Department of Pediatrics, University of Utah, Salt Lake City, Utah 84132
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ABSTRACT |
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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, -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
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INTRODUCTION |
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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.
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METHODS |
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Animal model.
We delivered fetal sheep prematurely (124 ± 3 days, term 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).
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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 NImmunoblot analyses of inducible NOS, -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).
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.
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RESULTS |
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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 · l1 · 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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Immunoblot analysis for iNOS, -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
-smooth muscle actin protein abundance in
homogenates of dissected arteries. We also assessed abundance of iNOS,
-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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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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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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DISCUSSION |
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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, -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,
-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.
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ACKNOWLEDGEMENTS |
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We thank Nancy Chandler (Health Sciences Center Research Microscopy Facility, University of Utah, Salt Lake City, UT) for technical assistance with these studies.
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FOOTNOTES |
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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.
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