Pulmonary nitric oxide synthases and nitrotyrosine: findings during lung development and in chronic lung disease of prematurity.

BACKGROUND: Nitric oxide mediates and modulates pulmonary transition from fetal to postnatal life. NO is synthesized by 3 nitric oxide synthase isoforms. One key pathway of nitric oxide metabolism results in nitrotyrosine, a stable, measurable marker of nitric oxide production. OBJECTIVE: The purpose of this study was to assess, by semiquantitative immunohistochemistry, nitric oxide synthase isoforms and nitrotyrosine at different airway and vascular tree levels in the lungs of neonates at different gestational ages and to compare results in control groups to those in infants with chronic lung disease. DESIGN/METHODS: Formalin-fixed, paraffin-embedded, postmortem lung blocks were prepared for immunohistochemistry using antibodies to each nitric oxide synthase isoform and to nitrotyrosine. Blinded observers evaluated the airway and vascular trees for staining intensity (0-3 scale) at 5 levels and 3 levels, respectively. The control population consisted of infants from 22 to 42 weeks' gestation who died in < 48 hours. Results were compared with gestation-matched infants with varying severity of chronic lung disease. RESULTS: In control and chronic lung disease groups, 22 to 42 weeks' gestation, staining for all 3 of the nitric oxide synthase isoforms was found in the airway epithelium from the bronchus to the alveolus or distal-most airspace. The abundance or distribution of nitric oxide synthase-3 staining in the airways did not show significant correlation with gestational age or severity of chronic lung disease. In the vascular tree, intense nitric oxide synthase-3 and moderate nitric oxide synthase-2 staining was found; nitric oxide synthase-1 was not consistently stained. Nitrotyrosine did stain in the pulmonary tree. Compared with controls where nitrotyrosine staining was minimal, regardless of gestation, in infants with chronic lung disease there was more than fourfold increase between severe chronic lung disease (n = 12) and either mild chronic lung disease or control infants (n = 16). CONCLUSIONS: All 3 of the nitric oxide synthase isoforms and nitrotyrosine are detectable by immunohistochemistry early in lung development. Nitric oxide synthase ontogeny shows no significant changes in abundance or distribution with advancing gestational age nor with chronic lung disease. Nitrotyrosine is significantly increased in severe chronic lung disease.

Hyperoxia and tidal volume: Independent and combined effects on neonatal pulmonary inflammation.

BACKGROUND: Hyperoxia and tidal volume mechanical ventilation are independent factors in the genesis of lung injury, but it remains unclear the extent to which each is responsible or contributes to this process in newborns. OBJECTIVES: To study the independent and combined effects of hyperoxia and tidal volume mechanical ventilation on the induction of lung inflammation in a newborn piglet model of ventilator-induced lung injury. METHODS: Following exposure to either ambient air or F(I)O2 = 1.0 for a period of 3 days, newborn piglets were randomized to receive mechanical ventilation with either high tidal volume (20 ml/kg) or low tidal volume (6 ml/kg) for 4 h while controlling for pH. RESULTS: Monocyte chemoattractant protein-1 level in the lungs of animals randomized to hyperoxia with high tidal volume ventilation was significantly elevated, compared to all other groups (p < 0.05). Myeloperoxidase assayed in lung homogenate was found to be significantly higher in nonventilated animals exposed to hyperoxia (p < 0.01). Only in animals previously exposed to hyperoxia did the addition of high tidal volume ventilation further increase the level of myeloperoxidase present (p < 0.05). Pulmonary vascular resistance was significantly elevated after 4 h of mechanical ventilation compared to 1 h (p < 0.001). CONCLUSIONS: We conclude that in neonatal piglets undergoing hyperoxic stress, superimposition of high tidal volume ventilation exacerbates the lung inflammation as assessed by lung monocyte chemoattractant protein-1 and level of myeloperoxidase.

Responses of pulmonary platelet-derived growth factor peptides and receptors to hyperoxia and nitric oxide in piglet lungs.

The peptides platelet-derived growth factor-A (PDGF-A) and especially -B have important roles in lung development. The effect of hyperoxic exposure with and without inhaled nitric oxide (iNO) on lung expression of PDGF and its receptors is unknown. We hypothesized that hyperoxia exposure would suppress mRNA expression and protein production of these ligands and their receptors. The addition of iNO to hyperoxia may further aggravate the effects of hyperoxia. Thirteen-day-old piglets were randomized to breathe 1) room air (RA); 2) 0.96 fraction of inspired oxygen (O2), or 3) 0.96 fraction of inspired oxygen plus 50 ppm of NO (O2+NO), for 5 d. Lungs were preserved for mRNA, Western immunoblot, and immunohistochemical analyses for PDGF-A and -B and their receptors PDGFR-alpha and -beta. PDGF-B mRNA expression was greater than that of PDGF-A or PDGFR-alpha and -beta in RA piglet lungs (p<0.05). Hyperoxia with or without iNO reduced lung PDGF-B mRNA and protein expression relative to the RA group lungs (p<0.01). PDGF-B immunostain intensity was significantly increased in the alveolar macrophages, which were present in greater numbers in the hyperoxia-exposed piglet lungs, with or without NO (p<0.01). PDGFR-beta immunostaining was significantly increased in airway epithelial cells in O2- and O2+NO-exposed piglets. PDGF-A and PDGFR-alpha immunostain intensity and distribution pattern were unchanged relative to the RA group. Sublethal hyperoxia decreases PDGF-B mRNA and protein expression but not PDGF-A or their receptors in piglet lungs. iNO neither aggravates nor ameliorates this effect.

Low levels of tissue inhibitors of metalloproteinases with a high matrix metalloproteinase-9/tissue inhibitor of metalloproteinase-1 ratio are present in tracheal aspirate fluids of infants who develop chronic lung disease.

OBJECTIVE: The pathogenesis of chronic lung disease (CLD) involves inflammation with proteolytic damage to lung extracellular matrix. Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that, acting in concert with their tissue inhibitors, tightly orchestrate extracellular matrix morphogenesis and repair after injury. Imbalances in their levels relative to that of their inhibitors have been implicated in diseases characterized by matrix disruption and remodeling. We investigated the possibility that imbalances in MMP-9 and MMP-2 relative to their tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2, respectively, in tracheal aspirates of preterm infants may be involved in the development of CLD. METHODS: Serial tracheal aspirates collected from birth until extubation in 49 ventilated preterm infants (24-32 weeks' gestations) were analyzed for MMP-2, MMP-9, TIMP-1, and TIMP-2. Data normalized by TA values of free secretory component of immunoglobulin A were compared for CLD (n = 22) versus no CLD (n = 27). Also, known clinical predictors of CLD (gestational age, birth weight, and sex) were assessed for both groups. Association of predictors with the outcome CLD was assessed by logistic regression. RESULTS: Mean gestational age was lower in CLD infants, but birth weight and gender were comparable for both groups. CLD infants had significantly lower TIMP-1 level with higher MMP-9/TIMP-1 ratio during the first 2 weeks of life and low TIMP-2 and MMP-2 levels during the first 3 days of life compared with no-CLD infants. Logistic regression analysis indicated that the findings are predictive of CLD. CONCLUSIONS: We conclude that low tracheal aspirate levels of TIMPs, with a high MMP-9/TIMP-1 ratio early in life, are associated with subsequent development of CLD.

Lung microvascular adaptation in infants with chronic lung disease.

Microvascular development is critical for normal lung maturation. The aims of this study were (1) to quantitatively and qualitatively assess lung microvascular growth in the human fetus, from 22 to 40 weeks' gestation, and (2) to compare development in these infants to those with mild, moderate and severe chronic lung disease (CLD). Using 1- and 4-microm thick sections and electron microscopy, lungs were morphometrically assessed for surface density of distal air spaces; volume density of parenchymal vessels having an air-blood barrier (ABB); percent of distal air space wall having an ABB, and capillary loading, defined as ABB/mm2 of epithelial surface area. The percent of vessels with ABB increased in controls during development in parallel with increasing lung parenchyma. Infants with severe CLD had fewer ABBs and less capillary loading than controls up to 34 weeks' post-conceptional age (PCA), but by 36-40 weeks, showed catch-up growth. Microvasculature vessel diameter, septal thickness, and air sac diameter at 36-40 weeks' PCA were increased with severe CLD, and vessels were more distant from the air surface. We conclude that infants with severe CLD have both stunted secondary septation and microvascular development, but over time, the primary septal wall adapts by thinning and increasing the number of ABBs, thereby taking on the function of secondary septa.

Acinar arterial changes with chronic lung disease of prematurity in the surfactant era.

Because echocardiographic studies on infants with chronic lung disease (CLD) suggest that pulmonary hypertension (PH) may contribute to its severity, we studied acinar arterial walls in the following surfactant-era infants: controls (n=38): 22-41 weeks of gestational age (GA), exposed briefly to oxygen and positive pressure ventilation, died within 48 hr of birth; prolonged rupture of fetal membranes (PROM) and persistent pulmonary hypertension (PPHN) (n=17); and SCORE (integrated area under curve of average daily FiO2 x average daily MAP) groups (<20, 20-69, and 70-500; mild, moderate, and severe clinical lung disease, respectively, n=35): 23-30 weeks GA, lived 7-79 days. Lungs were stained for elastic tissue and smooth muscle actin. Vessels were assessed for percent of vessel circumference with smooth muscle, extent of elastic laminae in the walls, and percent arterial wall thickness (%AWT) at three levels: terminal to respiratory bronchiole transition (TRB), alveolar duct, and saccule. At the alveolar ductal and saccular levels, percent arterial wall thickness (%AWT) in mild CLD (SCORE < 20) was less than controls (P < 0.05) and those with more severe CLD (SCORE 70-500), indicating that normal postnatal arterial wall thinning may be delayed, or there is remodeling associated with increased %AWT. Severe CLD infants also had a significantly higher percent of circumferential actin than those with milder disease (SCORE < or = 69) and controls. In moderate and severe CLD, there was an increase in extent of the elastic laminae compared to controls and mild CLD. These changes were also significantly greater in PROM and PPHN infants compared to even severe CLD. We conclude that PH is a real possibility in severe CLD infants after discharge at 36 weeks. Grading the severity of CLD at discharge, and echocardiographic studies, may guide subsequent oxygen therapy.

Exhaled nitric oxide and tracheal endothelin-1 in preterm infants with and without RDS.

We measured exhaled nitric oxide and tracheal aspirate endothelin-1 to determine relationships between these substances and alterations in pulmonary gas exchange during respiratory distress syndrome (RDS) in comparison to those obtained from control preterm infants without RDS. Eight infants with RDS had measurements made at 24 hr and again at 48-72 hr. Eight control infants were studied once at 24-48 hr of life. Exhaled gas was analyzed on-line, and minute excretion of NO (V(NO)) was calculated. ET-1 was determined by immunoassay. Median V(NO) at 24 hr in RDS was 0.405 nl/min/kg (range, 0.30 -0.79), which subsequently declined by 48-72 hr to 0.166 nl/min/kg (P < 0.01). The V(NO) in RDS infants was significantly higher than time-matched V(NO) in controls, with a median of 0.099 nl/min/kg (range, 0.03-0.27; P < 0.001). ET-1 was not correlated with initial V(NO) in the RDS or control patients. In conclusion, in RDS, V(NO) decreases as gas exchange improves. ET-1 is detectable in tracheal aspirate samples in both groups of infants.

Collagen scaffolding during development and its deformation with chronic lung disease.

OBJECTIVE: Infants with chronic lung disease (CLD) have an arrest of primary and secondary septation. We hypothesized that this may be related to damage or abnormal development of lung collagen secondary to positive pressure ventilation. Our aims were to identify the sites and quantity of collagen in control infants 22 to 72 weeks' postconceptional age and compare these with infants with various degrees of severity of CLD. METHODS: The controls were 22 to 42 weeks' gestation (n = 30), received minimal ventilator care, and died within 48 hours of birth, plus 5 term infants who died at 43 to 72 weeks' postconceptional age from nonpulmonary causes. Infants who were 23 to 30 weeks' gestation, were at risk for CLD, and lived 5 to 94 days (n = 33) were separated into 3 groups on the basis of respiratory score (score group; the integrated area under the curve of the average daily fraction of inspired oxygen x mean airway pressure [cm H2O] over the number of days lived). The score groups, <20, 20 to 69, and 70 to 500, related clinically to mild to moderate and severe lung disease. The lungs were tracheally perfused and formalin fixed. Total lung volume was determined by water displacement. The paraffin-embedded lung blocks were sectioned 5 micro m thick, stained with Gomori's reticulum stain, hematoxylin and eosin, and immunohistochemically for collagen IV. The parenchyma was point-counted, and the volume density of collagen was measured. The chord diameter of the peripheral airway saccules and alveoli was measured. Descriptive collagen data were assessed on en face 40- micro m-thick sections through the alveolar or saccular walls on all infants at risk for CLD and in selected controls. RESULTS: In the controls, the volume density of collagen decreased from a maximum of 9% at 22 weeks to 5% at term and 72 weeks. With Scores < or =69, the fraction of collagen was similar to controls, but in infants with scores 70 to 500, it was increased relative to controls. However, when collagen was expressed as the volume density of interstitial tissue, ie, excluding parenchymal air space, it increased from a low of 5% at 22 weeks to 25% at 72 weeks. In infants with scores 70 to 500, 79% of infants had collagens greater than controls. Saccular and alveolar diameter increased from 40 micro m at 23 weeks to 100 microm at 72 weeks. Most infants with severe CLD (scores > or =70) had diameters more than twice that of controls at the same age. The total lung parenchymal collagen had a similar pattern as the volume density of collagen in interstitial tissue, increasing from 0.4 cm3 at 23 weeks to 9.7 cm3 at 72 weeks in the controls. Eighty-five percent of infants with scores 70 to 500 had total parenchymal collagen greater than the 95% confidence interval of the controls. With en face sections, a fine collagen mesh was seen at 23 weeks, which progressively increased in fiber size and quantity until 72 weeks. With severe CLD, the secondary collagen fibers in the saccular wall were thickened, tortuous, and disorganized relative to same-aged controls. Under 30 weeks, in the controls, the interstitium contained a wide, delicate network of interconnected collagen fibers. After positive pressure ventilation, some saccules markedly increased their diameter, which compressed and obliterated the interstitial network. In contrast with severe CLD, the interstitium was wide, with coarse wavy collagen fibers. CONCLUSIONS: Parenchymal collagen increases throughout development. Before 30 weeks, there is a delicate complex interstitial collagen network, which may be important for primary septation and subsequent normal development. Positive pressure ventilation, if excessive, and depending on lung maturity and disease state, over a short time can severely compress the interstitium and damage this collagen network and prevent normal primary septation and arrest or distort future lung development. With severe CLD, distal air space diameter increases. There is a failure of primary and secondary septation, arrested lung development and remodeling, with thickened cnt and remodeling, with thickened collagenous saccular walls, and a wide interstitium with increased quantity and size of collagen fibers that can affect the mechanics of ventilation. We conclude that normal lung development is dependent on a normal interstitium and, perhaps, collagen architecture and that origins of CLD begin early in the course of positive pressure ventilation.

Endostatin and vascular endothelial cell growth factor (VEGF) in piglet lungs: effect of inhaled nitric oxide and hyperoxia.

Pulmonary hyperoxic injury manifests as widespread alveolar-epithelial and microvascular endothelial cell necrosis, resolution of which requires angiogenesis. We investigated the hypothesis that inhaled nitric oxide (iNO) and hyperoxia each decreases lung vascular endothelial growth factor (VEGF) expression but increases endostatin and that concurrent administration of both gases will show a greater effect. Piglets were randomized to breathe for 5 d room air (RA); RA + NO (RA + 50 ppm NO), O(2) (hyperoxia, F(I)O(2) >0.96), O(2) + NO, or O(2) + NO + REC (O(2) + NO plus recovery in 50% O(2) for 72 h. After the piglets were killed, we measured lung capillary leak, VEGF mRNA, VEGF, and endostatin protein in homogenates, plasma, and lavage. VEGF mRNA decreased significantly with O(2) and O(2) + NO compared with breathing RA (p < or = 0.05). VEGF protein declined in the experimental groups with a significant reduction in the recovery group compared with the RA group (p < or = 0.05). Similar but more dramatic, endostatin declined in all groups relative to the RA group (p < 0.001). Lavage fluid VEGF protein and lung capillary leak rose significantly with O(2) and O(2) + NO compared with RA, but endostatin was unchanged. At 72 h of recovery from hyperoxia, VEGF mRNA and lavage fluid VEGF but not lung VEGF protein had normalized. Hyperoxia and iNO suppresses lung endostatin expression, but iNO unlike hyperoxia alone does not alter lung VEGF production. Hyperoxia paradoxically raises lavageable VEGF levels. This latter effect and that on VEGF mRNA level but not protein is abrogated by recovery in reduced F(I)O(2) for 72 h.

Endogenous production of nitric oxide in endotoxemic piglets.

We sought to assess the relation between endotoxin-induced pulmonary hypertension and the production of nitric oxide (NO) in neonatal animals. Adult animals respond to endotoxin by increasing exhaled NO and plasma NO metabolites. The response of neonatal animals has not previously been reported. We administered 20 microg/kg of Escherichia coli lipopolysaccharide (LPS) to 12- to 18-day-old and to 5- to 7-week-old piglets. Pulmonary vascular resistance increased significantly in both age groups. Exhaled NO in the 12- to 18-day-old animals and in the 5- to 7-week-old piglets did not increase significantly. A similarly treated group of adult rats did show a significant increase in exhaled NO (2.6 +/- 1.0 to 109.5 +/- 54.3 ppb; p = 0.028). Plasma NO metabolite measurements followed the same pattern of no increase in both porcine groups, and a large increase in the rat group. However, immunostaining of lungs from 12- to 18-day-old piglets did reveal an increase in inducible NO synthase. These results suggest that piglets demonstrate a limited ability to modulate LPS-induced pulmonary hypertension by elevations in exhaled NO. They also demonstrate the differential response to LPS between species.

Fetal hydrops and familial pulmonary lymphatic hypoplasia.

Two siblings were born with pleural effusions and hydrops. The first infant was a 26-week-old gestation male and died at 8 hours of life with radiographically small lungs and pulmonary insufficiency. No lung tissue was obtainable. This pregnancy was followed by two normal term infants, a male and female. The fourth pregnancy resulted in a female born at 35 weeks' gestation with pleural effusion and hydrops who died at 32 months of age. This infant was discharged from hospital at 32 days of age with small pleural effusions, but needed supplemental oxygen and daily diuretics to control edema. There were 14 additional admissions until death-all for respiratory distress or infections. An open lung biopsy at 2 months, showed dilated pleural lymphatics, with hypoplasia of the acinar and terminal bronchiolar lymphatics. At postmortem examination there was a markedly thickened pleura, and slit-like hypoplastic lymphatics of the acinar and terminal bronchioles and interlobular septal regions. This is the second family reported with these distinctive pulmonary intra-acinar and peri-acinar hypoplastic lymphatics. This disease is compatible with an autosomal recessive mode of inheritance.

Crossover trial comparing pressure support with synchronized intermittent mandatory ventilation.

OBJECTIVE: To compare pressure support ventilation (PSV) with volume guarantee (VG) to synchronized intermittent mandatory ventilation (SIMV) in infants with respiratory distress syndrome (RDS). STUDY DESIGN: A randomized, crossover study design was used. We enrolled 14 infants [BW (mean+/-SD) 2.5+/-0.7 kg, GA 34+/-2 weeks, age 49+/-26 hours]. Subjects received 4 hours of each mode of ventilation, with the first mode selected randomly. End expiratory volume (EEV) was measured during both ventilatory modes. RESULTS: Minute ventilation was greater with PSV+VG than with SIMV (p=0.012). This occurred despite no difference in p(a)CO(2). Mean airway pressure was higher during PSV+VG (p=0.023). There was no difference in the arterial/alveolar oxygen tension (a/A) ratio or in the specific dynamic compliance (sCdyn). CONCLUSION: Because of an increase in V(E) with PSV+VG, and no difference in the a/A ratio or sCdyn, we do not recommend the routine use of PSV+VG for this population.

Platelet endothelial cell adhesion molecule-1 and capillary loading in premature infants with and without chronic lung disease.

It was our objective to quantify platelet endothelial cell adhesion molecule-1 (PECAM-1) by immunohistochemistry in control infants of 22-50 weeks postconceptual age, and to correlate it with varying degrees of neonatal chronic lung disease (CLD). We tested the hypothesis that the density of PECAM-1 staining will positively correlate with increasing gestational age (GA) and the inflammatory process in CLD. A library of postmortem lung tissue of infants receiving ventilator care was accessed. The population consisted of 35 control infants exposed briefly to oxygen and positive pressure ventilation, and 31 infants who were 23-30 weeks GA with mild to severe CLD. A monoclonal anti-human PECAM-1 antibody was used to stain 5-microm paraffin sections. The slides were viewed at a magnification of x 40 by a blinded examiner. Twenty consecutive fields from standardly expanded tissue samples were viewed, and the volume density of PECAM-1 (V(V PECAM)) per parenchyma was measured, using point counting. In addition, 1-microm sections from 15 controls and 5 infants with CLD were stained with Toluidine blue and viewed under oil at a magnification of x 100, and the volume density of capillaries (V(V CAP)) and capillary load (CL) were calculated. The V(V PECAM) increased significantly with GA in controls (r = 0.72, P < 0.001). There was no relationship between V(V PECAM) and severity of CLD. Both V(V CAP) and CL increased significantly with GA (r = 0.93, P < 0.001; r = 0.94, P < 0.001, respectively). The infants with CLD had a normal or increased V(V CAP) and CL compared to controls. In summary, V(V PECAM), V(V CAP), and CL increased significantly with gestational age in control infants, but the postconceptional age range in CLD infants was too short to determine whether the V(V PECAM) changed. Infants with CLD had normal or increased V(V CAP) and CL compared to controls. The PECAM-1 immunostain does not appear to be a sensitive method for assessing capillary density in infants with CLD. These findings of normal or increased capillary load may represent a vascular adaptation for the lack of secondary septation and decreased surface area in CLD.