-Type II epithelial cells are essential for lung development and remodeling, as they are precursors for type I cells and can produce vascular mitogens. Although type II cell proliferation takes place after hyperoxia, it is unclear why alveolar remodeling occurs normally in adults whereas it is permanently disrupted in newborns. Using a line of transgenic mice whose type II cells could be identified by their expression of enhanced green fluorescent protein and endogenous expression of surfactant proteins, we investigated the age-dependent effects of hyperoxia on type II cell proliferation and alveolar repair. In adult mice, type II cell proliferation was low during room air and hyperoxia exposure but increased during recovery in room air and then declined to control levels by day 7. Eight weeks later, type II cell number and alveolar compliance were indistinguishable from those in room air controls. In newborn mice, type II cell proliferation markedly increased between birth and postnatal day 7 before declining by postnatal day 14. Exposure to hyperoxia between postnatal days 1 and 4 inhibited type II cell proliferation, which resumed during recovery and was aberrantly elevated on postnatal day 14. Eight weeks later, recovered mice had 70% fewer type II cells and 30% increased lung compliance compared with control animals. Recovered mice also had higher levels of T1␣, a protein expressed by type I cells, with minimal changes detected in genes expressed by vascular cells. These data suggest that perinatal hyperoxia adversely affects alveolar development by disrupting the proper timing of type II cell proliferation and differentiation into type I cells. alveoli; cell proliferation; differentiation; enhanced green fluorescent protein; proliferating cell nuclear antigen THE ALVEOLUS IS COMPOSED OF two epithelial cell types that can be identified by their distinct morphology and expression of unique genes. Type I cells are thin, flat cells that cover pulmonary vascular endothelial cells and comprise 95% of the alveolar surface (56). These cells are important for gas exchange, regulation of alveolar fluid levels, and stretch-induced modulation of surfactant secretion. Type I cells can be identified by their expression of T1␣ (also known as RTI 40 ), aquaporin-5, caveolin-1, or the cyclin-dependent kinase inhibitor p15 (41,42). Type II cells, on the other hand, are large,

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RationaleInfants born to diabetic or obese mothers are at risk of respiratory distress and persistent pulmonary hypertension of the newborn (PPHN), conceivably through fuel-mediated pathogenic mechanisms. Prior research and preventative measures focus on controlling maternal hyperglycemia, but growing evidence suggests a role for additional circulating fuels including lipids. Little is known about the individual or additive effects of a maternal high-fat diet on fetal lung development.ObjectiveThe objective of this study was to determine the effects of a maternal high-fat diet, alone and alongside late-gestation diabetes, on lung alveologenesis and vasculogenesis, as well as to ascertain if consequences persist beyond the perinatal period.MethodsA rat model was used to study lung development in offspring from control, diabetes-exposed, high-fat diet-exposed and combination-exposed pregnancies via morphometric, histologic (alveolarization and vasculogenesis) and physiologic (echocardiography, pulmonary function) analyses at birth and 3 weeks of age. Outcomes were interrogated for diet, diabetes and interaction effect using ANOVA with significance set at p≤0.05. Findings prompted additional mechanistic inquiry of key molecular pathways.ResultsOffspring exposed to maternal diabetes or high-fat diet, alone and in combination, had smaller lungs and larger hearts at birth. High-fat diet-exposed, but not diabetes-exposed offspring, had a higher perinatal death rate and echocardiographic evidence of PPHN at birth. Alveolar mean linear intercept, septal thickness, and airspace area (D2) were not significantly different between the groups; however, markers of lung maturity were. Both diabetes-exposed and diet-exposed offspring expressed more T1α protein, a marker of type I cells. Diet-exposed newborn pups expressed less surfactant protein B and had fewer pulmonary vessels enumerated. Mechanistic inquiry revealed alterations in AKT activation, higher endothelin-1 expression, and an impaired Txnip/VEGF pathway that are important for vessel growth and migration. After 3 weeks, mortality remained highest and static lung compliance and hysteresis were lowest in combination-exposed offspring.ConclusionThis study emphasizes the effects of a maternal high-fat diet, especially alongside late-gestation diabetes, on pulmonary vasculogenesis, demonstrates adverse consequences beyond the perinatal period and directs attention to mechanistic pathways of interest. Findings provide a foundation for additional investigation of preventative and therapeutic strategies aimed at decreasing pulmonary morbidity in at-risk infants.

The most common form of newborn chronic lung disease, bronchopulmonary dysplasia (BPD), is thought to be caused by oxidative disruption of lung morphogenesis which results in decreased pulmonary vasculature and alveolar simplification. Although cellular redox status is known to regulate cellular proliferation and differentiation, redox-sensitive pathways associated with these processes in developing pulmonary epithelium are unknown. Redox sensitive pathways are commonly regulated by cysteine thiol modifications. Therefore two thiol oxidoreductase systems, thioredoxin and glutathione, were chosen in order to elucidate the roles of these pathways on cell death. Studies herein indicate thiol oxidation contributes to cell death through impaired activity of glutathione dependent (GSH-dependent) and thioredoxin (Trx) systems and altered signaling through redox sensitive pathways. Free thiol content decreased by 71% with hyperoxic (95% oxygen) exposure. Increased cell death was observed during oxygen exposure when either the thioredoxin (Trx) systems or glutathione dependent were pharmacologically inhibited with aurothioglucose (ATG) or buthionine sulphoximine respectively. However, inhibition of the Trx system yielded the smallest decrease in free thiol content (1.44% with ATG treatment vs 21.33% with BSO treatment). Although, Trx1 protein levels were unchanged, Trx1 function was impaired during hyperoxic treatment as indicated by progressive cysteine oxidation. Overexpression of Trx1 in H1299 cells utilizing an inducible construct increased cell survival during hyperoxia whereas siRNA knockdown of Trx1 during oxygen treatment reduced cell viability. Overall, this indicated a comparatively small pool of proteins rely on Trx redox functions to mediate cell survival in hyperoxia, and the protective functions of Trx1 are progressively lost by its oxidative inhibition. To further elucidate the role of Trx1, potential Trx1 redox protein-protein interactions mediating cytoprotection and cell survival pathways were determined by utilizing a substrate trap (mass action trapping) proteomics approach. With this method, known Trx1 targets were detected including peroxiredoxin-1 as well as novel targets, including two HSP90 isoforms (HSP90AA1 and HSP90AB1). Reactive cysteines within the structure of HSP90 are known to modulate its ATPase dependent chaperone activity through disulfide formation and S-nitrosylation. While HSP90 expression is unchanged at the protein level during hyperoxic exposure, siRNA knockdown significantly increased hyperoxic cell death by 2.5-fold, indicating cellular dependence on HSP90 chaperone functions in response of hyperoxic exposure. These data support the hypothesis that hyperoxic impairment of Trx1 negatively impacts HSP90-oxidative responses critical to cell survival with potential implications on pathways implicated in lung development and the pathogenesis of BPD.

Although it is well established that the cell cycle inhibitor p21 protects against genotoxic stress by preventing the replication of damaged DNA, recent studies have shown cytoplasmic forms can also protect. It protects by delaying the loss of the anti-apoptotic proteins, Mcl-1 and Bcl-XL; however, the mechanism of regulation is unknown. Utilizing hyperoxia as a model of chronic oxidative stress and DNA damage, p21 was detected in the nucleus and cytoplasm and cytoplasmic expression of p21 was sufficient for cytoprotection. P21 was enriched in a subcellular fraction containing mitochondria and endoplasmic reticulum (ER), suggesting that it may be coordinating ER and mitochondrial stress pathways. Consistent with this, p21 suppressed hyperoxic downregulation of BiP and subsequent activation ER stress signaling which effected Mcl-1, but not Bcl-XL; though both inhibited hyperoxic cell death. Taken together, these data show that p21 integrates the DNA damage response with ER stress signaling which then regulates mitochondrial death pathways during chronic genotoxic stress.

The cyclin-dependent kinase inhibitor p21Cip1/Waf1/Sdi1 protects the lung against hyperoxia, but the mechanism of protection remains unclear because loss of p21 does not lead to aberrant cell proliferation. Because some members of the Bcl-2 gene family have been implicated in hyperoxia-induced cell death, the current study investigated their expression as well as p21-dependent growth suppression and cytoprotection. Conditional overexpression of full-length p21, its amino-terminal cyclin-binding (p211-82NLS) domain or its carboxy-terminal PCNA-binding (p2176-164) domain inhibited growth of human lung adenocarcinoma H1299 cells, but only the full-length protein was cytoprotective. Low levels of p21 inhibited cell proliferation, whereas higher levels were required for protection. Expression of the anti-apoptotic protein Bcl-XL declined during hyperoxia but was maintained in cells expressing p21. RNA interference (RNAi) knockdown of Bcl-XL enhanced hyperoxic death of cells expressing p21, whereas overexpression of Bcl-XL increased cell survival. Consistent with growth suppression and cytoprotection requiring different levels of p21, hyperoxia inhibited PCNA expression in p21+/+ and p21+/- mice but not in p21-/- mice. In contrast, p21 was haplo-insufficient for maintaining expression of Bcl-XL and protection against hyperoxia. Taken together, these data show that p21-mediated cytoprotection against hyperoxia involves regulation of Bcl-XL and is uncoupled from its ability to inhibit proliferation.

Oxygen exposure in premature infants is a major risk factor for bronchopulmonary dysplasia and can impair the host response to respiratory viral infections later in life. Similarly, adult mice exposed to hyperoxia as neonates display alveolar simplification associated with a reduced number of alveolar epithelial type II cells and exhibit persistent inflammation, fibrosis, and mortality when infected with influenza A virus. Because type II cells participate in innate immunity and alveolar repair, their loss may contribute to oxygen-mediated sensitivity to viral infection. A genomewide screening of type II cells identified eosinophil-associated RNase 1 (Ear1). Ear1 was also detected in airway epithelium and was reduced in lungs of mice exposed to neonatal hyperoxia. Electroporation-mediated gene delivery of Ear1 to the lung before infection successfully reduced viral replication and leukocyte recruitment during infection. It also diminished the enhanced morbidity and mortality attributed to neonatal hyperoxia. These findings demonstrate that novel epithelial expression of Ear1 functions to limit influenza A virus infection, and its loss contributes to oxygen-associated epithelial injury and fibrosis after infection. People born prematurely may have defects in epithelial innate immunity that increase their risk for respiratory viral infections.

Prolonged exposure to hyperoxia inhibits cell proliferation in G1 via increased expression of p21. While p21 inhibits proliferating cell nuclear antigen (PCNA)-dependent DNA synthesis, it can also directly lower PCNA abundance; however, it is unclear whether loss of PCNA contributes to growth arrest. Here, we investigate how PCNA loss affects ability of p21 to exert G1 growth arrest of lung epithelial cells exposed to hyperoxia. In A549 cells that express p21 and growth arrest in G1 during hyperoxia, small interfering RNA (siRNA) knockdown of p21 led to G1 checkpoint bypass, increased cell death, and restoration of PCNA expression. Conditional overexpression of the PCNA binding domain of p21 in H1299 cells that do not normally express p21, or exposure to hyperoxia, caused a time-dependent loss of PCNA. Titrating PCNA levels using siRNA to approximate the low amount observed in cells expressing p21 resulted in S phase arrest. While lowering PCNA by itself caused S phase arrest, the combination of hyperoxia and siRNA against PCNA dramatically reduced PCNA abundance resulting in G1 arrest. G1 growth arrest was markedly enhanced upon the addition of p21 to these cells. Our findings suggest a model in which reducing expression of the abundant protein PCNA allows the less abundant protein p21 to be more effective at suppressing the processivity functions of remaining PCNA, thereby fully exerting the G1 checkpoint. Given that high p21 expression is often associated with lower PCNA abundance, our findings are suggestive of a global growth inhibitory mechanism involving p21-mediated PCNA suppression.