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. Author manuscript; available in PMC: 2011 May 1.
Published in final edited form as: Pediatr Res. 2010 May;67(5):521–525. doi: 10.1203/PDR.0b013e3181d4f20f

Opposing regulation of human alveolar Type II cell differentiation by nitric oxide and hyperoxia

Lindsay C Johnston 1, Linda W Gonzales 1, Richard T Lightfoot 1, Susan H Guttentag 1, Harry Ischiropoulos 1
PMCID: PMC3066065  NIHMSID: NIHMS279415  PMID: 20098340

Abstract

Clinical trials demonstrated decreasing rates of bronchopulmonary dysplasia in preterm infants with hypoxic respiratory failure treated with inhaled nitric oxide (iNO). However, the molecular and biochemical effects of iNO on developing human fetal lungs remain vastly unknown. Using a well-characterized model of human fetal alveolar type II cells, we assessed the effects of iNO and hyperoxia, independently and concurrently, on NO-cGMP signaling pathway and differentiation. Exposure to iNO increased cGMP levels by 40-fold after 3 days and by 8-fold after 5 days despite constant expression of phosphodiesterase-5 (PDE5). The levels of cGMP declined significantly upon exposure to iNO and hyperoxia at 3 and 5 days although expression of soluble guanylyl cyclase (sGC) was sustained. Surfactant proteins B and C (SP-B, SP-C) and Thyroid Transcription Factor-1 (TTF-1) mRNA levels increased in cells exposed to iNO in normoxia, but not upon exposure to iNO plus hyperoxia. Collectively these data indicate an increase in type II cell markers when undifferentiated lung epithelial cells are exposed to iNO in room air. However, hyperoxia overrides these potentially beneficial effects of iNO despite sustained expression of sGC.

INTRODUCTION

Alveolar type II cells are critical for normal lung development as the source of pulmonary surfactant. The components of surfactant, which are necessary to construct a surface-active film at the alveolar surface thereby preventing alveolar collapse, are developmentally regulated. Specifically, surfactant proteins B and C (SP-B and SP-C) undergo complex regulation involving transcriptional and post-transcriptional events occurring through the 2nd and 3rd trimesters of human gestation (1–4). Premature infants are at high risk for developing respiratory distress syndrome (RDS) in part due to interruption of this process. Despite advances in obstetrical and neonatal management, including antenatal glucocorticoid therapy and postnatal surfactant administration, RDS remains a major cause of morbidity and mortality in preterm infants (5), often because it is a risk factor for the development of bronchopulmonary dysplasia (BPD). There remains great interest in identifying additional therapeutic agents that will enhance alveolar type II cell maturation and surfactant production to further reduce the risk of RDS and BPD.

Nitric oxide (NO), a versatile endogenous mediator of vascular relaxation and signaling, has been effective in the treatment of term infants with pulmonary hypertension (6–8). The association of BPD with vascular pruning and pulmonary hypertension sparked interest in utilizing NO to improve outcomes in preterm infants with hypoxic respiratory failure who are at risk of developing BPD. Until recently, clinical trials revealed mixed results (9–12). However, two large multicenter randomized clinical trials indicated beneficial effects of NO in preterm infants. Ballard et al. reported significantly decreased rates of BPD and decreased need for supplemental oxygen in infants treated with iNO (13). Kinsella et al. also reported decreased BPD rates in infants with BW 1000–1250g, despite no overall difference in rates of BPD or death in the entire study population (14).

Studies performed in animal models revealed beneficial effects of iNO in alveolar epithelial physiology. Inhaled NO preserves airway structure and function, enhances development of alveolae (15), improves lung compliance and growth (16,17), decreases early lung inflammation and oxidant stress (18–19), and increases surfactant mRNA and protein levels (17,20–24). Endogenously produced by alveolar type II cells, NO participates in the transition from fluid-filled to air breathing lungs at birth (25). Despite these recognized beneficial effects of iNO in animal models, the molecular effects of exogenous NO have not been well-characterized in human fetal lung.

Alveolar type II cells perform many critical functions that include production of pulmonary surfactant, transport of water and ions across the epithelium barrier, production of trophic factors and molecules that modulate inflammatory responses, and regeneration type I cells upon injury (1–3). Alveolar type II cells differentiate from precursor epithelial cells during the second half of human gestation. Previously we developed and extensively characterized an in vitro model of differentiation of type II cells by exposing isolated epithelial cells derived from human fetal lung explants to glucocorticoid and/or cAMP in serum free media (26,27). Type II cell differentiation in this model is characterized by increased phosphatidylcholine synthesis, induction of surfactant protein synthesis, formation of lamellar bodies and secretion of surface-active surfactant. This model has been valuable for exploring factors and genes that regulate the differentiation of parenchymal epithelial cells to type II pneumocytes (26,27). In this study, this well-defined model system was employed to test the hypothesis that NO will induce the differentiation of human fetal alveolar type II cell and counter the potential harmful effects of hyperoxia. Experiments also examined for the first time the expression of the enzymes that mediate the NO response, specifically the NO-cGMP signaling pathway in human fetal type II cell cultures. Overall, the data provided new insights on the potential functions of NO in the developing lung, and a mechanistic rationale for the use of iNO in the treatment of extremely preterm infants at risk for BPD.

MATERIALS AND METHODS

Cell Culture

Enriched populations of epithelial cells from second- trimester (16–24 wk gestation) human fetal lung tissue were obtained from Advanced Bioscience Resources, Inc. (Alameda, CA) under Institutional Review Board-approved protocols, and were processed as described previously (26). After overnight culture, attached cells remained in serum-free Waymouth medium for 3 days. Next, the cells were cultured in 1 mL of serum-free Waymouth medium alone (control), or with 10 nM Dexamethasone + 0.1 mM 8-bromo-cAMP and 0.1 mM isobutylmethylxanthine (referred to as DCI) for 5 days to promote alveolar type II cell differentiation.

During the process of differentiation, cells were cultured in normoxia (RA) + 5% CO2 in a standard incubator (control), or in one of 3 test conditions (normoxia plus 20 ppm NO (Matheson Tri-Gas, Bridgeport, NJ), 95% oxygen alone, or 95% oxygen plus 20 ppm NO) in a Proox Model 110 chamber (BioSpherix, Redfield, NY) to maintain stable concentrations of oxygen, CO2 and NO. Because of the limited number of cells generated from each fetal lung tissue, only one experimental condition and a corresponding air exposed control were analyzed at a time. This experimental design also accounts for biological variance derived from the different human fetal lung tissues. The difference between the experimental treatment condition and the corresponding control group was used to determine the statistical significance of the data. Therefore the data was presented as fold change as compared to the corresponding air exposed group.

Cyclic GMP Enzyme Immunoassay and Nitric oxide metabolite analysis

cGMP and NO-derived metabolites were quantified by established methodologies described previously (28).

DNA Analysis

DNA concentration was measured using fluorimetry with Hoechst dye (Molecular Probes, Eugene, OR). The standard curve of DNA fluorescence units was used to determine sample DNA concentration, which was normalized to room air controls.

Real-time reverse transcriptase polymerase chain reaction (RT-PCR)

The two-step PCR protocol was performed as detailed elsewhere (29).

Western Blot Analysis

Immunoblotting for Surfactant Protein-B, Pepsinogen C, soluble Guanylyl Cyclase, Phosphodiesterase 5, and GAPDH was performed using previously described procedures (3).

Statistical Analysis

Results are given as mean ± standard error of the mean (SEM). To determine the statistical significance we used aired t-tests (performed using GraphPad Prism 4.00 GraphPad, San Diego, CA). All protein results were normalized to GAPDH, and RNA results were normalized to 18s rRNA.

RESULTS

Cellular Responses to Nitric Oxide and Hyperoxia

Cells cultured in hyperoxia exhibited altered viability compared to cells cultured in normoxia reflected by protein and DNA levels. Total protein recovered after 5 d of hyperoxia, with or without nitric oxide, was significantly decreased (hyperoxia 0.70 ± 0.06, hyperoxia plus NO 0.50 ± 0.04 values represent percent of normoxia control value, n =15 for normoxia and n=4 for hyperoxia and hyperoxia plus NO, p < 0.05). A modest but not significant decrease in protein recovery from the cells exposed to normoxia plus NO (0.73 ± 0.09 n=3, p=NS). Total cellular DNA recovery decreased moderately in cells exposed to normoxia plus NO (0.44 ± 0.02), hyperoxia plus NO (0.73 ± 0.4) and hyperoxia alone (0.64 ± 0.17) when compared to normoxia controls, (values represent percent of normoxia control value, n=11 for normoxia and n=3 for all other exposures, p= NS). These results are consistent with the known toxicity of hyperoxia and the duality of nitric oxide as both a contributor to cell toxicity as well as a protector of cell viability (30). The effects of NO in cell survival are a function of duration and level of NO exposure as well as the concomitant exposure to hyperoxia (30). Consequently, all assays were performed using equal amounts of total protein, and densitometry-based assays were normalized to well-recognized housekeeping genes and proteins.

Activation of soluble guanylyl cyclase (sGC) and production of cGMP directly report on the delivery and utilization of iNO after exposure. The levels of cGMP were quantified in cells after 3 and 5 d of culture (Fig. 1A). A significant elevation in cGMP levels occurred on day 3 in both NO exposed groups when compared to controls in normoxia and hyperoxia alone. However, at day 3 the levels of cGMP were significantly lower in the iNO plus hyperoxia exposed cells as compared to cells exposed to iNO under normoxic conditions. Moreover, the cGMP levels in the NO treated groups (both normoxia and hyperoxia) decreased significantly after 5 days of exposure, despite continuous exposure of the cells (Fig. 1A).

Figure 1.

Figure 1

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Panel A, cGMP in cell lysates from day 3 and 5 DCI treated cells. Panel B and C, stable NO-metabolites in cell media (B), and cell lysates (C). Exposures include: normoxia (□), hyperoxia (■), normoxia plus NO (Inline graphic), or hyperoxia plus NO (Inline graphic); *p < 0.05 compared to normoxia on day 3 and †p < 0.05 compared to corresponding treatment group on day 3, n=4 for all treatment groups.

The levels of stable products of NO metabolism were also significantly elevated in the media from cells cultured in DCI media and exposed to nitric oxide, with or without hyperoxia (Fig. 1B). However, no difference was detected in the cell lysates (Fig. 1C). Therefore, NO was delivered to the cells and a corresponding activation of sCG accompanied the exposure.

Expression of sGC and phosphodiesterase 5

The changes in cGMP levels prompted the investigation of the levels of sGC, the molecular target of NO and the enzyme responsible for cGMP production. No significant differences in the expression of sGC were measured between exposure groups and the corresponding air exposed controls on days 3 and 5 (Fig. 2). The expression of phosphodiesterase 5 (PDE5), the major enzyme responsible for the metabolism of cGMP, was also assessed. Similarly, the levels of PDE5 did not vary significantly in the three treatment groups as compared to corresponding air exposed controls (Fig. 3).

Figure 2.

Figure 2

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Near-infrared quantification of sGC levels in cells exposed to various gases in DCI media at day 3 (Panel A) and at day 5 (Panel B). Exposure conditions: normoxia (□), hyperoxia (■), normoxia plus NO (Inline graphic), or hyperoxia plus NO (Inline graphic).

Figure 3.

Figure 3

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The levels of PDE5 were quantified by western blotting and near-infrared detection at day 3 (Panel A) and day 5 (Panel B). Exposure groups: normoxia (□), hyperoxia (■), normoxia NO (Inline graphic), or hyperoxia plus NO (Inline graphic).

Thyroid transcription factor-1 (TTF-1) and Surfactant Protein mRNA with iNO and hyperoxic exposure

Real-time RT-PCR revealed increased expression of Type II cell markers on day 5 in cells exposed to normoxia plus nitric oxide. Cells cultured with normoxia plus iNO had a 2-fold increase in SP-B mRNA levels (Fig. 4A) and a 10-fold increase in SP-C mRNA levels (Fig. 4B) as compared to normoxia controls. Hyperoxia alone had no effect on SP-B or SP-C RNA. The effect of iNO on SP-B and SP-C mRNA did not extend to cells cultured in hyperoxia plus nitric oxide. The mRNA for TTF-1, an essential transcription factor regulating surfactant protein synthesis, increased 5-fold in cells exposed to normoxia plus iNO (Fig. 4C). This increase was not apparent in cells exposed to iNO plus hyperoxia.

Figure 4.

Figure 4

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mRNAs for Type II cell SP-B (A), SP-C (B), and TTF-1 (C) were measured after 5 days of culture in DCI media and either normoxia (□), hyperoxia (■), normoxia plus NO (Inline graphic), or hyperoxia plus NO (Inline graphic); *p < 0.05, n=3 for all treatment groups.

Type II Cell Markers with iNO and hyperoxia

Mature SP-B protein was quantified by immunoblotting after exposure to various gas conditions (Fig. 5A). As expected, a significant increase in mature SP-B was detected in cells cultured with the DCI media as compared to cells cultured in control media (not shown). The SP-B protein levels were not significantly different in cells exposed to normoxia plus iNO compared to normoxia controls despite the significant increase in SP-B mRNA. Mature SP-B levels declined by approximately 80% in cells exposed to hyperoxia as compared to normoxia controls (Fig. 5B). Exposure to iNO in the presence of hyperoxia did not prevent the decline in the levels of SP-B. The levels of pepsinogen C (PGC), an enzyme involved in SP-B post-translational proteolytic processing (31), were not significantly altered in the different groups as compared to normoxia controls (data not shown).

Figure 5.

Figure 5

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Panel A, Representative SP-B blots. Lanes: (1–2) normoxia controls with DCI media at days 3 and 5 respectively; (3–4) cells exposed to respective gases as indicated without DCI media at days 3 and 5; (5–6) cells exposed to respective gases as indicated with DCI media at days 3 and 5 respectively. Panel B, SP-B protein levels normalized to GAPDH loading controls after 5 days of culture in DCI media and either normoxia (□), hyperoxia (■), normoxia plus NO (Inline graphic), or hyperoxia plus NO (Inline graphic); data is expressed as percent of the corresponding normoxia control *p < 0.05, n=5 for all treatment groups.

DISCUSSION

Inhaled nitric oxide has been proven to be useful in the treatment of a variety of respiratory diseases of neonates and adults (6,7,13,14). Motivated by the encouraging results of the most recent clinical trials in neonates, we explored for the first time the responses of human fetal alveolar epithelial cells to iNO under normoxic and hyperoxic conditions. Since the production of surfactant is critically important for normal lung development, we hypothesized that nitric oxide/cGMP-induced differentiation of alveolar type II cells might contribute to the pulmonary benefits found in preterm infants treated with nitric oxide. Employing a well established in vitro model of fetal alveolar type II cell differentiation, the data revealed opposing regulatory functions of hyperoxia and iNO in the differentiation of human fetal type II pneumocytes.

By quantifying the levels of cGMP, the data indicated that exposure to iNO results in the activation of sGC. The activation of sGC and production of cGMP is one of the major pathways by which NO influences a variety of biological functions including cellular differentiation (32–34). The data also document for the first time that sGC is expressed by human fetal type II pneumocytes and, interestingly, the expression is maintained during hyperoxia. Previously, sGC and PDE5 were shown to increase during the perinatal period in rodents and were thought to be important in the transition to air breathing (35,36). However, the NO-stimulated activation of sGC and resultant production of cGMP significantly declined in the presence of hyperoxia. The diminished response to iNO in the presence of hyperoxia is not explained by a decline in the levels of sGC (Fig. 2). The decrease in cGMP levels on day 3 of exposure to NO plus hyperoxia is also not derived from an increase in PDE5 protein, the main lung phosphodiesterase that removes cGMP, the expression of which is actually decreased (Fig. 3). The decline in cGMP levels may be due to inactivation of sGC or the reaction of nitric oxide with superoxide or other radical intermediates formed during hyperoxic exposures. It has been relatively well-documented that hyperoxia increases the production of superoxide and other radicals, and that exposure to hyperoxia and iNO alters the reactive nitrogen species formed in rodent lungs (37,38). Evidence for the formation of reactive nitrogen species derived from iNO are also present in the plasma of premature infants exposed to hyperoxia with or without iNO (37,38). Consistent with these findings are the documentation that hyperoxia decreased intracellular cGMP levels in ovine fetal pulmonary artery smooth muscle cells (39). Compared to day 3, a significant decline in the levels of cGMP was noted on day 5 despite continuous exposure to iNO. The unexpected decline in cGMP levels after 5 days of iNO exposure may be explained by decreased responsiveness of sGC after prolonged iNO exposure despite stable protein levels similarly to the suggested tolerance induced by the use of systemic vasodilators (34). Alternatively, it may indicate a rapid non-PDE5 regulated consumption of cGMP for signaling or other regulatory functions.

Significant increases in SP-B, SP-C, and TTF-1 mRNA were measured in cells treated with normoxia plus nitric oxide. This suggests that either more cells are differentiating to a type II phenotype, or that increased levels of these products are being made by existing type II cells. Interestingly, these changes were not observed in conditions of hyperoxia plus nitric oxide, suggesting that high levels of oxygen may oppose the beneficial effects of nitric oxide. The levels of mature SP-B increased in cells treated with hormones but not upon exposure to iNO despite increased levels of SP-B mRNA.

The present study offers a number of new insights in the human fetal lung responses to hyperoxia and nitric oxide. As expected, exposure to iNO increased the levels of cGMP by activating sGC. Exposure of premature human fetal epithelial cells to normoxia plus NO leads to increased markers for Type II pneumocytes, but these beneficial effects are not observed when NO is administered concomitantly with hyperoxia. Although rodent and other animal models of inhaled nitric oxide, such as full term lamb and premature baboon, demonstrated attenuation of certain aspects of hyperoxic lung injury, the data presented herein indicate that potential beneficial effects of NO in the maturation and differentiation of alveolar type II cells are mitigated by hyperoxia. Although this study did not explore the dose-dependency of the interaction between hyperoxia and nitric oxide, the data support that efforts to lower exposure to hyperoxia in the clinical setting may help offset the detrimental effects of hyperoxia and promote the beneficial biological consequences of inhaled nitric oxide.

Acknowledgements

The authors would like to thank Dr. C. Foster for assistance in study design and reading of the manuscript, P. Wang for preparing the cell monolayers, P. Zhang and L. Varghese for technical assistance, Dr. S.A. Lorch, for statistical support, and Dr. T. Greco and E. Tsika for assistance with figure preparation.

Statement of Financial Support: The work was supported by the Gisela and Dennis Alter Chair in Pediatric Neonatology, the Joseph Strokes Jr. Investigator program at the Children's Hospital of Philadelphia, the ES013508 NIEHS Center of Excellence in Environmental Toxicology, and NIH HL059959.

Abbreviations

cGMP

cyclic guanosine monophosphate

PGC

Pepsinogen C

PDE5

Phosphodiesterase 5

sGC

soluble Guanylyl Cyclase

SP-B (C)

Surfactant Protein B (C)

TTF-1

Thyroid Transcription Factor-1

Footnotes

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