Early abnormalities of pulmonary vascular development in the Fawn-Hooded rat raised at Denver's altitude

Timothy D. Le Cras1, Dug-Ha Kim2, Neil E. Markham1, and And Steven H. Abman1

1 Pediatric Heart Lung Center, Department of Pediatrics, University of Colorado School of Medicine and The Children's Hospital, Denver, Colorado 80262; and 2 Department of Pediatrics, Hallym University, 150-030 Seoul, South Korea


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The Fawn-Hooded rat (FHR) is a genetic strain that has been extensively studied as a model of primary pulmonary hypertension in adult rats. Based on our recent observations that alveolar number and pulmonary arterial density are reduced in FHRs raised at Denver's altitude, we hypothesized that early abnormalities in pulmonary vascular development contribute to the progression of pulmonary hypertension in the FHR. We found that endothelial nitric oxide synthase (eNOS) protein content was lower in the lungs of fetal, 1- and 7-day-old, 3-week-old, and adult FHRs compared with that in the normal Sprague-Dawley (SDR) and Fischer rat strains, all raised at Denver's altitude. In contrast, lung expression of the endothelial proteins kinase insert domain-containing receptor/fetal liver kinase-1 (KDR/Flk-1) and platelet endothelial cell adhesion molecule-1 (CD31) was not different between strains. Barium arteriograms showed that pulmonary arterial density was reduced in 3-week-old FHRs compared with SDRs. Perinatal treatment of FHRs with mild hyperbaria to simulate sea-level alveolar PO2 improved lung eNOS content and pulmonary vascular growth and reduced right ventricular hypertrophy. We conclude that the development of pulmonary hypertension in Denver-raised FHRs is characterized by reductions in lung eNOS expression and abnormal pulmonary vascular growth during the fetal, neonatal, and postnatal periods.

alveolarization; lung development; lung hypoplasia; endothelial nitric oxide synthase


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

PRIMARY PULMONARY HYPERTENSION (PPH) is a severe disease of high morbidity and mortality that often presents in young adults without apparent risk factors (8). Despite extensive clinical and pathological studies, the pathogenesis of PPH remains poorly understood. Multiple experimental animal models have been developed to study pulmonary hypertension, including chronic hypoxia (21), monocrotaline treatment (18), and hemodynamic stress (19). In these models, exposure to these adverse stimuli causes pulmonary hypertension with altered pulmonary vascular tone and structure (1). Because genetic factors are also likely to contribute to PPH, several investigators (26, 29, 30) have studied the pulmonary circulation of the Fawn-Hooded rat (FHR) to better understand the mechanisms that increase susceptibility to pulmonary hypertension. The FHR is a genetic strain in which adults develop severe pulmonary hypertension (11), especially after exposure to mild decreases in alveolar PO2 (26).

Although the primary genetic abnormality in the FHR is unknown, several mechanisms may contribute to the development of pulmonary hypertension in this strain. These include abnormal vasoreactivity and vascular smooth muscle growth due to altered serotonin metabolism and increased lung endothelin-1 (ET-1) (2, 30). A recent study (33) has also demonstrated that endothelial nitric oxide (NO) synthase (eNOS), a major source of the potent vasodilator and antiproliferative product NO, is downregulated in the adult FHR. This is in contrast to other models of pulmonary hypertension in adult rats, including chronic hypoxic pulmonary hypertension in which lung eNOS expression is upregulated (15, 28). Mechanisms that reduce lung eNOS in the adult FHR are unknown, but these findings suggest that altered lung eNOS regulation and expression may be unique in the FHR compared with other adult models of pulmonary hypertension. However, the timing of reduced lung eNOS expression in the FHR and whether early reductions contribute to the development of pulmonary hypertension in the FHR are unknown.

Recently, Le Cras et al. (16) reported that the adult lung in Denver-raised FHRs is characterized by fewer and larger alveoli and reduced pulmonary arterial density. They observed decreased lung growth in the FHR during the perinatal period. Perinatal treatment of FHRs with mild hyperbaria to simulate sea-level alveolar PO2 reduced the severity of lung hypoplasia and right ventricular hypertrophy (RVH) (16). These findings led to our speculation that the high risk for pulmonary hypertension in adult FHRs at Denver's altitude may be due to early abnormalities of lung maturation and pulmonary vascular development. Therefore, we hypothesized that early abnormalities in pulmonary vascular growth and function increase the risk for pulmonary hypertension in Denver-raised FHRs. To examine this hypothesis, we asked five questions. 1) Is lung eNOS protein expression reduced in the FHR compared with normal rat strains raised at Denver's altitude? 2) Is reduction of eNOS protein evident in the systemic circulation? 3) What is the maturational timing of the altered pattern of lung eNOS expression, and do other endothelial cell proteins such as kinase insert domain-containing receptor/fetal liver kinase-1 (KDR/Flk-1) and platelet endothelial cell adhesion molecule-1 (PECAM; CD31) show similar patterns of expression? 4) Does the FHR have early abnormalities in pulmonary vascular growth? 5) Does perinatal treatment with mild hyperbaria to simulate sea-level alveolar PO2 alter lung eNOS expression and pulmonary vascular growth?


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. All procedures and protocols were approved by the Animal Care and Use Committee at the University of Colorado Health Sciences Center (Denver, CO). FHRs were a gift from Dr. T. Stelzner (Department of Medicine, University of Colorado School of Medicine, Denver, CO), and a breeding colony was established. Sprague-Dawley (SDRs) and Fischer (FSRs) rats (Harlan Laboratories, Indianapolis, IN) were used as the normal rat strains for these studies. Pregnant SDRs and FSRs were purchased and maintained at Denver's altitude for at least 1 wk before giving birth. Animals were fed ad libitum and exposed to a 12:12-h light-dark cycle. Fetal rat lung tissue was obtained at 20 days of gestation (term = 21 days) after euthanasia of pregnant females by lethal intraperitoneal injection of pentobarbital sodium (1.25 mg/g). Neonatal, infant, and adult rats were also euthanized by lethal injection of pentobarbital sodium, and body weights were measured.

Study design. Several experimental protocols were used in this study. 1) To determine whether lung eNOS protein expression is reduced in adult FHRs in comparison with normal rat strains, Western blot analysis was performed on lung homogenates from 10-wk-old FHRs, SDRs, and FSRs (n = 6 animals). 2) To determine whether eNOS expression is reduced in the systemic circulation, Western blot analysis was performed on homogenates of aortas obtained from adult FHRs and SDRs (n = 6). 3) To characterize the maturational timing of the altered pattern of lung eNOS expression in the FHRs compared with that in SDRs and FSRs, Western blot analysis was performed on lung tissue from late-gestation fetuses, 1- and 7-day-old neonates, and 3-wk-old infants as described in Western blot analysis for eNOS, KDR/Flk-1, and PECAM (n = 6 animals). Lung eNOS expression was localized by immunohistochemistry to determine whether the pattern of eNOS expression differed between the rat strains (n = 4 animals). To determine whether other endothelial cell proteins such as KDR/Flk-1 and PECAM show similar patterns of expression in the FHR lung, Western blot analysis was performed for KDR/Flk-1 and PECAM on lung tissue from the same 3-wk-old FHRs and SDRs (n = 6). 4) To determine whether pulmonary vascular growth is disturbed in infant FHRs, barium arteriograms and vessel density counts were performed on 3-wk-old FHRs and SDRs (n = 5). 5) To determine whether perinatal treatment with mild hyperbaria to simulate sea-level alveolar PO2 improves pulmonary vascular growth and lung eNOS expression, barium arteriograms, pulmonary arterial density counts, and Western blot analysis for eNOS were performed on FHRs treated with mild hyperbaria, and the results were compared with similar measurements taken from Denver-raised FHRs and SDRs (n = 4-6). Body weights, weight of right ventricle (RV) and left ventricle plus septum (LV+S), lung histology, and radial alveolar counts were obtained on 3-wk-old FHRs (Denver raised and hyperbaria treated) and Denver-raised SDRs (n = 5-12). In addition, lung eNOS content, body weights, and RV and LV+S weights were compared between SDRs raised at Denver's altitude and SDRs treated with perinatal hyperbaria to determine whether, in normal rats, Denver's altitude would alter lung eNOS content and increase the risk for pulmonary hypertension (n = 7 animals).

Mild hyperbaria treatment. Rats were placed in a hyperbaric chamber in which the pressure was increased to 18.5-19 kPa to simulate sea-level alveolar PO2. The air in the chamber changed 42 times every hour. Carbon dioxide and ammonia buildup were prevented by absorbent systems present in the chamber. A 12:12-h light-dark cycle was provided and controlled by timers. Food and water were supplied ad libitum. Pregnant FHRs and SDRs were exposed to mild hyperbaria to simulate sea-level conditions for 1 wk before and 3 wk after giving birth. Rats were briefly exposed to Denver's altitude for <10 min twice a week while the cages were changed and fresh water and food were supplied.

Western blot analysis for eNOS, KDR/Flk-1, and PECAM. Western blot analysis was performed with the following primary antibodies: a monoclonal antibody to eNOS (Transduction Laboratories, Lexington, KY) diluted 1:500, a rabbit polyclonal antibody to KDR/Flk-1 (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:200, and a goat polyclonal antibody to PECAM (Santa Cruz) diluted 1:200, as previously published (15). Lung homogenates (25 µg) were separated by SDS-PAGE and then transferred to nitrocellulose membranes by electroblotting. Blots were blocked overnight at 4°C in 5% nonfat dried milk (for eNOS and KDR/Flk-1) or 5% BSA (for PECAM). Immunodetection was performed with the primary antibody diluted in blocking buffer for 1 h at room temperature. After the blots were washed to remove unbound antibody, a secondary antibody [horseradish peroxidase-conjugated anti-mouse (eNOS), anti-rabbit (KDR/Flk-1), or anti-goat (PECAM; Vector Laboratories)] was diluted in blocking buffer (1:10,000) and applied for 30-60 min. After three washes, ECL Plus detection (Amersham, Piscataway, NJ) was performed. Densitometry of X-ray films was performed with a scanner and NIH Image software.

Immunohistochemical staining for eNOS protein. Localization of lung eNOS protein was performed using previously published methods (17). Paraffin sections (5 µm) were serially mounted onto slides. Slides were dewaxed in xylene, and sections were rehydrated by immersion in 100% ethanol, then 95% ethanol-5% water, 70% ethanol-30% water, and finally 100% water. Antigen retrieval was performed by boiling the slides in citric acid (pH 6.0). Slides were then washed in PBS. Endogenous biotin in the tissue sections was blocked by glucose-glucose oxidase treatment, and the slides were again washed in PBS. Sections were blocked with Super Block (SkyTek, Logan, UT) and then incubated with anti-eNOS monoclonal antibody diluted 1:10,000 or an IgG1 negative control antibody. After incubation with the primary antibodies, sections were washed in PBS. Biotin-labeled anti-mouse secondary antibody was incubated with the sections, and the slides were once again washed in PBS. Slides were incubated in streptavidin-biotin-horseradish peroxide solution and developed with diaminobenzidine and hydrogen peroxide, with nickel chloride for enhancement. The diaminobenzidine-nickel chloride enhancement color development reaction was stopped by washing with water. The slides were sequentially dehydrated in 70% ethanol + 30% water, 95% ethanol + 5% water, 100% ethanol, and finally 100% xylene before coverslips were added.

Barium-gelatin arteriograms and arterial density counts. Three-week-old FHRs and SDRs were anesthetized with intraperitoneal pentobarbital sodium (1.25 mg/g), and a thoracotomy was rapidly performed. Rats were injected with heparin (50 U) into the RV. A tracheostomy was performed, and the lungs were inflated with air. Blood was flushed from the lungs with heparinized saline (1 U heparin/ml saline) through a main pulmonary artery catheter. A barium-gelatin mixture was infused at 73 mmHg pressure into the main pulmonary artery catheter for 3-4 min until filling of vessels with barium was seen uniformly over the surface of the lung as described by deMello et al. (4). The main pulmonary artery was tied off under pressure, and the lungs were inflation fixed with Formalin at constant pressure (10 cmH2O) (16). Left lungs were excised after fixation and imaged to radiography. After radiography, left lungs were paraffin embedded, and sections were cut and stained with hematoxylin and eosin. Barium-filled pulmonary arteries were counted per high-power field (×100 magnification). Five fields were counted per animal. High-power fields of peripheral lung next to the pleural surface were counted. Fields containing large airways or major vessels were avoided.

Lung histology and radial alveolar counts. Rat lungs were fixed for histology by tracheal instillation of 10% buffered Formalin under constant pressure (10 cmH2O) (16). The trachea was ligated after sustained inflation, and the lungs were excised and immersed in Formalin overnight. Formalin-fixed lung tissue was cut into 4- to 5-mm-thick sections, placed in 10% buffered Formalin, and embedded in paraffin. Paraffin sections (5 µm thick) were serially mounted onto Superfrost Plus slides (Fisher Scientific, Fair Lawn, NJ) and stained with hematoxylin and eosin. At least three lung sections from each animal were used for analysis. Alveolarization was assessed by the radial alveolar count methods of Emery and Mithal (5) and Cooney and Thurlbeck (3). Radial counts were performed by identifying respiratory bronchioles as previously described (22). From the center of the bronchiole, a perpendicular line was taken to the edge of the acinus (connective tissue septum or pleura), and the number of alveoli that intersected the line was counted. At least 10 counts/animal were performed.

RVH measurements. At death, hearts were removed and dissected to isolate the free wall of the RV from the LV+S. The ratio of RV weight to LV+S weight (RV/LV+S) was used as an index of RVH (7).

Statistical analysis. Data are presented as means ± SE. Statistical analysis was performed with the Statview software package (Abacus Concepts, Berkeley, CA). Statistical comparisons were made with ANOVA and Fisher's protected least significant difference test. P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Protocol 1: Lung eNOS expression in adult FHRs compared with SDRs and FSRs. Western blot analysis detected a single band for eNOS protein in adult FHR, SDR, and FSR lung homogenates at 135 kDa (Fig. 1). Compared with control strains, FHR lung eNOS content was reduced, attaining only 24 ± 7% of SDR values (P < 0.05) as assessed by densitometry. Lung eNOS content in adult FSR lungs was not different from that in SDRs.


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 1.   Western blot analysis comparing lung endothelial nitric oxide synthase (eNOS) protein content in Denver-raised adult rats from 3 different rat strains: Fawn-Hooded (FHR), Sprague-Dawley (SDR), and Fischer (FSR). Lung eNOS protein content was significantly lower in adult FHR compared with SDR and FSR. * P < 0.05 vs. SDR and FSR.

Protocol 2: eNOS expression in the aortas from adult FHRs and SDRs. To determine whether the reduction in lung eNOS protein content was also evident in the systemic circulation, we compared eNOS protein content in aortas that were obtained from adult FHRs and SDRs. There was no difference in eNOS protein content in aortas from FHRs and SDRs (P = NS; Fig. 2).


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 2.   Western blot analysis of the eNOS protein content of aortas harvested from adult FHR and SDR. Aortic eNOS content was not different between FHR and SDR.

Protocol 3: Characterization of lung eNOS and KDR/Flk-1 and PECAM expression in fetal, neonatal, and 3-wk-old FHRs. To determine the maturational timing of the reduction in lung eNOS expression in FHRs, we measured lung eNOS protein by Western blot analysis in samples from late-gestation fetal, neonatal (1- and 7-day-old), and 3-wk-old (infant) FHRs, SDRs, and FSRs. Compared with that in SDRs, FHR lung eNOS protein was decreased to 69 ± 8, 56 ± 8, and 68 ± 7% in fetal and postnatal day 1 and animals, respectively (P < 0.05 at each age; Fig. 3). Immunostaining studies demonstrated selective staining for eNOS in vascular endothelium at all ages studied: fetal, days 1 and 7, and 3 and 10 wk (Fig. 4, thick arrows). Weak staining of bronchial epithelium was found in adult rats of both strains (Fig. 4, thin arrows) but not at earlier ages. The pattern of eNOS expression was not different between FHRs and SDRs.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 3.   Comparison of lung eNOS protein content in Denver-raised late-gestation fetal and neonatal (1- and 7-day) FHR, SDR, and FSR. Lung eNOS content was lower in the fetal and neonatal FHR compared with that in SDR and was lower than that in FSR at 1 and 7 days of age. * P < 0.05 vs. SDR. # P < 0.05 vs. FSR.



View larger version (75K):
[in this window]
[in a new window]
 
Fig. 4.   Immunohistochemical localization of eNOS protein in lung sections taken from Denver-raised fetal and 10-wk-old adult FHR and SDR. Lung eNOS protein staining was primarily detected in vascular endothelium in fetal and 10-wk-old adult FHR and SDR lungs (thick arrows). Weak eNOS immunoreactivity was detected in the bronchial epithelium of 10-wk-old adult FHR and SDR (thin arrows) but not at earlier ages. Staining is shown for IgG negative controls. Original magnification, ×200.

At 3 wk of age, lung eNOS content of FHRs was 51 ± 16% that of SDRs (P < 0.05; Fig. 5A). FSR lungs had a similar eNOS content to those of SDRs (P = NS). Western blot analysis detected KDR/Flk-1 and PECAM proteins in 3-wk-old lung homogenates at 200-220 and 130 kDa, respectively (Fig. 5, B and C, respectively). KDR/Flk-1 and PECAM protein contents were not different in 3-wk-old FHR lungs compared with those of SDRs and FSRs (P = NS).


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 5.   Comparison of lung eNOS content (A) and endothelial cell proteins kinase insert domain-containing receptor/fetal liver kinase-1 (KDR/Flk-1; B) and platelet endothelial cell adhesion molecule-1 (PECAM; C) in 3-wk-old Denver-raised FHR, SDR, and FSR. In contrast with KDR/Flk-1 and PECAM, lung eNOS protein content was significantly lower in FHR lungs compared with that in SDR and FSR. * P < 0.05 vs. SDR and FSR.

Protocol 4: Pulmonary vascular development in 3-wk-old FHRs. Barium-gelatin angiography showed a reduction in barium filling as reflected by a reduction in the background density of small pulmonary arteries in 3-wk-old Denver-raised FHRs (Fig. 6). Pulmonary arterial vessel density counts were 33% lower in 3-wk-old Denver-raised FHRs compared with those in SDRs (P < 0.05; Fig. 7).


View larger version (43K):
[in this window]
[in a new window]
 
Fig. 6.   Barium-gelatin arteriograms of the left lungs of 3-wk-old SDR and FHR raised at Denver's altitude (SDR Denver and FHR Denver, respectively) and of hyperbaria-treated FHR (FHR HB Tx). The arteriograms demonstrated narrowing of the large conduit arteries and decreased background filling, suggesting a reduction in small pulmonary artery number. Treatment with mild hyperbaria to simulate sea-level alveolar PO2 improved pulmonary arterial filling as assessed by arteriograms.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 7.   Pulmonary arterial density in 3-wk-old SDR compared with FHR. Pulmonary arterial density was reduced in 3-wk-old FHR Denver compared with SDR Denver. Treatment with mild hyperbaria to simulate sea-level alveolar PO2 increased pulmonary arterial density in 3-wk-old FHR HB Tx. * P < 0.05 vs. SDR Denver. # P < 0.05 vs. FHR Denver.

Protocol 5: Effects of perinatal hyperbaria treatment on pulmonary vascular growth and lung eNOS expression in 3-wk-old FHRs and SDRs. Perinatal hyperbaria treatment of FHRs improved barium filling and small pulmonary arterial density (Fig. 6). Pulmonary arterial vessel density counts increased by 28% after perinatal hyperbaria treatment of FHRs (P < 0.05; Fig. 7). Perinatal hyperbaria treatment increased lung eNOS protein content by 39% in 3-wk-old FHRs compared with that in Denver-raised FHRs (P < 0.05; Fig. 8). There was no difference in lung eNOS protein content between Denver-raised SDRs and SDRs treated with perinatal hyperbaria or between SDRs and FHRs treated with perinatal hyperbaria (P > 0.05; Fig. 8).


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 8.   Lung eNOS protein content in 3-wk-old SDR Denver, SDR HB Tx, FHR Denver, and FHR HB Tx. Treatment with perinatal mild hyperbaria to simulate sea-level alveolar PO2 increased lung eNOS protein content in 3-wk-old FHR HB Tx but did not change lung eNOS protein in 3-wk-old SDR HB Tx compared with SDR Denver. * P < 0.05 vs. SDR Denver. # P < 0.05 vs. FHR Denver. Lung eNOS content in SDR Denver, SDR HB Tx, and FHR HB Tx was not different (P > 0.05).

Body weight, RV and LV+S weights, lung histology, and radial alveolar counts in 3-wk-old FHRs and SDRs. Body weight was 60% lower in 3-wk-old Denver-raised FHRs compared with that in Denver-raised SDRs, but perinatal hyperbaria treatment increased body weight by 38% above that in Denver-raised FHRs (P < 0.05; Table 1). The ratio of RV weight to body weight (RV/BW) was 6.8-fold higher in Denver-raised FHRs compared with that in Denver-raised SDRs (P < 0.05). RV/BW in hyperbaria-treated FHRs was not different from that in Denver-raised SDRs (P = NS). The ratio of LV+S weight to body weight (LV+S/BW) was 2.4-fold higher in Denver-raised FHRs compared with that in Denver-raised SDRs but was decreased to SDR levels by perinatal hyperbaria treatment (P < 0.05). RVH, expressed as RV/LV+S, was 2.6-fold higher in Denver-raised FHRs compared with that in Denver-raised SDRs but was decreased to SDR levels by perinatal hyperbaria treatment (P < 0.05). Body weight, RV/BW, LV+S/BW, and RVH were not different between Denver-raised SDRs and SDRs treated with perinatal hyperbaria to simulate sea-level conditions (P > 0.05). Compared with SDRs, the lung histology of 3-wk-old Denver-raised FHRs showed a pattern of alveolar simplification, including increased alveolar size and decreased alveolar number (Fig. 9). Radial alveolar counts were 38% lower in Denver-raised FHRs compared with those in SDRs (P < 0.05; Fig. 10). Perinatal hyperbaria treatment increased radial alveolar counts by 24% in FHRs (P < 0.05; Fig. 10).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Body weights, RV/BW, LV+S, and right ventricular hypertrophy in 3-wk-old rats



View larger version (45K):
[in this window]
[in a new window]
 
Fig. 9.   Effects of mild hyperbaria on lung histology in SDR and FHR at 3 wk. Pulmonary arteries were injected with barium and appear green in color. Compared with SDR Denver, lung histology shows a pattern of alveolar simplification, characterized by reduced alveolar numbers and larger distal air spaces (FHR Denver). Treatment with mild hyperbaria to simulate sea-level alveolar PO2 improved lung histology in the FHR HB Tx. Micrographs are representative and are shown at the same magnification (×100).



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 10.   Alveolarization in 3-wk-old SDR and FHR. FHR Denver group showed reduced radial alveolar counts compared with SDR Denver. Treatment with mild hyperbaria to simulate sea-level alveolar PO2 increased radial alveolar counts in 3-wk-old FHR HB Tx. * P < 0.05 vs. SDR Denver. # P < 0.05 vs. FHR Denver. P = NS for SDR Denver vs. FHR HB Tx.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Although the FHR strain has been used to explore mechanisms of adult pulmonary hypertension (26, 29, 30), Le Cras et al. (16) have recently reported that Denver-raised FHRs displayed an abnormal pattern of lung growth, as reflected by reduced lung weight and alveolarization, in addition to development of severe pulmonary hypertension. Because altered lung growth is evident during the perinatal period and NO plays an important role in lung vascular regulation, we hypothesized that early abnormalities in lung vascular development and reductions in lung eNOS expression may increase the risk for pulmonary hypertension in the FHR. In the present study, we found that lung eNOS is reduced in adult FHRs compared with that in two normal rat strains, Sprague-Dawley and Fischer, all raised at Denver's altitude. This reduction may be specific to the pulmonary circulation because aortic eNOS content was not different between FHR and SDR strains.

In addition, maturational studies demonstrated that lung eNOS expression was lower in FHRs throughout development, including during the fetal and neonatal periods. Although lung eNOS expression was lower in infant FHRs, expression of other endothelial markers, KDR/Flk-1 and PECAM, was not different from age-matched control strains, suggesting a specific downregulation of eNOS expression in FHR lungs. At 3 wk, lung histology and barium angiography revealed reduced alveolar and vascular development in Denver-raised FHRs compared with SDRs. Denver-raised infant FHRs had lower body weights and higher RV and LV+S weights than SDRs, indicating that pulmonary and systemic hypertension were already present in 3-wk-old FHRs. Systemic hypertension has previously been reported in this strain by others (11, 14). Perinatal treatment of FHRs with mild hyperbaria to simulate sea-level alveolar PO2 increased lung eNOS content, alveolar number, and vascular density to levels that were similar to those in SDR lungs at 3 wk of age. Lung eNOS content was not different between FHRs and SDRs treated with perinatal hyperbaria and Denver-raised SDRs, suggesting that exposure to Denver's altitude leads to a de novo deficiency in lung eNOS expression in the FHR that is not present at sea level. Perinatal hyperbaria also increased body weight and reduced RV and LV+S weights in the FHR, suggesting a reduction in pulmonary and systemic hypertension.

These findings are interesting because they suggest that abnormal growth and development of the pulmonary circulation and reduced eNOS expression in the perinatal period are associated with an increased risk for pulmonary hypertension in the FHR. The role of perinatal events in the development of adult pulmonary diseases in humans has been suggested by several investigators (25, 27), but few experimental studies have examined this question, and the potential mechanisms are uncertain. We suggest that the FHR provides a unique model to explore the relationship between genetic factors, perinatal events, and abnormal lung and vascular development with the subsequent risk for pulmonary hypertension. Interestingly, our studies have shown that treatment of FHRs with mild hyperbaria to simulate sea-level alveolar PO2 during early infancy improved lung growth, vascular development, and eNOS expression and reduced the risk for pulmonary hypertension. Whether reductions in lung eNOS in the FHR contribute to the abnormal vascular development and the risk for pulmonary hypertension at Denver's altitude is unknown. Inhaled NO has been shown to reduce pulmonary hypertension in neonatal and adult SDRs exposed to chronic hypoxia (13, 23, 24), but whether NO produced by eNOS also plays a role in pulmonary vascular development is uncertain. In addition to its effects on smooth muscle cell proliferation (32), NO may also contribute to angiogenesis (20), especially in response to vascular endothelial growth factor (35). However, although eNOS-deficient [eNOS(-/-)] mice have been shown to develop pulmonary hypertension (6, 31) that is exacerbated at Denver's altitude (6), they have not been reported to show evidence of abnormal vascular development or lung hypoplasia.

In this study, perinatal treatment with mild hyperbaria to simulate sea-level alveolar PO2 increased barium filling of the pulmonary circulation, seen as an increase in the "background haze" on left lung arteriograms, indicating that vascular as well as alveolar development was improved in the hyperbaria-treated 3-wk-old FHRs. Western blot analysis showed that although lung eNOS content was reduced, levels of endothelial cell proteins KDR/Flk-1 and PECAM were not, suggesting that reductions in lung eNOS expression in FHRs were not due to reduced vascular development but represent reduced lung eNOS expression. Recently, Tyler et al. (33) reported reduced eNOS levels in the lungs of adult FHRs compared with those in SDRs. In our study, FHRs showed evidence of pulmonary and systemic hypertension at 3 wk of age, whereas, in a previous study (26), the earliest reported development of pulmonary hypertension in FHRs was at 1 mo of age. Also, similar to a previous study by Le Cras et al. (16) in adult FHRs, we found that perinatal treatment with mild hyperbaria reduced RVH, an index of pulmonary hypertension, in 3-wk-old FHRs. In this study, we also report that 3-wk-old FHRs had increased LV+S weight, which is consistent with the presence of systemic hypertension, and that perinatal hyperbaria treatment also reduced LV+S weight.

Several mechanisms can contribute to the development of pulmonary hypertension, including altered production of or responsiveness to vasoactive substances. The role of vasoconstrictive factors and vasodilators in the development of pulmonary hypertension has been suggested by several experimental models (1). NO is a potent vasodilator that can also modulate cell proliferation and gene expression (9, 12, 32). NO is produced by three NOSs. Reduced expression of lung eNOS (type III) has been observed in patients with pulmonary hypertension (10) and in fetal sheep with pulmonary hypertension induced by ligation of the ductus arteriosus (34). In eNOS(-/-) and eNOS heterozygote [eNOS(+/-)] mice, baseline pulmonary arterial pressure is mildly elevated (6, 31), and the severity of pulmonary hypertension is increased during exposure to even mild reductions in alveolar PO2 experienced at Denver's altitude, suggesting that even partial loss (50%) of eNOS expression is sufficient to increase the risk for pulmonary hypertension (6).

Several factors may play a role in the development of pulmonary hypertension in the FHR. First, early reductions in lung eNOS expression may lead to reduced NO production, and because NO is a potent vasodilator, this would favor increased vascular tone and increased risk for smooth muscle cell proliferation. Second, reduced vascular density decreases the cross-sectional area of the pulmonary circulation, leading to increased hemodynamic stress that may cause altered vascular tone and structure. Similarly, reduced lung surface area secondary to decreased alveolarization might increase the risk for pulmonary hypertension over time. Finally, Stelzner et al. (30) have previously reported that FHR lungs have an elevated expression of the vasoconstrictor peptide ET-1, which would also favor increased pulmonary vascular tone and increased smooth muscle cell proliferation (26). Because NO has been described as having antiproliferative effects on vascular smooth muscle cells (9), increased ET-1 expression in the setting of reduced endothelial NO production might be expected to further promote vascular smooth muscle cell hyperplasia as well as favor increased pulmonary vascular tone. Similarly, downregulation of NOS and decreased NO production would increase the risk for pulmonary hypertension in the setting of other risk factors such as increased ET-1 and reduced vascular surface area.

The precise genetic basis for differences in lung development and pulmonary hypertension in the FHR is unknown. A preliminary report by Stelzner et al. (29) localized high pulmonary arterial pressure in the FHR to the PH1 locus on chromosome 1. Whether this locus also contains genes that modulate oxygen sensing in the FHR lung and regulate basic mechanisms of lung vascular development is unknown. Determination of these genetic differences may be important for understanding pathophysiological mechanisms of pulmonary hypertension in this strain. A potential limitation of our study is that we compared lung eNOS expression with aortic eNOS expression and suggested that eNOS was not reduced in the systemic circulation of the FHR. However, eNOS expression may differ between conduit and resistance vessels, and our study was not able to differentiate these because total lung homogenates were used, and these would have contained both conduit and resistance vessels.

We conclude that lung eNOS content of the FHR at Denver's altitude was lower than that in normal rat strains and that reduction of eNOS expression was already present in the fetal and neonatal lung. In contrast, other endothelial proteins (KDR/Flk-1 and PECAM) were not different from those in age-matched control strains. In addition, Denver-raised FHRs showed early decreases in lung vascular growth, but perinatal treatment with mild hyperbaria to simulate sea-level alveolar PO2 improved lung structure, increased arterial density and eNOS expression, and decreased RVH.


    ACKNOWLEDGEMENTS

We thank Charles R. Ahrens for performing radiography for the barium angiograms and the Caitlyn Whitley Pediatric Cardiology Research Fund for donating the Zeiss Axioskop 2 microscope and ProgRes 3008 digital camera used to take the photomicrographs.


    FOOTNOTES

This work was supported by a National American Heart Association Scientist Development Grant (T. D. Le Cras), a Grant-In-Aid (S. H. Abman), and National Heart, Lung, and Blood Institute Specialized Center of Research Grant HL-57144 (S. H. Abman).

Address for reprint requests and other correspondence: T. D. Le Cras, Department of Pediatrics, Box C218, University of Colorado Health Sciences Center, 4200 E. Ninth Ave., Denver, CO 80262 (E-mail: Timothy.Lecras{at}uchsc.edu).

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. §1734 solely to indicate this fact.

Received 13 September 1999; accepted in final form 11 February 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abman, SH, Kinsella JP, Parker TA, Storme L, and Le Cras TD. Physiologic roles of nitric oxide in the perinatal pulmonary circulation. In: The Fetal and Neonatal Pulmonary Circulations, edited by Weir EK, Archer SL, and Reeves JT.. Armonk, NY: Futura, 1999, p. 239-260.

2.   Ashmore, RC, Rodman DM, Sato K, Webb SA, O'Brien RF, McMurtry IF, and Stelzner TJ. Paradoxical constriction to platelets by arteries from rats with pulmonary hypertension. Am J Physiol Heart Circ Physiol 260: H1929-H1934, 1991[Abstract/Free Full Text].

3.   Cooney, TP, and Thurlbeck WM. The radial alveolar count method of Emery and Mithal: a reappraisal 1-postnatal lung growth, 2-intrauterine and early postnatal lung growth. Thorax 37: 572-579, 1982[Abstract], 580-583.

4.   DeMello, DE, Sawyer D, and Reid LM. Early fetal development of lung vasculature. Am J Respir Cell Mol Biol 16: 568-581, 1997[Abstract].

5.   Emery, JL, and Mithal A. The number of alveoli in the terminal respiratory unit of man during intrauterine life and childhood. Arch Dis Child 35: 483-485, 1960.

6.   Fagan, K, Fouty BW, Tyler RC, Morris KG, Hepler LK, Sato K, Le Cras TD, Abman SH, Weinberger HD, Huang PL, McMurtry IF, and Rodman DM. The pulmonary circulation of mice with either homozygous or heterozygous disruption of the endothelial nitric oxide synthase is hyper-responsive to chronic hypoxia. J Clin Invest 103: 291-299, 1999[Abstract/Free Full Text].

7.   Fulton, RM, Hutchinson EC, and Jones AM. Ventricular weight in cardiac hypertrophy. Br Heart J 14: 413-420, 1952.

8.   Gaine, SP, and Rubin LJ. Primary pulmonary hypertension. Lancet 352: 291-299, 1999[ISI].

9.   Garg, UC, and Hassid A. NO-generating vasodilators and 8-bromo-cGMP inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J Clin Invest 83: 17744-17747, 1989.

10.   Giaid, A, and Saleh D. Reduced expression of endothelial nitric oxide synthase in the lungs of patients with pulmonary hypertension. N Engl J Med 333: 214-221, 1995[Abstract/Free Full Text].

11.   Kentera, D, Susic D, Veljkovic V, Tucakovic G, and Koko V. Pulmonary arterial pressure in rats with hereditary platelet function defect. Respiration 54: 110-114, 1988[ISI][Medline].

12.   Kourembanas, S, McQuillan LP, Leung GK, and Faller DV. Nitric oxide regulates the expression of vasoconstrictors and growth factors by vascular endothelium under both normoxia and hypoxia. J Clin Invest 92: 99-104, 1993[ISI][Medline].

13.   Kouyoumdjian, C, Adnot S, Levame M, Eddahibi S, Bousbaa H, and Raffestin B. Continuous inhalation of nitric oxide protects against development of pulmonary hypertension in chronically hypoxic rats. J Clin Invest 94: 578-584, 1994[ISI][Medline].

14.   Kuijpers, MH, and de Jong W. Spontaneous hypertension in the Fawn-Hooded rat: a cardiovascular disease model. J Hypertens 4, Suppl: S41-S44, 1986[ISI].

15.   Le Cras, TD, Xue C, Rengasamy A, and Johns RA. Chronic hypoxia upregulates endothelial and inducible NO synthase gene and protein expression in rat lung. Am J Physiol Lung Cell Mol Physiol 270: L164-L170, 1996[Abstract/Free Full Text].

16.   Le Cras, TD, Kim D, Gebb S, Markham NE, Shannon JM, Ruder RM, and Abman SH. Abnormal lung growth and the development of pulmonary hypertension in the Fawn-Hooded rat. Am J Physiol Lung Cell Mol Physiol 277: L709-L718, 1999[Abstract/Free Full Text].

17.   Le Cras, TD, Tyler RC, Horan MP, Morris KG, Tuder RM, McMurtry IF, Johns RA, and Abman SH. Effects of chronic hypoxia and altered hemodynamics on endothelial nitric oxide synthase expression in the adult rat lung. J Clin Invest 101: 796-801, 1998.

18.   Meyrick, B, Gamble W, and Reid L. Development of Crotalaria pulmonary hypertension: hemodynamic and structural study. Am J Physiol Heart Circ Physiol 239: H692-H702, 1980[ISI][Medline].

19.   Okada, K, Tanaka Y, Bernstein M, Zhang W, Patterson GA, and Botney MD. Pulmonary hemodynamics modify the rat pulmonary artery response to injury. A neointimal model of pulmonary hypertension. Am J Pathol 151: 1019-1025, 1997[Abstract].

20.   Pipli-Synetos, E, Sakkoula E, and Maragoudakis ME. Nitric oxide is involved in the regulation of angiogenesis. Br J Pharmacol 108: 855-857, 1993[Abstract].

21.   Rabinovitch, M, Gamble W, Nadas AS, Miettinen OS, and Reid L. Rat pulmonary circulation after chronic hypoxia: hemodynamic and structural features. Am J Physiol Heart Circ Physiol 236: H818-H827, 1979[Abstract/Free Full Text].

22.   Randell, SH, Mercer RR, and Young SL. Postnatal growth of pulmonary acini and alveoli in normal and oxygen-exposed rats studied by serial section reconstructions. Am J Anat 186: 55-68, 1989[ISI][Medline].

23.   Roberts, JD, Jr, Roberts CT, Jones RC, Zapol WM, and Bloch KD. Continuous nitric oxide inhalation reduces pulmonary arterial structural changes, right ventricular hypertrophy, and growth retardation in the hypoxic newborn rat. Circ Res 76: 215-222, 1995[Abstract/Free Full Text].

24.   Roos, CM, Frank DU, Xue C, Johns RA, and Rich GF. Chronic inhaled nitric oxide: effects on pulmonary vascular endothelial function and pathology in rats. J Appl Physiol 80: 252-260, 1996[Abstract/Free Full Text].

25.   Sartori, C, Allemann Y, Delabays A, Nicod P, and Scherrer U. Augmented vasoreactivity in adult life associated with perinatal vascular insult. Lancet 353: 2205-2207, 1999[ISI][Medline].

26.   Sato, K, Webb S, Tucker A, Rabinovitch M, O'Brien RF, McMurtry IF, and Stelzner TJ. Factors influencing the idiopathic development of pulmonary hypertension in the Fawn-Hooded rat. Am Rev Respir Dis 145: 793-797, 1992[ISI][Medline].

27.   Shaheen, S, and Barker DJ. Early lung growth and chronic airflow obstruction. Thorax 49: 533-536, 1994[ISI][Medline].

28.   Shaul, PW, North AJ, Brannon TS, Ujiie K, Wells LB, Nisen PA, Lowenstein CJ, Snyder SH, and Star RA. Prolonged in vivo hypoxia enhances nitric oxide synthase type I and type III gene expression in adult rat lung. Am J Respir Cell Mol Biol 13: 167-174, 1995[Abstract].

29.  Stelzner T, Hofman TA, Brown D, Deng A, and Jacob HJ. Genetic determinants of pulmonary hypertension in Fawn-Hooded rats (Abstract). Chest 111, Suppl: 96S, 1997.

30.   Stelzner, TJ, O'Brien RF, Yanagisawa M, Sakurai R, Sato K, Webb S, Zamora M, McMurtry IF, and Fisher JH. Increased lung endothelin-1 production in rats with idiopathic pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 262: L614-L620, 1992[Abstract/Free Full Text].

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.   Thomae, KR, Nakayama DK, Billiar TR, Simmons RL, Pitt BR, and Davies P. The effect of nitric oxide on fetal pulmonary artery smooth muscle growth. J Surg Res 59: 337-343, 1995[ISI][Medline].

33.   Tyler, RC, Muramatsu M, Abman SH, Stelzner TJ, Rodman DM, Bloch KD, and McMurtry IF. Variable expression of endothelial NO synthase in three forms of rat pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 276: L297-L303, 1999[Abstract/Free Full Text].

34.   Villamor, E, Le Cras TD, Horan MP, Halbower AC, Tuder RM, and Abman SH. Chronic intrauterine pulmonary hypertension impairs endothelial nitric oxide synthase in the ovine fetus. Am J Physiol Lung Cell Mol Physiol 272: L1013-L1020, 1997[Abstract/Free Full Text].

35.   Ziche, M, Morbidelli L, Masini E, Amerini S, Granger HJ, Maggi CA, Geppetti P, and Ledda F. Nitric oxide mediates angiogenesis in vivo and endothelial cell growth and migration in vitro promoted by substance P. J Clin Invest 94: 2036-2044, 1994[ISI][Medline].


Am J Physiol Lung Cell Mol Physiol 279(2):L283-L291
1040-0605/00 $5.00 Copyright © 2000 the American Physiological Society