was not found in eNOS–/– animals, confirming the Western blot (Fig. 7). Immunostaining for iNOS was not present on either the endothelial cells or smooth muscle cells of the pulmonary arteries in wild-type or eNOS or iNOS–/– mice (Fig. 7).



View larger version (124K):
[in this window]
[in a new window]
 
Fig. 7. Photomicrographs of mouse pulmonary arteries after immunostaining with eNOS or iNOS. No eNOS is seen on the endothelium of pulmonary arteries in eNOS–/– (KO) mice. It is clearly seen in wt and iNOS KO mice. iNOS reactivity was not seen in pulmonary arteries of any of the mice studied. Positive iNOS stain was found in smooth muscle cells of the aorta from an LPS-treated mouse. Bar = 25 µm.

 

    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The present study demonstrates that genetic deletion of eNOS results in increased pulmonary artery smooth muscle in both male and female mice during fetal life. Within the first 15 h after birth there had already been a decrease in medial muscle in all eNOS–/– animals, and there was a further decrease by 14 days of age. Thus all the eNOS–/– mice showed structural evidence of adaptation to extrauterine life, and all survived. The early postnatal reduction in muscularity was greater in females than males, and by 14 days of age pulmonary arterial muscle was excessive in the male animals and normal in female animals. This increase in pulmonary artery smooth muscle in males was associated with RV hypertrophy, indicating pulmonary hypertension. These structural sex differences persisted into adulthood when RV pressure was significantly greater in male than in female eNOS–/– mice. These results suggest that eNOS-derived NO plays an important role in intrauterine development but that it is not essential for survival. The striking finding of this study was that in female mice, compensation for the lack of eNOS started very soon after birth. Even in animals <15 h old, the most peripheral arteries had significantly less muscle in female than in male animals. In male eNOS–/– and all wild-type mice there was a 33% decrease in percentage of arterial medial area between birth and 14 days of age, but the female eNOS–/– mice showed a 50% decrease in muscularity, achieving a normal percentage of arterial medial area.

By contrast, we found no evidence of any increase in pulmonary arterial muscle in the iNOS–/– animals, male or female, at any age, and deletion of iNOS did not influence the pressure in either the pulmonary or systemic circulation in either sex in our adult mice. Previous reports on iNOS–/– mice showed a normal systemic pressure (14) with a modest increase in RV systolic pressure (9). Thus iNOS was not essential for the normal development of the pulmonary arteries.

In our eNOS–/– mice we did not see evidence of abnormal airway or alveolar development in the lungs nor an increase in fetal or neonatal mortality, as has recently been reported (13). This is an important observation since it would appear that mice from different colonies with the same genotype do not necessarily show the same phenotype. Others have noted structural differences in alveolar size in inbred mouse strains (29). Our animals were from a pure breeding line, and we demonstrated by Western blot and immunohistochemistry that there was indeed no eNOS present in the lungs of the eNOS–/– mice. Thus, although pulmonary arterial muscle was increased, it would seem that eNOS was not essential for normal lung morphogenesis in the strain of mice we used.

Previous reports on adult eNOS–/– mice concluded that the animals had mild pulmonary hypertension in a normoxic environment (8, 32). The RV pressure was elevated, but there was no echocardiographic or morphological evidence of pulmonary hypertension. These studies did not, however, separate the sexes, and therefore the structural changes that we report in adult males may have been masked. They also found that adult eNOS–/– mice showed an exaggerated pulmonary hypertensive response to chronic hypoxia, an observation that is in keeping with our own study in which pulmonary arterial muscle was increased in utero. Early life events can compromise the response to stress in later life in normal animals. Newborn rats that were exposed to hypoxia developed pulmonary hypertension and allowed to recover showed an enhanced response to hypoxic stress in later life (34). Augmented pulmonary vasoreactivity has also been described in human adults who had perinatal pulmonary hypertension (26). Exposure to mild hypoxia in the neonatal period led to a failure of capillary and alveolar growth in eNOS–/– mice that was not seen in normal mice, suggesting that the eNOS–/– mice are more susceptible to hypoxic stress (5).

The increase in pulmonary arterial smooth muscle during fetal life in eNOS–/– mice may have been caused by reduced inhibition of smooth muscle cell multiplication. An in vitro study on neonatal porcine pulmonary artery smooth muscle cells demonstrated that proliferation could be inhibited by NO donors and that proliferation rate was greatest at between 5 and 10% oxygen (fetal levels) (3). Thus eNOS may be an important determinant of fetal pulmonary artery muscle area.

Compensation for eNOS deficiency was sex specific throughout postnatal life but not during fetal life when both male and female animals were equally severely affected. The compensatory mechanism appeared in the females immediately after birth, since even at <15 h of age females had significantly less pulmonary arterial smooth muscle than males in the smallest arteries. We considered the possibility that a non-eNOS-derived NO might help compensate for eNOS deficiency, but on the Western blots we found no consistent evidence of an increase of either iNOS or nNOS in the eNOS–/– animals, female or male at any age. Nor did there appear to be any difference in the amount of either iNOS or nNOS between females and males in mice deficient in eNOS or in wild-type mice. Others have reported an increase in iNOS but not nNOS in adult lungs from eNOS–/– mice from a different colony. No separation by sex was described (7). iNOS immunoreactivity was not seen on the blood vessels of any eNOS or iNOS–/– or wild-type animals in our study.

To identify and characterize the mechanism(s) that compensate for the eNOS deficiency in both sexes and to explain the sex-related difference in outcome is a complex challenge. Work on the systemic circulation of eNOS–/– mice has implicated sex-related differences in vasodilator prostaglandin release, activation of large-conductance calcium-activated potassium channels, and endothelium-derived hyperpolarizing factor (EDHF) (16, 18, 35). EDHF may itself show estrogen dependence (24). Pharmacological studies showed that nNOS-derived NO, together with enhanced prostaglandin synthesis, maintained flow-induced dilatation in the coronary arteries of adult male eNOS–/– mice (18). In female eNOS–/– mice, metabolites of P-450 mediated flow-induced dilatation in skeletal muscle via activation of large-conductance Ca2+-activated potassium channels, a finding that indicated substitution of EDHF for NO (16). The skeletal muscle of male eNOS–/– mice showed enhanced release of endothelial dilator prostaglandins (33). The same group (35) found that in rats treated with N{omega}-nitro-L-arginine methyl ester for 4 wk, flow-mediated dilatation of gracilis muscle arterioles was considerably greater in females than in males. Upregulation of a metabolite of cytochrome P-450 was responsible for the vasodilator response in the females, whereas enhanced release of endothelial prostaglandins accounted for the response in the males. Similar mechanisms may account for the sex differences in the pulmonary circulation that we report here. It is known that at birth there is an abrupt increase in EDHF activity as pulmonary blood flow increases (20, 23). If females have greater reliance on EDHF than males in normal mice this might explain why they were more protected from the absence of eNOS. EDHF activity is influenced by estrogen in systemic vessels (21), and estradiol improves vascular remodeling in perinatal pulmonary hypertension in lambs (25). All these intriguing possibilities for mechanisms, together with any sex differences in receptor density for agonists known to stimulate NO release, will require detailed pharmacological and molecular study in the future.

In regard to the relevance of sex-specific differences in the eNOS pathway, our observations on the better outcome in female eNOS–/– mice appear to run contrary to clinical experience in that the incidence of primary pulmonary hypertension is considerably higher in women than men (4). There is, however, evidence of early male attrition in utero in humans and of a higher postnatal morbidity and mortality in males (19).

In conclusion, this study shows that absence of eNOS can compromise normal arterial growth and adaptation to postnatal life and that such compromise can lead to sustained pulmonary hypertension in adulthood. Male mice failed to adapt, but female mice did so successfully, and understanding why they were able to do so offers the potential to explore alternative treatment strategies in babies with PPHN. This work emphasizes that future studies on intact animals or even on derived cells should consider the possibility of sex-related differences.


    GRANTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by the British Heart Foundation.


    ACKNOWLEDGMENTS
 
The authors thank Ray Stidwill and the Medical Research Council Sepsis Cooperative for assistance with the hemodynamic measurements and Elaine Philips for technical assistance with the immunohistochemical studies.

Present address of A. A. Miller: Howard Florey Institute, Melbourne, Australia.


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. A. Hislop, Inst. of Child Health, University College London, 30 Guilford St., London WC1N 1EH, UK (e-mail: a.hislop{at}ich.ucl.ac.uk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Abman SH. Abnormal vasoreactivity in the pathophysiology of persistent pulmonary hypertension of the newborn. Pediatr Rev 20: e103–e109, 1999.
  2. Abman SH, Chatfield BA, Hall SL, and McMurtry IF. Role of endothelium-derived relaxing factor during transition of pulmonary circulation at birth. Am J Physiol Heart Circ Physiol 259: H1921–H1927, 1990.[Abstract/Free Full Text]
  3. Ambalavanan N, Mariani G, Bulger A, and Philips JB III. Role of nitric oxide in regulating neonatal porcine pulmonary artery smooth muscle cell proliferation. Biol Neonate 76: 291–300, 1999.[CrossRef][ISI][Medline]
  4. Archer S and Rich S. Primary pulmonary hypertension: a vascular biology and translational research "Work in progress". Circulation 102: 2781–2791, 2000.[Abstract/Free Full Text]
  5. Balasubramaniam V, Tang JR, Maxey A, Plopper CG, and Abman SH. Mild hypoxia impairs alveolarization in the endothelial nitric oxide synthase-deficient mouse. Am J Physiol Lung Cell Mol Physiol 284: L964–L971, 2003.[Abstract/Free Full Text]
  6. Berkenbosch JW, Baribeau J, and Perreault T. Decreased synthesis and vasodilation to nitric oxide in piglets with hypoxia-induced pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 278: L276–L283, 2000.[Abstract/Free Full Text]
  7. Cook S, Vollenweider P, Menard B, Egli M, Nicod P, and Scherrer U. Increased eNO and pulmonary iNOS expression in eNOS null mice. Eur Respir J 21: 770–773, 2003.[Abstract/Free Full Text]
  8. Fagan KA, Fouty BW, Tyler RC, Morris KG Jr, Hepler LK, Sato K, LeCras TD, Abman SH, Weinberger HD, Huang PL, McMurtry IF and Rodman DM. The pulmonary circulation of homozygous or heterozygous eNOS-null mice is hyperresponsive to mild hypoxia. J Clin Invest 103: 291–299, 1999.[Abstract/Free Full Text]
  9. Fagan KA, Tyler RC, Sato K, Fouty BW, Morris KG Jr, Huang PL, McMurtry IF, and Rodman DM. Relative contributions of endothelial, inducible, and neuronal NOS to tone in the murine pulmonary circulation. Am J Physiol Lung Cell Mol Physiol 277: L472–L478, 1999.[Abstract/Free Full Text]
  10. Fike CD, Kaplowitz MR, Thomas CJ, and Nelin LD. Chronic hypoxia decreases nitric oxide production and endothelial nitric oxide synthase in newborn pig lungs. Am J Physiol Lung Cell Mol Physiol 274: L517–L526, 1998.[Abstract/Free Full Text]
  11. Fineman JR, Wong J, Morin FC III, Wild LM, and Soifer SJ. Chronic nitric oxide inhibition in utero produces persistent pulmonary hypertension in newborn lambs. J Clin Invest 93: 2675–2683, 1994.[ISI][Medline]
  12. Halbower AC, Tuder RM, Franklin WA, Pollock JS, Forstermann U, and Abman SH. Maturation-related changes in endothelial nitric oxide synthase immunolocalization in developing ovine lung. Am J Physiol Lung Cell Mol Physiol 267: L585–L591, 1994.[Abstract/Free Full Text]
  13. Han RN, Babaei S, Robb M, Lee T, Ridsdale R, Ackerley C, Post M, and Stewart DJ. Defective lung vascular development and fatal respiratory distress in endothelial NO synthase-deficient mice: a model of alveolar capillary dysplasia? Circ Res 94: 1115–1123, 2004.[Abstract/Free Full Text]
  14. Hickey MJ, Sharkey KA, Sihota EG, Reinhardt PH, Macmicking JD, Nathan C, and Kubes P. Inducible nitric oxide synthase-deficient mice have enhanced leukocyte-endothelium interactions in endotoxemia. FASEB J 11: 955–964, 1997.[Abstract/Free Full Text]
  15. Hislop AA, Springall DR, Oliveira H, Pollock JS, Polak JM, and Haworth SG. Endothelial nitric oxide synthase in hypoxic newborn porcine pulmonary vessels. Arch Dis Child Fetal Neonatal Ed 77: F16–F22, 1997.[Abstract/Free Full Text]
  16. Huang A, Sun D, Carroll MA, Jiang H, Smith CJ, Connetta JA, Falck JR, Shesely EG, Koller A, and Kaley G. EDHF mediates flow-induced dilation in skeletal muscle arterioles of female eNOS-KO mice. Am J Physiol Heart Circ Physiol 280: H2462–H2469, 2001.[Abstract/Free Full Text]
  17. Huang A, Sun D, Koller A, and Kaley G. Gender difference in flow-induced dilation and regulation of shear stress: role of estrogen and nitric oxide. Am J Physiol Regul Integr Comp Physiol 275: R1571–R1577, 1998.[Abstract/Free Full Text]
  18. Huang A, Sun D, Shesely EG, Levee EM, Koller A, and Kaley G. Neuronal NOS-dependent dilation to flow in coronary arteries of male eNOS-KO mice. Am J Physiol Heart Circ Physiol 282: H429–H436, 2002.[Abstract/Free Full Text]
  19. Ingemarsson I. Gender aspects of preterm birth. BJOG 110, Suppl 20: 34–38, 2003.[ISI][Medline]
  20. Levy M, Souil E, Sabry S, Favatier F, Vaugelade P, Mercier JC, Dall'Ava-Santucci J, and Dinh-Xuan AT. Maturational changes of endothelial vasoactive factors and pulmonary vascular tone at birth. Eur Respir J 15: 158–165, 2000.[Abstract/Free Full Text]
  21. Liu MY, Hattori Y, Sato A, Ichikawa R, Zhang XH, and Sakuma I. Ovariectomy attenuates hyperpolarization and relaxation mediated by endothelium-derived hyperpolarizing factor in female rat mesenteric artery: a concomitant decrease in connexin-43 expression. J Cardiovasc Pharmacol 40: 938–948, 2002.[CrossRef][ISI][Medline]
  22. Lloyd-Jones DM and Bloch KD. The vascular biology of nitric oxide and its role in atherogenesis. Annu Rev Med 47: 365–375, 1996.[CrossRef][ISI][Medline]
  23. Pak KJ, Geary GG, Duckles SP, and Krause DN. Male-female differences in the relative contribution of endothelial vasodilators released by rat tail artery. Life Sci 71: 1633–1642, 2002.[CrossRef][ISI][Medline]
  24. Parker TA, Afshar S, Kinsella JP, Grover TR, Gebb S, Geraci M, Shaul PW, Cryer CM, and Abman SH. Effects of chronic estrogen-receptor blockade on ovine perinatal pulmonary circulation. Am J Physiol Heart Circ Physiol 281: H1005–H1014, 2001.[Abstract/Free Full Text]
  25. Parker TA, Ivy DD, Galan HL, Grover TR, Kinsella JP, and Abman SH. Estradiol improves pulmonary hemodynamics and vascular remodeling in perinatal pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 278: L374–L381, 2000.[Abstract/Free Full Text]
  26. Sartori C, Allemann Y, Trueb L, Delabays A, Nicod P, and Scherrer U. Augmented vasoreactivity in adult life associated with perinatal vascular insult. Lancet 353: 2205–2207, 1999.[CrossRef][ISI][Medline]
  27. Shaul PW, Yuhanna IS, German Z, Chen Z, Steinhorn RH, and Morin FC III. Pulmonary endothelial NO synthase gene expression is decreased in fetal lambs with pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 272: L1005–L1012, 1997.[Abstract/Free Full Text]
  28. Sherman TS, Chen Z, Yuhanna IS, Lau KS, Margraf LR, and Shaul PW. Nitric oxide synthase isoform expression in the developing lung epithelium. Am J Physiol Lung Cell Mol Physiol 276: L383–L390, 1999.[Abstract/Free Full Text]
  29. Soutiere SE, Tankersley CG, and Mitzner W. Differences in alveolar size in inbred mouse strains. Respir Physiol Neurobiol 140: 283–291, 2004.[CrossRef][ISI]
  30. Spitzer AR, Davis J, Clarke WT, Bernbaum J, and Fox WW. Pulmonary hypertension and persistent fetal circulation in the newborn. Clin Perinatol 15: 389–413, 1988.[ISI][Medline]
  31. Steudel W, Ichinose F, Huang PL, Hurford WE, Jones RC, Bevan JA, Fishman MC, and Zapol WM. Pulmonary vasoconstriction and hypertension in mice with targeted disruption of the endothelial nitric oxide synthase (NOS 3) gene. Circ Res 81: 34–41, 1997.[Abstract/Free Full Text]
  32. Steudel W, Scherrer-Crosbie M, Bloch KD, Weimann J, Huang PL, Jones RC, Picard MH, and Zapol WM. Sustained pulmonary hypertension and right ventricular hypertrophy after chronic hypoxia in mice with congenital deficiency of nitric oxide synthase 3. J Clin Invest 101: 2468–2477, 1998.[Abstract/Free Full Text]
  33. Sun D, Huang A, Smith CJ, Stackpole CJ, Connetta JA, Shesely EG, Koller A, and Kaley G. Enhanced release of prostaglandins contributes to flow-induced arteriolar dilation in eNOS knockout mice. Circ Res 85: 288–293, 1999.[Abstract/Free Full Text]
  34. Tang JR, Le Cras TD, Morris KG Jr, and Abman SH. Brief perinatal hypoxia increases severity of pulmonary hypertension after reexposure to hypoxia in infant rats. Am J Physiol Lung Cell Mol Physiol 278: L356–L364, 2000.[Abstract/Free Full Text]
  35. Wu Y, Huang A, Sun D, Falck JR, Koller A, and Kaley G. Gender-specific compensation for the lack of NO in the mediation of flow-induced arteriolar dilation. Am J Physiol Heart Circ Physiol 280: H2456–H2461, 2001.[Abstract/Free Full Text]