Marye H. Godinez

Dysfunction of pulmonary surfactant in chronically ventilated premature infants

Infants of <30 wk gestation often require respiratory support for several weeks and may develop bronchopulmonary dysplasia (BPD), which is associated with long-term pulmonary disability or death in severe cases. To examine the status of surfactant in infants at high risk for BPD, this prospective study analyzed 247 tracheal aspirate samples from 68 infants of 23–30 wk gestation who remained intubated for 7–84 d. Seventy-five percent of the infants had one or more surfactant samples with abnormal function (minimum surface tension 5.1–21.7 mN/m by pulsating bubble surfactometer), which were temporally associated with episodes of infection (p = 0.01) and respiratory deterioration (p = 0.005). Comparing normal and abnormal surfactant samples, there were no differences in amount of surfactant phospholipid, normalized to total protein that was recovered from tracheal aspirate, or in relative content of phosphatidylcholine and phosphatidylglycerol. Contents of surfactant proteins (SP) A, B, and C, measured in the surfactant pellet by immunoassay, were reduced by 50%, 80%, and 72%, respectively, in samples with abnormal surface tension (p ≤ 0.001). On multivariable analysis of all samples, SP-B content (r = −0.58, p < 0.0001) and SP-C content (r = −0.32, p < 0.001) were correlated with surfactant function. We conclude that most premature infants requiring continued respiratory support after 7 d of age experience transient episodes of dysfunctional surfactant that are associated with a deficiency of SP-B and SP-C.

Surfactant composition and function in a primate model of infant chronic lung disease: Effects of inhaled nitric oxide

Bronchopulmonary dysplasia, or chronic lung disease (CLD), of premature infants involves injury from hyperoxia and mechanical ventilation to an immature lung. We examined surfactant and nitric oxide (NO), which are developmentally deficient in premature infants, in the baboon model of developing CLD. Fetuses were delivered at 125 d gestation and were managed for 14 d with ventilation and oxygen prn without (controls) or with inhaled NO at 5 ppm. Compared with term infants, premature control infants had reduced maximal lung volume, decreased tissue content of surfactant proteins SP-A, -B, and -C, abnormal lavage surfactant as assessed by pulsating bubble surfactometer, and a low concentration of SP-B/phospholipid. NO treatment significantly increased maximal lung volume and tissue SP-A and SP-C, reduced recovery of lavage surfactant by 33%, decreased the total protein:phospholipid ratio of surfactant by 50%, and had no effect on phospholipid composition or SP content except for SP-C (50%). In both treatment groups, levels of SP-B and SP-C in surfactant were negatively correlated with STmin, with a 5-fold greater SP efficiency for NO versus control animals. By contrast, lung volume and compliance were not correlated with surfactant function. We conclude that surfactant is often dysfunctional in developing CLD secondary to SP-B deficiency. NO treatment improves the apparent ability of hydrophobic SP to promote low surface tension, perhaps secondary to less protein inactivation of surfactant, and improves lung volume by a process unrelated to surfactant function.

Lipidomics of cellular and secreted phospholipids from differentiated human fetal type II alveolar epithelial cells

Maturation of fetal alveolar type II epithelial cells in utero is characterized by specific changes to lung surfactant phospholipids. Here, we quantified the effects of hormonal differentiation in vitro on the molecular specificity of cellular and secreted phospholipids from human fetal type II epithelial cells using electrospray ionization mass spectrometry. Differentiation, assessed by morphology and changes in gene expression, was accompanied by restricted and specific modifications to cell phospholipids, principally enrichments of shorter chain species of phosphatidylcholine (PC) and phosphatidylinositol, that were not observed in fetal lung fibroblasts. Treatment of differentiated epithelial cells with secretagogues stimulated the secretion of functional surfactant-containing surfactant proteins B and C (SP-B and SP-C). Secreted material was further enriched in this same set of phospholipid species but was characterized by increased contents of short-chain monounsaturated and disaturated species other than dipalmitoyl PC (PC16:0/16:0), principally palmitoylmyristoyl PC (PC16:0/14:0) and palmitoylpalmitoleoyl PC (PC16:0/16:1). Mixtures of these PC molecular species, phosphatidylglycerol, and SP-B and SP-C were functionally active and rapidly generated low surface tension on compression in a pulsating bubble surfactometer. These results suggest that hormonally differentiated human fetal type II cells do not select the molecular composition of surfactant phospholipid on the basis of saturation but, more likely, on the basis of acyl chain length.

Surfactant Function and Composition in Premature Infants Treated With Inhaled Nitric Oxide

OBJECTIVES. We hypothesized that inhaled nitric oxide treatment of premature infants at risk for bronchopulmonary dysplasia would not adversely affect endogenous surfactant function or composition. METHODS. As part of the Nitric Oxide Chronic Lung Disease Trial of inhaled nitric oxide, we examined surfactant in a subpopulation of enrolled infants. Tracheal aspirate fluid was collected at specified intervals from 99 infants with birth weights <1250 g who received inhaled nitric oxide (20 ppm, weaned to 2 ppm) or placebo gas for 24 days. Large-aggregate surfactant was analyzed for surface activity with a pulsating bubble surfactometer and for surfactant protein contents with an immunoassay. RESULTS. At baseline, before administration of study gas, surfactant function and composition were comparable in the 2 groups, and there was a positive correlation between minimum surface tension and severity of lung disease for all infants. Over the first 4 days of treatment, minimum surface tension increased in placebo-treated infants and decreased in inhaled nitric oxide–treated infants. There were no significant differences between groups in recovery of large-aggregate surfactant or contents of surfactant protein A, surfactant protein B, surfactant protein C, or total protein, normalized to phospholipid. CONCLUSIONS. We conclude that inhaled nitric oxide treatment for premature infants at risk of bronchopulmonary dysplasia does not alter surfactant recovery or protein composition and may improve surfactant function transiently.

Carcinoembryonic cell adhesion molecule 6 in human lung: regulated expression of a multifunctional type II cell protein

Carcinoembryonic cell adhesion molecule 6 (CEACAM6) is a glycosylated, glycosylphosphatidylinositol (GPI)-anchored protein expressed in epithelial cells of various human tissues. It binds gram-negative bacteria and is overexpressed in cancers, where it is antiapoptotic and promotes metastases. To characterize CEACAM6 expression in developing lung, we cultured human fetal lung epithelial cells and examined responses to differentiation-promoting hormones, adenovirus expressing thyroid transcription factor-1 (TTF-1), and silencing of TTF-1 with small inhibitory RNA. Glucocorticoid and cAMP had additive stimulatory effects on CEACAM6 content, and combined treatment maximally increased transcription rate, mRNA, and protein approximately 10-fold. Knockdown of TTF-1 reduced hormone induction of CEACAM6 by 80%, and expression of recombinant TTF-1 increased CEACAM6 in a dose-dependent fashion. CEACAM6 content of lung tissue increased during the third trimester and postnatally. By immunostaining, CEACAM6 was present in fetal type II cells, but not mesenchymal cells, and localized to both the plasma membrane and within surfactant-containing lamellar bodies. CEACAM6 was secreted from cultured type II cells and was present in both surfactant and supernatant fractions of infant tracheal aspirates. In functional studies, CEACAM6 reduced inhibition of surfactant surface properties by proteins in vitro and blocked apoptosis of electroporated cultured cells. We conclude that CEACAM6 in fetal lung epithelial cells is developmentally and hormonally regulated and a target protein for TTF-1. Because CEACAM6 acts as an antiapoptotic factor and stabilizes surfactant function, in addition to a putative role in innate defense against bacteria, we propose that it is a multifunctional alveolar protein.

Hormonal regulation of alveolarization: structure-function correlation

BackgroundDexamethasone (Dex) limits and all-trans-retinoic acid (RA) promotes alveolarization. While structural changes resulting from such hormonal exposures are known, their functional consequences are unclear.MethodsNeonatal rats were treated with Dex and/or RA during the first two weeks of life or were given RA after previous exposure to Dex. Morphology was assessed by light microscopy and radial alveolar counts. Function was evaluated by plethysmography at d13, pressure volume curves at d30, and exercise swim testing and arterial blood gases at both d15 and d30.ResultsDex-treated animals had simplified lung architecture without secondary septation. Animals given RA alone had smaller, more numerous alveoli. Concomitant treatment with Dex + RA prevented the Dex-induced changes in septation. While the results of exposure to Dex + RA were sustained, the effects of RA alone were reversed two weeks after treatment was stopped. At d13, Dex-treated animals had increased lung volume, respiratory rate, tidal volume, and minute ventilation. On d15, both RA- and Dex-treated animals had hypercarbia and low arterial pH. By d30, the RA-treated animals resolved this respiratory acidosis, but Dex-treated animals continued to demonstrate blood gas and lung volume abnormalities. Concomitant RA treatment improved respiratory acidosis, but failed to normalize Dex-induced changes in pulmonary function and lung volumes. No differences in exercise tolerance were noted at either d15 or d30. RA treatment after the period of alveolarization also corrected the effects of earlier Dex exposure, but the structural changes due to RA alone were again lost two weeks after treatment.ConclusionWe conclude that both RA- and corticosteroid-treatments are associated with respiratory acidosis at d15. While RA alone-induced changes in structure andrespiratory function are reversed, Dex-treated animals continue to demonstrate increased respiratory rate, minute ventilation, tidal and total lung volumes at d30. Concomitant treatment with Dex + RA prevents decreased septation induced by Dex alone and results in correction of hypercarbia. However, these animals continue to have abnormal pulmonary function and lung volumes. Increased septation as a result of RA treatment alone is reversed upon discontinuation of treatment. These data suggest that Dex + RA treatment results in improved gas exchange likely secondary to normalized septation.

Inspired oxygen concentration alters the phospholipids and protein content in the bronchoalveolar lavage-accessible space.

OBJECTIVE To examine the effect of FIO2 on the contents of total protein, total phospholipids, phosphatidylcholine, and phosphatidylglycerol in the bronchoalveolar lavage-accessible space in male and female rats in vivo. DESIGN Prospective, controlled trial. SETTING Research laboratory. SUBJECTS Adult male and female Sprague-Dawley rats. INTERVENTIONS After animals were anesthetized with an intraperitoneal injection of pentobarbitol (50 mg/kg), a 24-gauge catheter was placed in the femoral artery. Determinations of arterial pH and PaO2 and PaCO2 were performed before tracheostomy, and all animals were then ventilated for 3 mins with an FIO2 of 0.21, followed by a reduction bronchoalveolar lavage. The animals were randomly divided equally by gender and given either an FIO2 of 0.21, 0.50, or 1.00. All subjects were ventilated in the same manner. Sampling bronchoalveolar lavage was performed 80 and 160 mins after institution of the variable FIO2. Bronchoalveolar lavage samples were analyzed for protein and phospholipid content. Arterial blood was obtained for determination of arterial pH and the PaO2 and PaCO2 immediately and 40 mins after each sampling bronchoalveolar lavage. MEASUREMENTS AND MAIN RESULTS At the times of bronchoalveolar sampling lavage, the PaCO2 increased and the PaO2 decreased, as did the pH. In the 40-min samples obtained between sampling lavages, the arterial pH and PaCO2 and PaO2 returned to pretracheostomy values (animals ventilated with an FIO2 of 0.21) and/or higher pO2 values (animals ventilated with an FIO2 of 0.5 or 1.0). No differences were found between genders in amounts of total protein and phospholipid content in reduction and zero time bronchoalveolar lavage fluid. Males and females ventilated with an FIO2 of 0.21 differed in the amounts of total protein, total phospholipids, phosphatidylcholine, and phosphatidylglycerol found in sampling bronchoalveolar lavage at 80 and 160 mins. Amounts of total protein and total phospholipids also demonstrated gender differences with the administration of an FIO2 of 1.0, but no differences with the administration of and FIO2 of 0.5. In terms of the phospholipids, males had greater amounts in the sampling bronchoalveolar lavage at 80 mins, and females at 160 mins. Administration of an FIO2 of 0.5 or 1.0 resulted in decreased amounts of total phospholipids in both males and females when compared with and FIO2 of 0.21. In males, administration of both FIO2 of 0.5 and 1.0 resulted in decreased amounts of phosphtidylcholine found in the bronchoalveolar lavage-accessible space; in females, amounts of phosphatidylcholine were only decreased when and FIO2 of 1.0 was administered. In males, administration of and FIO2 of 1.0 also resulted in decreased amounts of phosphatidylglycerol. The decreased amount of phosphatidylglycerol occurred in females given an FIO2 of 0.5. Amounts of total protein in males and females given an FIO2 of 0.5 and in females given an FIO2 of 1.0 were found to be increased. CONCLUSIONS Our findings support the hypothesis that hyperoxia alters surfactant composition. Further investigation is warranted to determine the mechanisms affecting secretion of phosphatidylcholine and phosphatidylglycerol into the bronchoalveolar space and to explore the gender difference in secretion.

Hyaluronan Binding Peptide Limits Surfactant Dysfunction, Respiratory Distress and Surfactant Protein-B and -C Deficiency after Acute Lung Injury

Hyaluronan Binding Peptide Limits Surfactant Dysfunction, Respiratory Distress and Surfactant Protein-B and -C Deficiency after Acute Lung Injury