Reduced endothelial nitric oxide synthase in lungs of chronically ventilated preterm lambs

Amy N. MacRitchie, Kurt H. Albertine, Jiancheng Sun, Paul S. Lei, Suzanne C. Jensen, Allen A. Freestone, Philip M. Clair, Mar Janna Dahl, Emily A. Godfrey, David P. Carlton, and Richard D. Bland

Department of Pediatrics, University of Utah, Salt Lake City, Utah 84132


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Nitric oxide (NO), produced in lung vascular endothelium and airway epithelium, has an important role in regulating smooth muscle cell growth and tone. Chronic lung disease, a frequent complication of premature birth, is characterized by excess abundance, tone, and reactivity of smooth muscle in the pulmonary circulation and conducting airways, leading to increased lung vascular and airway resistance. Whether these structural and functional changes are associated with diminished pulmonary expression of endothelial nitric oxide synthase (eNOS) protein is unknown. Both quantitative immunoblot analysis and semiquantitative immunohistochemistry showed that there was less eNOS protein in the endothelium of small intrapulmonary arteries and epithelium of small airways of preterm lambs that were mechanically ventilated for 3 wk compared with control lambs born at term. No significant differences were detected for other proteins (inducible NOS, alpha -smooth muscle actin, and pancytokeratin). Lung vascular and respiratory tract resistances were greater in the chronically ventilated preterm lambs compared with control term lambs. These results support the notion that decreased eNOS in the pulmonary circulation and respiratory tract of preterm lambs may contribute to the pathophysiology of chronic lung disease.

chronic lung disease of prematurity; bronchopulmonary dysplasia; pulmonary vascular resistance; airway resistance; pulmonary circulation; respiratory failure; immunohistochemistry


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

NITRIC OXIDE (NO) has an important role in regulating smooth muscle tone in pulmonary blood vessels and conducting airways of newborn animals. Inhibition of NO production attenuates the normal postnatal decrease of pulmonary vascular resistance in newborn lambs (1, 12) and results in increased respiratory tract resistance and decreased dynamic lung compliance in newborn piglets (34). Several studies (9-11, 13, 16, 22, 27, 38-40, 51, 52) have shown that inhaled NO causes an abrupt and profound decrease in pulmonary vascular resistance, with an associated increase in arterial oxygenation in newborn animals and in human infants with pulmonary hypertension. Inhaled NO also leads to a substantial decrease in respiratory tract resistance in newborn piglets (34). Thus there is considerable evidence that NO can influence smooth muscle tone in pulmonary blood vessels as well as in conducting airways during postnatal development.

NO also acts as a signaling molecule to inhibit the growth of both vascular and airway smooth muscle cells in culture (18, 47). An in vivo study (41) has shown that inhibition of endogenous NO production results in increased smooth muscle growth in response to injury. Conversely, providing exogenous NO, either by inhalation or via a NO donor, inhibits smooth muscle growth after mechanical injury (17, 25, 42).

NO is produced through the conversion of L-arginine to L-citrulline by the enzyme NO synthase (NOS) (43). In the lung, endothelial NOS (eNOS) is found in both vascular endothelium and airway epithelium (45). Studies (6, 44) have shown decreased abundance of eNOS protein in lungs of newborn lambs, with elevated pulmonary vascular resistance after in utero closure of the ductus arteriosus. Whether there is a similar relationship between decreased eNOS protein abundance and elevated airway resistance is not known.

Our laboratory (3, 7) previously showed that both pulmonary vascular and airway resistances are increased in preterm lambs with chronic lung disease (CLD) compared with those in control lambs born at term. These pathophysiological changes are associated with increased abundance of smooth muscle in pulmonary arterioles and bronchioles. The physiological and morphological abnormalities seen in chronically ventilated preterm lambs are similar to those reported in infants with CLD (2, 19, 28). These observations led us to consider the possibility that NO production might be reduced in evolving CLD and that this might be the result of diminished pulmonary expression of eNOS protein.

We used immunoblot analysis to measure eNOS protein abundance in homogenates of dissected intrapulmonary arteries and airways, and we used immunohistochemistry to examine the cellular distribution of eNOS protein in tissue sections from the same lungs of chronically ventilated preterm lambs and two groups of control lambs that were born at term gestation. eNOS protein was less in both pulmonary arterial endothelium and airway epithelium of chronically ventilated preterm lambs compared with that in control lambs. We detected eNOS protein in both vascular endothelial and airway epithelial cells but not in other cell types.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animal model. We delivered fetal sheep prematurely (124 ± 3 days, term approx 147 days) by cesarean section as previously described (3, 7, 33). Before delivery, we placed catheters in a jugular vein and carotid artery of the fetus. We then intubated the fetus, withdrew lung liquid, and instilled surfactant into the lung lumen (350 mg of Infasurf; generous gift of ONY, Amherst, NY). Next, we removed the fetus from the uterus and placed it on a neonatal bed beneath a radiant warmer. We initiated mechanical ventilation with 100% O2. We adjusted peak inflation pressure to maintain arterial PCO2 between 35 and 45 mmHg, and we adjusted the fraction of inspired O2 to maintain arterial PO2 between 60 and 90 mmHg. We gave the preterm lambs buprenorphine (0.03 mg/kg iv) soon after birth and as needed to prevent agitation. The preterm lambs initially received an intravenous glucose-saline solution (3% glucose, 25 meq/l of NaHCO3, and 50 meq/l of NaCl) after birth and subsequently received parenteral nutrition with solutions containing both protein and glucose to supplement enteral tube feedings. Penicillin and gentamicin were given soon after birth and were continued for at least the first week. If signs and symptoms of sepsis developed thereafter, alternative broad-spectrum antibiotics were given. We adjusted the heat output of the radiant warmer to maintain body temperature between 37 and 38.5°C (normal for newborn sheep). Blood glucose concentrations were monitored with an Exactech glucose-measuring device (Medisense, Waltham, MA), urine output was determined from diaper weights before and after each voiding, and arterial blood was sampled hourly for measurement of pH and arterial PO2 and PCO2 with a calibrated blood gas machine (model 178, Chiron Diagnostics, Norwood, MA). We weighed the preterm lambs daily to monitor fluid balance and nutritional status. Serum electrolytes were measured with ion-selective electrodes (Na/K/Cl Stat Analyzer, model 644, Ciba Corning Diagnostics, Medfield, MA). Chest radiographs were obtained periodically to assess lung inflation. After the preterm lambs were stable on mechanical ventilation, we performed two thoracotomies 2-3 days apart using fentanyl anesthesia for surgical ligation of the ductus arteriosus and placement of catheters in the pulmonary artery and left atrium, a thermister wire in the pulmonary artery for subsequent measurement of pulmonary blood flow, and a cannula in the main lymphatic vessel draining the lungs (7).

All preterm lambs had evidence of severe lung disease after birth that persisted throughout the 3- to 4-wk study period (Table 1). They received mechanical ventilation at 20 breaths/min and an end-expiratory pressure of 5 cmH2O, with considerable variability in peak inflation pressure and fraction of inspired O2. After 3-4 wk of continuous mechanical ventilation, we gave the lambs intravenous pentobarbital sodium (30 mg/kg body wt), opened the chest, and removed the lungs. We dissected the intrapulmonary arteries and airways from the caudal lobe of the right lung and processed the right middle lobe for immunohistochemistry (formalin fixation, paraffin embedment).

                              
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Table 1.   Body weight and physiological variables of chronically ventilated preterm lambs

We also obtained lungs from two groups of control lambs. One group of control lambs was born at term gestation and killed 1 day after birth (newborn lambs). These lambs were of the same postconceptional age as the chronically ventilated preterm lambs at death. The other group of control lambs was born at term and killed 3 wk later. These lambs were the same postnatal age as the chronically ventilated preterm lambs at death. Lung tissue from the control lambs was obtained and processed in the same manner as that used for the preterm lambs.

These studies were approved by the University of Utah Committee for Animal Care.

Immunoblot analysis of eNOS protein. We used immunoblot analysis to measure eNOS protein abundance in third- to fifth-generation intrapulmonary arteries and airways that had been dissected from the lungs of the chronically ventilated preterm lambs and from both groups of control lambs. We dissected the intrapulmonary arteries and airways from the right caudal lobe. The dissections were performed at 4°C. We rinsed the lungs with cold sterile saline, dissected third-, fourth-, and fifth-generation intrapulmonary arteries and adjacent airways, and placed them immediately into liquid nitrogen for later processing.

Segments of the pulmonary arteries and airways were placed in a tissue homogenizer containing Tris · HCl buffer (50 mM Tris, pH 7.5, 4°C) containing 10 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), 3 mM dithiothreitol, 20 µM tetrahydrobiopterin, and protease inhibitors (2 µg/ml of pepstatin A, 20 µg/ml of leupeptin, 40 µg/ml of Nalpha -p-tosyl-L-lysine chloromethylketone, and 20 µg/ml of aprotinin). We centrifuged the homogenized samples and saved aliquots of the supernatant at -80°C. Protein content was determined by bicinchoninic acid assay (46) (Pierce Chemical, Rockford, IL) with bovine serum albumin as the standard. We performed SDS-PAGE on 50 µg total protein/sample with the method of Laemmli (24). The proteins were electrophoretically transferred to nitrocellulose filters and blocked for 1.5 h in buffer containing 150 mM NaCl, 50 mM Tris · HCl, and 0.1% Tween 20. Next, we incubated the filters overnight at 4°C in the presence of a primary antibody specific for eNOS (1:500 dilution in blocking buffer; Transduction Laboratories, Lexington, KY). After incubation with the primary antibody, we washed the nitrocellulose filters with 150 mM NaCl buffer containing 50 mM Tris · HCl and 0.1% Tween 20 and incubated them for 90 min with a secondary antibody that was linked to horseradish peroxidase (Amersham, Little Chalfont, UK).

Immunoblot analyses of inducible NOS, alpha -smooth muscle actin, and pancytokeratin proteins. We used immunoblot analysis to determine whether other proteins in arteries and airways changed in the chronically ventilated preterm lambs. For measuring inducible NOS (iNOS) protein abundance, frozen segments of the same airways that were analyzed for eNOS protein were ground in a porcelain mortar that was cooled in liquid nitrogen. The tissue powder was placed in lysis buffer (10 mM Tris · HCl buffer, pH 7.4) containing 1% SDS and protease inhibitors (protease inhibitor cocktail tablets; Roche Molecular Biochemicals, Indianapolis, IN). Other procedures were similar to those described for eNOS protein except that SDS-PAGE was done on 100 µg total protein/sample and nitrocellulose filters were incubated for 1.5-2 h at room temperature in the presence of a primary antibody specific for iNOS (1:250 dilution in blocking buffer; Transduction Laboratories).

Immunoblot analysis for iNOS protein in dissected intrapulmonary arteries was attempted. However, we were unable to detect iNOS protein in the archived homogenates of arteries that were used for the eNOS protein immunoblot analysis. A reason for the lack of detection of iNOS protein was that the method given by the manufacturer for preparing tissue lysates for immunoblot analysis with their iNOS antibody required different conditions than the ones we used for preparing the homogenates for eNOS immunoblots. Because we used all of the dissected arteries for the immunoblot analysis for eNOS protein, we had no frozen tissue left to prepare new lysates for immunoblot analysis for iNOS protein. Therefore, we did not obtain immunoblot results for iNOS protein in intrapulmonary arteries.

We used immunoblot analysis to measure alpha -smooth muscle actin protein in the archived homogenates of the dissected pulmonary arteries and alpha -smooth muscle actin and pancytokeratin proteins in lysates of the dissected airways. For alpha -smooth muscle actin protein, SDS-PAGE was performed on 1 µg total protein/sample. For pancytokeratin protein, SDS-PAGE was performed on 2.5 µg total protein/sample. The filters were incubated overnight at 4°C in the presence of primary antibodies specific for alpha -smooth muscle actin (1:20,000 dilution in blocking buffer; Sigma, St. Louis, MO) and pancytokeratin (1:100,000 dilution in blocking buffer; Sigma). Otherwise the samples were processed as described above for immunoblot analysis.

Densitometry of protein bands. We visualized the bands by chemiluminescence (ECL Western blotting analysis system; Amersham) and quantified them by densitometry (NIH Image software).

Immunohistochemical localization of eNOS protein. Immunohistochemistry was used to localize eNOS protein expression in pulmonary arterioles and bronchioles of lung tissue sections prepared from the same lambs. We analyzed pulmonary arterioles next to terminal bronchioles because both are numerous and contribute to pulmonary vascular and airway resistances. We also observed central arteries and airways when they appeared in the tissue sections. Because central arteries and airways were infrequent in the tissue sections, however, we did not compare their immunostaining density among the groups of lambs.

Briefly, we double-clamped a large piece of the ventral portion of the left caudal lobe (~3-4 cm3) at the prevailing peak inspiratory pressure (3). This procedure retained the gas and blood volume of the lobe, thereby preserving the three-dimensional configuration of the lung. We placed the clamped lobe in 10% neutral buffered formalin (4°C) for 24 h and cut each clamped piece into 3-mm-thick slabs along the parasagittal planes (8). Large tissue blocks (2-4 cm3, 2-3 blocks/lamb) were dehydrated in a graded ethanol series, embedded in paraffin, and sectioned at 4-µm thickness. We treated deparaffinized tissue sections with blocking serum and with methanol-H2O2 to block endogenous peroxidase activity. To improve antigen detection, we employed antigen retrieval using microwave irradiation in citrate buffer (BioGenex, San Ramon, CA) (45). We used the same primary antibody specific for eNOS (Transduction Laboratories) as described in Immunoblot analysis of eNOS protein. The optimal dilution was 1:800. For antigen detection, we used a standard peroxidase method (Elite ABC kit; Vector Laboratories, Burlingame, CA). Immunohistochemical staining controls included substitution of the primary antibody with an irrelevant, species-matched, immunoglobulin isotype-matched primary antibody (anti-insulin); omission of the primary antibody (replaced with blocking buffer); and omission of the secondary antibody (replaced with blocking buffer). The tissue sections were counterstained with Gill's no. 3 hematoxylin diluted 1:10 with water.

Immunohistochemical localization of iNOS protein. We immunolocalized iNOS protein in tissue sections that were adjacent to those that we used for eNOS immunohistochemistry to see if the eNOS results were specific or perhaps indicative of more global inhibition of NOS. We used the same immunohistochemical method described for eNOS except that the optimal dilution of the iNOS primary antibody was 1:100. We also immunolocalized neuronal NOS (nNOS) in some tissue sections, but the immunoperoxidase reaction product was uniformly faint among the groups of lambs (data not shown).

Immunostain densitometry of eNOS and iNOS proteins. To assess the immunostaining density for eNOS and iNOS in endothelium and epithelium among the groups of lambs, we used a computer-assisted true-color imaging system (BIOQUANT True-Color Windows; R & M Biometrics, Nashville, TN). An observer who was unaware of the group from which each tissue section was obtained placed five uniformly sized enclosed rectangular frames over the full height (lumen to basement membrane) of the endothelial and epithelial cytoplasm, excluding the nucleus. Four of the enclosed frames were placed at clock positions 12, 3, 6, and 9; the fifth enclosed frame was randomly placed between two of those positions. The relative density of the brown peroxidase reaction product within each enclosed frame was determined automatically by computer. For each tissue section (1 random section/lamb), 75 framed areas were analyzed for 15-20 pulmonary arterioles and the neighboring terminal bronchioles.

Minimum relative density was established from the tissue sections that were not treated with primary antibody. Maximum relative density was determined from the most intensely immunostained endothelial and epithelial cells in duplicate tissue sections from the newborn (1-day-old) control lambs. These duplicate tissue sections were processed and imaged with the other sections. We used tissue sections from the newborn control lambs to determine maximum relative density because they demonstrated the most intense staining among the groups. The observer who set the minimum and maximum density levels did not perform the densitometry measurements on the randomized digital images for the three groups of lambs.

Because immunohistochemical comparisons may be influenced by processing protocols and observer bias, we took several precautions to minimize such influences. First, we cut 4-µm-thick sections from all of the tissue blocks on 1 day with the same microtome. Second, we performed the immunostaining steps on all of the tissue sections at once using master batches of reagent mixtures of sufficient volume to treat all of the microscope slides. The microscope slides were assigned random numbers by an individual who was not involved with the analysis. Third, we used a true-color video camera (DEI-470; Optronics Engineering, Goleta, CA) mounted on a light microscope (Olympus BHTU-F; Scientific Instruments, Aurora, CO) connected to a computer-aided image analysis system (BIOQUANT True-Color Windows; R & M Biometrics) to capture high-resolution, calibrated, true-color digital images. A ×100 oil-immersion lens was used, and the images were projected on a 21-inch, high-resolution color monitor (final image enlargement was ×1,200). Fourth, a regulated power supply was used for the illumination system of the microscope, and the digital images for each primary antibody were captured consecutively on 1 day. Fifth, we analyzed 15-20 profiles of pulmonary arterioles and terminal bronchioles per tissue section (1 tissue section/lamb). To be eligible for analysis, arterioles had to satisfy two histological criteria: they had to be immediately adjacent to a terminal bronchiole that was 75-100 µm in external diameter, and they had to have a circular profile with smooth contours of both the endothelium and smooth muscle. We used terminal bronchioles as independent landmarks so that similar generations of pulmonary arterioles were analyzed. The circular profile assured cross-sectional views of all of the arterioles. We also analyzed circular profiles of terminal bronchioles. The smooth contour of the arteriolar endothelium, bronchiolar epithelium, and underlying smooth muscle ensured that the pulmonary arterioles and terminal bronchioles were not constricted.

Statistics. Results for the immunoblot and immunohistochemistry analyses are reported in arbitrary densitometry units as means ± SD. Statistical significance for the immunoblot results was determined by ANOVA followed by the Student-Newman-Keuls correction for multiple comparisons (50). Statistical significance for the immunohistochemical results was determined by ANOVA followed by the Kruskal-Wallis rank test for multiple comparisons (50). Differences were considered statistically significant when P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Body weight and physiological variables for the chronically ventilated preterm lambs are summarized in Table 1. The preterm lambs gained a slight amount of weight between weeks 1 and 3, although the change was not significant. There were no significant changes over time in respiratory variables, pulmonary vascular resistance, or airway resistance. For comparison, we measured pulmonary vascular resistance (PVR) and airway resistance (Raw) at weekly intervals after birth in eight term control lambs. PVR in these term lambs averaged 12 ± 4 mmHg · l-1 · min at week 1 vs. 7 ± 2 mmHg · l-1 · min at week 3 (significant decrease over time; significant difference compared with preterm lambs at weeks 1 and 3, P < 0.05). Raw in these term lambs averaged 55 ± 14 cmH2O · l-1 · s at week 1 vs. 40 ± 17 cmH2O · l-1 · s at week 3 (no significant change over time; significant difference compared with preterm lambs at week 3, P < 0.05). Thus both PVR and Raw were greater in chronically ventilated preterm lambs than in control lambs that were born at term.

Immunoblot analysis for eNOS protein. Results of immunoblot analysis for eNOS protein in third- to fifth-generation intrapulmonary arteries that were dissected from the chronically ventilated preterm lambs and both groups of control lambs are shown in Fig. 1. A single band was detected at the expected size of 135 kDa (Fig. 1A). Quantitative densitometry showed that arteries from chronically ventilated preterm lambs, when compared with newborn and 3-wk-old control lambs, had ~45-60% less eNOS protein in the intrapulmonary arteries (P < 0.05; Fig. 1B).


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Fig. 1.   A: immunoblot analysis for endothelial nitric oxide synthase (eNOS) in dissected intrapulmonary arteries from 1-day-old (newborn) control lambs, 3-wk-old control lambs, and chronically ventilated preterm lambs. Signal for eNOS protein was detected as a single band at the expected size of 135 kDa. B: summary data as determined by densitometry. Values are means ± SD for 3-4 lambs/group. *P < 0.05 vs. newborn control and 3-wk-old control lambs.

Figure 2 shows the results of immunoblot analysis for eNOS protein in third- to fifth-generation airways that were dissected from the same chronically ventilated preterm lambs and control lambs. A prominent band was detected at the expected size of 135 kDa (Fig. 2A). Quantitative densitometry showed that the chronically ventilated preterm lambs, when compared with newborn and 3-wk-old control lambs, had ~35-40% less eNOS protein in the airways (P < 0.05; Fig. 2B).


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Fig. 2.   A: immunoblot analysis for eNOS in dissected intrapulmonary airways from 1-day-old (newborn) and 3-wk-old control lambs and chronically ventilated preterm lambs. Signal for eNOS protein was detected as a prominent band at the expected size of 135 kDa. B: summary data as determined by densitometry. Values are means ± SD for 3-4 lambs/group. *P < 0.05 vs. newborn control and 3-wk-old control lambs.

Immunoblot analysis for iNOS, alpha -smooth muscle actin, and pancytokeratin proteins. To see if the decrease in lung eNOS protein expression in lambs with CLD reflected a general loss of proteins in cells of arteries and airways (i.e., vascular or airway smooth muscle cells or airway epithelial cells), we performed immunoblot analysis on homogenates of lung tissue. We assessed alpha -smooth muscle actin protein abundance in homogenates of dissected arteries. We also assessed abundance of iNOS, alpha -smooth muscle actin, and pancytokeratin proteins in homogenates of dissected airways. There were no differences between the chronically ventilated preterm lambs and the two groups of control lambs (Table 2).

                              
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Table 2.   Immunoblot results for intrapulmonary arteries and airways

Immunohistochemical localization of eNOS protein. Immunohistochemical localization of eNOS protein in pulmonary arterioles of chronically ventilated preterm lambs and both groups of control lambs is shown in Fig. 3. Immunostaining was detected in the endothelium of all generations of arteries. We did not find immunostaining for eNOS protein in either vascular smooth muscle or adventitial cells. Immunostaining density for eNOS protein in the endothelium of the chronically ventilated preterm lambs appeared to be less than that seen in the two groups of control lambs. Relative immunostaining density for eNOS protein in arteriolar endothelium from 5-6 lamb lungs/group, as determined by semiquantitative computer-aided densitometry, corroborated this impression. Immunostaining density (in arbitrary densitometry units) for eNOS protein in arteriolar endothelium was significantly less in lung tissue sections from chronically ventilated preterm lambs (82 ± 41) than in lung tissue sections from control lambs that were either newborn (149 ± 10; P < 0.05) or 3 wk old (134 ± 10; P < 0.05). These results support the quantitative immunoblot results for third- to fifth-generation intrapulmonary arteries from the lungs of the same lambs (see Fig. 1).


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Fig. 3.   Immunohistochemistry for eNOS protein in pulmonary arterioles in lung tissue sections from a 1-day-old (newborn; a) and a 3-wk-old (b) control lamb and a preterm lamb that was mechanically ventilated for 3 wk (c). eNOS protein is detected by the brown immunostaining (arrows) in endothelial cells lining pulmonary arterioles (PA). eNOS immunostaining appears greatest in the endothelium of the newborn control lamb (a). eNOS immunostaining appears less in the endothelium of the chronically ventilated preterm lamb (c) compared with both control lambs (a and b). All photographs are the same magnification.

Immunolocalization of eNOS protein in terminal bronchioles of the same chronically ventilated preterm lambs and control lambs is shown in Fig. 4. Immunostaining was detected in the epithelium of all generations of airways. Neither airway smooth muscle nor adventitial cells exhibited eNOS protein immunostaining. Immunostaining density for eNOS protein in the bronchiolar epithelium of chronically ventilated preterm lambs appeared to be less than that in the control lambs. Semiquantitative computer-aided densitometry of terminal bronchiolar epithelium from 5-7 lambs/group showed significantly less eNOS immunostaining density (in arbitrary densitometry units) in bronchiolar epithelium of chronically ventilated preterm (111 ± 9) compared with newborn control (133 ± 9; P < 0.05) lambs. These results support the quantitative immunoblot results for intrapulmonary airways from the same chronically ventilated preterm and newborn control lambs. There was no significant difference in eNOS immunostaining density in bronchiolar epithelium of chronically ventilated preterm (111 ± 9) compared with 3-wk-old control (97 ± 19) lambs. The reason for this apparent discrepancy between immunoblot and immunohistochemistry results for central airways is unclear. It is possible that the expression of eNOS protein in airway epithelial cells may differ along the length of the respiratory tract, a notion that we have yet to evaluate.


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Fig. 4.   Immunohistochemistry for eNOS protein in terminal bronchiolar epithelium in lung tissue sections from a 1-day-old (newborn; a) and a 3-wk-old (b) control lamb and from a preterm lamb that was mechanically ventilated for 3 wk (c). eNOS protein is detected by the brown immunostaining (arrows) in epithelial cells lining terminal bronchioles (TB). eNOS immunostaining appears greatest in the epithelium of the newborn control lamb (a). eNOS immunostaining in the chronically ventilated preterm lamb (c) appears similar to that in the epithelium of the control lamb that was 3 wk old (b). All photographs are the same magnification.

Immunohistochemical localization of iNOS protein. Immunohistochemistry results for iNOS protein localization in pulmonary arterioles of chronically ventilated preterm lambs and in both groups of control lambs are shown in Fig. 5. Immunostaining was evident in the endothelium of all generations of arteries but not in their smooth muscle or adventitial cells. Alveolar macrophages were intensely immunostained (data not shown) as expected. Immunostaining density for iNOS protein in the endothelium of pulmonary arterioles appeared the same among the chronically ventilated preterm and both groups of control lambs. This impression was supported by semiquantitative computer-aided densitometry. Immunostaining densitometry for iNOS protein in endothelium of pulmonary arterioles of chronically ventilated preterm lambs (122 ± 9) was the same as in control lambs that were either newborn (117 ± 8) or 3 wk old (112 ± 8).


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Fig. 5.   Immunohistochemistry for inducible NOS (iNOS) protein in PA in lung tissue sections from a 1-day-old (newborn; a) and a 3-wk-old (b) control lamb and from a preterm lamb that was mechanically ventilated for 3 wk (c). iNOS protein is detected by the brown immunostaining (arrows) in endothelial cells lining PA. iNOS immunostaining appears greatest in the endothelium of the newborn control lamb (a). iNOS immunostaining appears less in the endothelium of the chronically ventilated preterm lamb (c) compared with both control lambs (a and b). All photographs are the same magnification.

Immunohistochemical localization of iNOS protein in terminal bronchioles of the same chronically ventilated preterm lambs and control lambs is shown in Fig. 6. Immunostaining was detected in the epithelium but not in smooth muscle or adventitial cells of all generations of airways. The epithelium of terminal bronchioles appeared to have the same immunostaining density for iNOS protein in the chronically ventilated preterm lambs as in both groups of control lambs. Semiquantitative computer-aided densitometry of terminal bronchiolar epithelium showed no significant difference in iNOS immunostaining density between the chronically ventilated preterm lambs (92 ± 19) and the control lambs that were newborn (93 ± 19) or 3 wk old (86 ± 22).


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Fig. 6.   Immunohistochemistry for iNOS protein in terminal bronchiolar epithelium in lung tissue sections from 1-day-old (newborn; a) and a 3-wk-old (b) control lamb and a preterm lamb that was mechanically ventilated for 3 wk (c). iNOS protein is detected by the brown immunostaining (arrows) in epithelial cells lining terminal bronchioles (TB). iNOS immunostaining appears greatest in the epithelium of the newborn control lamb (a). iNOS immunostaining in the chronically ventilated preterm lamb (c) appears similar to that in the epithelium of the control lamb that was 3 wk old (b). All photographs are the same magnification.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We have shown that eNOS protein content is decreased in arteries and airways in the lungs of chronically ventilated preterm lambs compared with that in control lambs of similar postconceptional and postnatal ages. In contrast to the reduced eNOS expression in the lungs of lambs with CLD, there was no difference in the abundance of other proteins (iNOS, alpha -smooth muscle actin, or pancytokeratin) in either arteries or airways of these chronically ventilated preterm lambs compared with control lambs. To our knowledge, this is the first study to show diminished eNOS protein expression in lungs of animals with CLD of prematurity.

The lambs used in this study were a subset of lambs that has been used to examine the physiological and pathological effects of prolonged mechanical ventilation after premature birth (3, 7, 33). Pulmonary vascular resistance in the chronically ventilated preterm lambs did not decrease significantly during the 3-wk study period and was significantly greater than that in 3-wk-old control lambs (7). There also was persistent muscularization of pulmonary arterioles (landmark reference was terminal bronchioles) in the same chronically ventilated preterm lambs compared with control lambs (7). The diminished abundance of eNOS protein in pulmonary arterial vessels, shown by immunoblot and immunohistochemical analyses of arterial vessels from the lungs of the same lambs and with the same anti-eNOS antibody, provides a potential explanation for the persistent elevation of pulmonary vascular resistance seen in chronically ventilated preterm lambs.

NO production in the lungs is important in facilitating the normal decrease in pulmonary vascular resistance that occurs at birth. Studies (1, 12) have shown that inhibition of NO production attenuated ~50% of the normal fall in pulmonary vascular resistance at birth in fetal lambs. Another study (31) has shown that the expression of eNOS protein increases in rat lung during late gestation, becoming maximal near term. A similar pattern of increasing eNOS protein expression during development was seen in fetal sheep, although maximal expression was seen earlier in gestation (32). These studies indicate that NO has a role in regulating pulmonary vascular resistance during development.

The role of NO in regulating pulmonary vascular resistance has been studied in other models of neonatal lung disease. Congenital diaphragmatic hernia is a clinical condition that is associated with elevated pulmonary vascular resistance and increased muscularization of pulmonary arteries (26). North et al. (30) found that the abundance of eNOS protein and mRNA was decreased in the lungs of rats with pharmacologically induced congenital diaphragmatic hernia compared with levels in control rats. In another study, Shaul et al. (44) discovered that fetal lambs born 7-14 days after mechanical constriction of the ductus arteriosus had persistent pulmonary hypertension that was associated with less pulmonary eNOS activity, protein content, and mRNA abundance compared with control lambs. In contrast, Black et al. (5) found that eNOS expression was increased in intrapulmonary arteries of lambs that had increased lung blood flow through a surgically produced aorta-to-pulmonary shunt. These authors (5) attributed this difference in eNOS to increased shear stress in the pulmonary circulation, which also exhibited increased pulmonary vascular resistance and increased smooth muscle in intrapulmonary arteries (37). These findings taken together indicate that decreased NO production may be important in the development of pulmonary hypertension in some but not all neonatal cardiopulmonary disorders.

Previous studies have shown that NO may affect proliferation of vascular smooth muscle cells both in vivo and in vitro. Several studies (17, 25, 42) have shown that adult rats exposed to exogenous NO, either by inhalation of the gas or by treatment with a NO donor, have less smooth muscle mass in their arteries after mechanical injury than rats that did not receive NO. Other reports indicate that inhibition of endogenous NO production leads to increased accumulation of vascular smooth muscle. For example, Rudic et al. (41) found that mice with a targeted disruption of the eNOS gene displayed an increased arterial wall thickness after ligation of the external carotid artery. In vitro studies (14, 49) with cultured aortic smooth muscle cells have shown a dose-dependent inhibition of cell growth by NO-releasing vasodilators and solutions saturated with NO gas. In a study with cultured pulmonary artery smooth muscle cells, Thomae et al. (47) found a biphasic effect of NO on cell growth. High concentrations of NO inhibited pulmonary artery smooth muscle cell growth, whereas low concentrations stimulated growth. Our in vivo observations of reduced eNOS abundance associated with greater pulmonary vascular resistance and arteriolar smooth muscle are consistent with the above observations in rodent smooth muscle cells. Thus diminished expression of eNOS protein in the pulmonary arterial tree of preterm lambs with CLD might account, at least in part, for the persistent muscularization of the resistance arterioles in the lungs of these lambs (7). That is, diminished expression of eNOS protein might contribute to failure of the regression of pulmonary vascular smooth muscle that normally occurs postnatally.

We also found that eNOS protein abundance was decreased in airway epithelium of lambs with CLD compared with that in healthy term lambs. As previously reported (3), airway resistance did not change significantly in these preterm lambs during the 3 wk of mechanical ventilation; airway resistance was significantly greater in our lambs with CLD than it was in our control lambs. There was also increased smooth muscle accumulation around terminal bronchioles in the same chronically ventilated lambs compared with control lambs (3). Our observation of increased airway resistance and smooth muscle accumulation around small airways in chronically ventilated preterm lambs is consistent with the notion that CLD may be associated with decreased endogenous production of NO due to diminished abundance of eNOS protein in airway epithelium.

A physiological role for NO in regulating airway resistance is supported by both in vitro and in vivo studies. NO is produced in the airway by epithelial cells (43). In the mature airway, NO induces smooth muscle relaxation, participates in neurotransmission and bacteriostasis, and modulates ciliary motility and mucin secretion (4, 15). There is also evidence for the role of epithelium-derived NO in the regulation of bronchomotor tone in the developing lung (20, 34). Sherman et al. (45) recently showed that eNOS is expressed in bronchial and bronchiolar epithelium of fetal and newborn sheep, indicating that this enzyme may be important in the generation of NO in the airway during development. Similar to the effect of NO on vascular smooth muscle growth, NO donors inhibit serum- and thrombin-induced proliferation of cultured human airway smooth muscle cells (18).

In this study, we measured neither NO production nor NOS activity. Previous studies (29, 44), however, have shown that alterations in eNOS protein content are accompanied by parallel changes in NOS activity in both cultured cells and isolated tissues. We therefore measured eNOS protein content as a marker of the ability to produce NO.

In the pulmonary vasculature and airways, eNOS is but one of multiple sources of NO production. Recent studies (35-37) showed that both iNOS and nNOS may contribute to the regulation of pulmonary vascular resistance in the developing fetus. Sherman et al. (45) described expression of both iNOS and nNOS, in addition to eNOS, in airway epithelial cells of fetal, newborn, and adult sheep.

We examined the expression of iNOS protein in lung tissue from the same preterm lambs with CLD and control lambs. Our immunoblot results, obtained on dissected airways that were available for only two of the chronically ventilated preterm lambs, indicated a trend toward diminished iNOS protein in intrapulmonary airways. The immunohistochemical results indicated that iNOS protein expression in terminal bronchiolar epithelial cells was the same between the chronically ventilated preterm lambs and the two groups of control lambs. Likewise, endothelial cell expression of iNOS protein, detected immunohistochemically, was the same between the chronically ventilated preterm lambs and the two groups of control lambs. Another pulmonary source of iNOS protein expression, and therefore of endogenous NO generation in the lung, is alveolar macrophages (21, 23, 48). Their contribution to NO production in CLD compared with that of vascular endothelium and airway epithelium was not assessed in our study.

In summary, we found that eNOS protein expression is decreased in central and peripheral pulmonary arteries and airways from chronically ventilated preterm lambs compared with term lambs of similar developmental and postnatal ages. This decrease in eNOS protein expression was not associated with a change in iNOS abundance, alpha -smooth muscle actin, or pancytokeratin within the same anatomic structures. This decrease of eNOS protein abundance, therefore, may contribute to the excess accumulation of smooth muscle around pulmonary arterioles and bronchioles and to the increase in pulmonary vascular and airway resistance seen in chronically ventilated preterm lambs. These structural and functional abnormalities of the pulmonary vasculature and airways are similar to those seen in human infants with CLD of prematurity (3, 7). Our results suggest that decreased eNOS in the pulmonary circulation and respiratory tract of preterm lambs may play a role in the pathophysiology of CLD.


    ACKNOWLEDGEMENTS

We thank Nancy Chandler (Health Sciences Center Research Microscopy Facility, University of Utah, Salt Lake City, UT) for technical assistance with these studies.


    FOOTNOTES

This work was supported in part by March of Dimes Birth Defects Foundation Grant 6-FY97-0138 (to R. D. Bland), American Heart Association Grant 96014370 (to K. H. Albertine), and National Heart, Lung, and Blood Institute (NHLBI) Grants HL-62512 (to R. D. Bland) and HL-62875 (to K. H. Albertine).

Studies included in this report were conducted during the tenure of a Fellowship-to-Faculty Transition Award (to A. N. MacRitchie) supported in part by the Howard Hughes Medical Institute under the Research Resources Program for Medical Schools and NHLBI Research Training Grant T35-HL-07744 for medical students (to S. C. Jensen and A. A. Freestone).

Address for reprint requests and other correspondence: R. D. Bland, Dept. of Pediatrics, Univ. of Utah School of Medicine, 50 North Medical Dr., Salt Lake City, UT 84132.

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.

Received 13 October 2000; accepted in final form 6 June 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   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].

2.   Abman, SH, and Groothius JR. Pathophysiology and treatment of bronchopulmonary dysplasia. Pediatr Clin North Am 41: 277-315, 1994[ISI][Medline].

3.   Albertine, KH, Jones GP, Starcher BC, Bohnsack JF, Davis PL, Cho S, Carlton DP, and Bland RD. Chronic lung injury in preterm lambs: disordered respiratory tract development. Am J Respir Crit Care Med 159: 945-958, 1999[Abstract/Free Full Text].

4.   Barnes, PJ. Nitric oxide and airway disease. Ann Med 27: 380-393, 1995.

5.   Black, SM, Fineman JR, Steinhorn RH, Bristow J, and Soifer SJ. Increased endothelial NOS in lambs with increased pulmonary blood flow and pulmonary hypertension. Am J Physiol Heart Circ Physiol 275: H1643-H1651, 1998[Abstract/Free Full Text].

6.   Black, SM, Johengen MJ, and Soifer SJ. Coordinated regulation of genes of the nitric oxide and endothelin pathways during the development of pulmonary hypertension in fetal lambs. Pediatr Res 44: 821-830, 1998[Abstract].

7.   Bland, RD, Albertine KH, Carlton DP, Kullama L, Davis P, Cho S, Kim B, Dahl M, and Tabatabaie N. Chronic lung injury in preterm lambs: abnormalities of the pulmonary circulation and lung fluid balance. Pediatr Res 48: 64-74, 2000[Abstract/Free Full Text].

8.   Bolender, RP, Hyde DM, and Dehoff RT. Lung morphometry: a new generation of tools and experiments for organ, tissue, cell, and molecular biology. Am J Physiol Lung Cell Mol Physiol 265: L521-L548, 1993[Abstract/Free Full Text].

9.   Clark, R, Kueser T, Walker M, Southgate W, Huckaby J, Perez J, Roy B, Keszler M, and Kinsella J. Low-dose nitric oxide therapy for persistent pulmonary hypertension of the newborn. N Engl J Med 342: 469-474, 2000[Abstract/Free Full Text].

10.   Cornfield, D, Maynard R, deRegnier R, Guiang S, Barbato J, and Milla C. Randomized, controlled trial of low-dose inhaled nitric oxide in the treatment of term and near-term infants with respiratory failure and pulmonary hypertension. Pediatrics 104: 1089-1094, 1999[Abstract/Free Full Text].

11.   Day, R, Lynch J, White K, and Ward J. Acute response to inhaled nitric oxide in newborns with respiratory failure and pulmonary hypertension. Pediatrics 98: 698-705, 1996[Abstract].

12.   Fineman, J, Wong J, Morin F, Wild L, and Soifer S. Chronic nitric oxide inhibition in utero produces persistent pulmonary hypertension in newborn lambs. J Clin Invest 93: 2675-2683, 1994[ISI][Medline].

13.   Frostell, C, Fratacci MD, Wain JC, Jones R, and Zapol WM. Inhaled nitric oxide, a selective pulmonary vasodilator reversing hypoxic pulmonary vasoconstriction. Circulation 83: 2038-2047, 1991[Abstract].

14.   Garg, UC, and Hassid A. Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J Clin Invest 83: 1774-1777, 1989[ISI][Medline].

15.   Gaston, B, Drazen JM, Loscalzo J, and Stamler JS. The biology of nitrogen oxides in the airways. Am J Respir Crit Care Med 149: 538-551, 1994[Abstract].

16.   Group Neonatal Inhaled Nitric Oxide Study. Inhaled nitric oxide in full-term and nearly full-term infants with hypoxic respiratory failure. N Engl J Med 336: 597-604, 1997[Abstract/Free Full Text].

17.   Guo, JP, Panday MM, Consigny PM, and Lefer AM. Mechanisms of vascular preservation by a novel NO donor following rat carotid artery intimal injury. Am J Physiol Heart Circ Physiol 269: H1122-H1131, 1995[Abstract/Free Full Text].

18.   Hamad, AM, Johnson SR, and Knox AJ. Antiproliferative effects of NO and ANP in cultured human airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 277: L910-L918, 1999[Abstract/Free Full Text].

19.   Hislop, AA, and Haworth SG. Pulmonary vascular damage and the development of cor pulmonale following hyaline membrane disease. Pediatr Pulmonol 9: 152-161, 1990[ISI][Medline].

20.   Jakupaj, M, Martin RJ, Dreshaj IA, Potter CF, Haxhiu MA, and Ernsberger P. Role of endogenous NO in modulating airway contraction mediated by muscarinic receptors during development. Am J Physiol Lung Cell Mol Physiol 273: L531-L536, 1997[Abstract/Free Full Text].

21.   Jorens, PG, Van Overveld FJ, Bult H, Vermeire PA, and Herman AG. L-Arginine-dependent production of nitrogen oxides by rat pulmonary macrophages. Eur J Pharmacol 200: 205-209, 1991[ISI][Medline].

22.   Kinsella, JP, Neish SR, Shaffer E, and Abman SH. Low-dose inhalational nitric oxide in persistent pulmonary hypertension of the newborn. Lancet 340: 819-820, 1992[ISI][Medline].

23.   Kobzik, L, Bredt DS, Lowenstein CJ, Drazen H, Gaston B, Sugarbaker D, and Stamler JS. Nitric oxide synthase in human and rat lung: immunocytochemical and histochemical localization. Am J Respir Cell Mol Biol 9: 371-377, 1993[ISI][Medline].

24.   Laemmli, UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970[ISI][Medline].

25.   Lee, JS, Adrie C, Jacob HJ, Roberts JD, Jr, Zapol WM, and Bloch KD. Chronic inhalation of nitric oxide inhibits neointimal formation after balloon-induced arterial injury. Circ Res 78: 337-342, 1996[Abstract/Free Full Text].

26.   Levin, DL. Morphologic analysis of the pulmonary vascular bed in congenital left-sided diaphragmatic hernia. J Pediatr 92: 805-809, 1978[ISI][Medline].

27.   Lonnqvist, P, Winberg P, Lundell B, Sellden H, and Olsson G. Inhaled nitric oxide in neonates and children with pulmonary hypertension. Acta Paediatr 83: 1132-1136, 1994[ISI][Medline].

28.   Margraf, LR, Tomashefski JF, Jr, Bruce MC, and Dahms BB. Morphometric analysis of the lung in bronchopulmonary dysplasia. Am Rev Respir Dis 143: 391-400, 1991[ISI][Medline].

29.   North, AJ, Lau KS, Brannon TS, Wu LC, Wells LB, German Z, and Shaul PW. Oxygen upregulates nitric oxide synthase gene expression in ovine fetal pulmonary artery endothelial cells. Am J Physiol Lung Cell Mol Physiol 270: L643-L649, 1996[Abstract/Free Full Text].

30.   North, AJ, Moya FR, Mysore MR, Thomas VL, Wells LB, Wu LC, and Shaul PW. Pulmonary endothelial nitric oxide synthase gene expression is decreased in a rat model of congenital diaphragmatic hernia. Am J Respir Cell Mol Biol 13: 676-682, 1995[Abstract].

31.   North, AJ, Star RA, Brannon TS, Ujiie K, Wells LB, Lowenstein CJ, Snyder SH, and Shaul PW. Nitric oxide synthase type I and type III gene expression are developmentally regulated in rat lung. Am J Physiol Lung Cell Mol Physiol 266: L635-L641, 1994[Abstract/Free Full Text].

32.   Parker, TA, Le Cras TD, Kinsella JP, and Abman SH. Developmental changes in endothelial nitric oxide synthase expression and activity in ovine fetal lung. Am J Physiol Lung Cell Mol Physiol 278: L202-L208, 2000[Abstract/Free Full Text].

33.   Pierce, RA, Albertine KH, Starcher BC, Bohnsack JF, Carlton DP, and Bland RD. Chronic lung injury in preterm lambs: disordered pulmonary elastin deposition. Am J Physiol Lung Cell Mol Physiol 272: L452-L460, 1997[Abstract/Free Full Text].

34.   Potter, CF, Dreshaj IA, Haxhiu MA, Stork EK, Chatburn RL, and Martin RJ. Effect of exogenous and endogenous nitric oxide on the airway and tissue components of lung resistance in the newborn piglet. Pediatr Res 41: 886-891, 1997[Abstract].

35.   Rairigh, RL, Le Cras TD, Ivy D, Kinsella JP, Richter G, Horan MP, Fan I, and Abman SH. Role of inducible nitric oxide synthase in regulation of pulmonary vascular tone in the late gestation ovine fetus. J Clin Invest 101: 15-21, 1998[Abstract/Free Full Text].

36.   Rairigh, RL, Storme L, Parker TA, Le Cras TD, Kinsella JP, Jakkula M, and Abman SH. Inducible NO synthase inhibition attenuates shear stress-induced pulmonary vasodilation in the ovine fetus. Am J Physiol Lung Cell Mol Physiol 276: L513-L521, 1999[Abstract/Free Full Text].

37.   Reddy, VM, Meyrick B, Wong J, Khoor A, Liddicoat JR, Hanley FL, and Fineman JR. In utero placement of aortopulmonary shunts. A model of postnatal pulmonary hypertension with increased pulmonary blood flow in lambs. Circulation 92: 606-613, 1995[Abstract/Free Full Text].

38.   Roberts, JD, Chen TY, Kawai N, Wain J, Dupuy P, Shimouchi A, Bloch K, Polaner D, and Zapol WM. Inhaled nitric oxide reverses pulmonary vasoconstriction in the hypoxic and acidotic newborn lamb. Circ Res 72: 246-254, 1993[Abstract].

39.   Roberts, JD, Jr, Fineman JR, Morin FC, III, Shaul PW, Rimar S, Schreiber MD, Polin RA, Zwass MS, Zayek MM, Gross I, Heymann MA, and Zapol WM. Inhaled nitric oxide and persistent pulmonary hypertension of the newborn. The Inhaled Nitric Oxide Study Group. N Engl J Med 336: 605-610, 1997[Abstract/Free Full Text].

40.   Roberts, JD, Polander DM, Lang P, and Zapol WM. Inhaled nitric oxide in persistent pulmonary hypertension of the newborn. Lancet 340: 818-819, 1992[ISI][Medline].

41.   Rudic, RD, Shesely EG, Maeda N, Smithies O, Segal SS, and Sessa WC. Direct evidence for the importance of endothelium-derived nitric oxide in vascular remodeling. J Clin Invest 101: 731-736, 1998[Abstract/Free Full Text].

42.   Seki, J, Nishio M, Kato Y, Motoyama Y, and Yoshida K. FK409, a new nitric-oxide donor, suppresses smooth muscle proliferation in the rat model of balloon angioplasty. Atherosclerosis 117: 97-106, 1995[ISI][Medline].

43.   Shaul, PW. Nitric oxide in the developing lung. Adv Pediatr 42: 367-414, 1995[Medline].

44.   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].

45.   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].

46.   Smith, PK, Krohn RI, Hermanson GT, Mallia AK, and Gartner FH. Measurement of protein using bicinchoninic acid. Anal Biochem 150: 76-85, 1985[ISI][Medline].

47.   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].

48.   Tracey, WR, Xue C, Klinghofer V, Barlow J, Pollock JS, Förstermann U, and Johns RA. Immunochemical detection of inducible NO synthase in human lung. Am J Physiol Lung Cell Mol Physiol 266: L722-L727, 1994[Abstract/Free Full Text].

49.   Yang, W, Ando J, Korenaga R, Toyo-oka T, and Kamiya A. Exogenous nitric oxide inhibits proliferation of cultured vascular endothelial cells. Biochem Biophys Res Commun 203: 1160-1167, 1994[ISI][Medline].

50.   Zar, J. Biostatistical Analysis (2nd ed.). Englewood Cliffs, NJ: Prentice-Hall, 1984, p. 162-235.

51.   Zayek, M, Cleveland D, and Morin F. Treatment of persistent pulmonary hypertension in the newborn lamb by inhaled nitric oxide. J Pediatr 122: 743-750, 1993[ISI][Medline].

52.   Zayek, M, Wild L, Roberts J, and Morin F. Effect of inhaled nitric oxide on the survival rate and incidence of lung injury in newborn lambs with persistent pulmonary hypertension. J Pediatr 123: 947-952, 1993[ISI][Medline].


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