Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2017 May 1.
Published in final edited form as: Stem Cells. 2016 Mar 7;34(5):1396–1406. doi: 10.1002/stem.2330

The oxygen environment at birth specifies the population of alveolar epithelial stem cells in the adult lung

Min Yee 1, Robert Gelein 2, Thomas J Mariani 1, B Paige Lawrence 2, Michael A O’Reilly 1,3
PMCID: PMC4860080  NIHMSID: NIHMS758446  PMID: 26891117

Abstract

Alveolar epithelial type II cells (AEC2) maintain pulmonary homeostasis by producing surfactant, expressing innate immune molecules, and functioning as adult progenitor cells for themselves and alveolar epithelial type I cells (AEC1). How the proper number of alveolar epithelial cells is determined in the adult lung is not well understood. Here, BrdU labeling, genetic lineage tracing, and targeted expression of the anti-oxidant extracellular superoxide dismutase (ECSOD) in AEC2s are used to show how the oxygen environment at birth influences postnatal expansion of AEC2s and AEC1s in mice. Birth into low (12%) or high (≥60%) oxygen stimulated expansion of AEC2s through self-renewal and differentiation of the airway Scgb1a1+ lineage. This non-linear or hormesis response to oxygen was specific for the alveolar epithelium because low oxygen stimulated and high oxygen inhibited angiogenesis as defined by changes in V-cadherin and PECAM (CD31). Although genetic lineage tracing studies confirmed adult AEC2s are stem cells for AEC1s, we found no evidence that postnatal growth of AEC1s were derived from self-renewing Sftpc+ or the Scbg1a1+ lineage of AEC2s. Taken together, our results show how a non-linear response to oxygen at birth promotes expansion of AEC2s through two distinct lineages. Since neither lineage contributes to the postnatal expansion of AEC1s, the ability of AEC2s to function as stem cells for AEC1s appears to be restricted to the adult lung.

Keywords: Adult stem cells, Cre-loxP system, Differentiation, Lung, Self-Renewal

Graphical Abstract

The oxygen environment at birth regulates the postnatal expansion of alveolar epithelial cells. AEC2s progressively expand in room air through self-renewal until the lung has reached maximal size. Exposure to low or high oxygen at birth promotes expansion of AEC2s by increasing self-renewal and promoting differentiation of the airway Scgb1a1+ lineage. Contrary to their ability to function as adult stem cells, neonatal AEC2s do not contribute to the postnatal expanding population of AEC1s.

INTRODUCTION

The lung is a complex branched organ that terminates in a highly vascularized alveolar epithelium designed to efficiently exchange inspired oxygen with carbon dioxide. Because it is exposed to the external environment, the lung must also maintain tight barrier function, defend against pathogens and inhaled pollutants, and efficiently repair when injured. Size, shape, chemical reactivity, and host cell tropism influences where oxidant gases, particulates and pathogens deposit and injure the lung [1]. Distinct stem and progenitor cell niches have been identified along the respiratory epithelium that protect and maintain homeostasis in the lung [2]. Understanding how these resident stem and progenitor cell niches are defined and maintained is therefore of great scientific and therapeutic importance.

Gas exchange takes place in alveoli that are lined by cuboidal alveolar epithelial type II (AEC2) and squamous alveolar epithelial type I (AEC1) cells. AEC2s express surfactant protein C (proSP-C), the ATP-binding cassette subfamily A member 3 transporter (ABCA3), and various phospholipids that lower surface tensions allowing for respiration to occur at normal intrapulmonary pressures. AEC2s also express genes that are vital for maintaining fluid homeostasis and host defense [3, 4]. Genetic lineage tracing and 3H-thymidine labeling studies in experimental animals have established AEC2s are long-term adult stem cells, capable of both self-renewal and differentiation into AEC1s following alveolar injury [5–8]. AEC1s have historically been considered to be a terminally differentiated alveolar cell of the lung and not capable of self-renewal or differentiation [9]. However, this conclusion is challenged by recent studies showing rat AEC1 can proliferate in culture and by genetic lineage-labeling suggesting mouse AEC1s can differentiate into AEC2s during post-pneumonectomy lung growth [10, 11].

Growing evidence suggests perturbations of the alveolar epithelium correlates with a variety of lung pathologies. Reduced number of alveoli is seen in humans with chronic obstructive pulmonary disease (COPD) [12] and complete alveolar destruction is seen in idiopathic pulmonary fibrosis (IPF) [13]. Lung disease has also been attributed to defects in specific populations of alveolar cells. For example, depletion of AEC2s has been observed in humans with emphysema [14], mutations in surfactant proteins that activate the unfolded protein responses in AEC2s exist in familial forms of IPF [15], and alveolar apoptosis and senescence is seen in COPD [12]. Targeted depletion of AEC2s in adult mice expressing diphtheria toxin receptor causes mild fibrotic lung disease [16]. Given AEC2s play an essential role in maintaining and defending the alveolus, it is important to understand how the optimal number of AEC2s is defined.

The percentage of AEC2s present in alveoli of several adult species has been determined using morphometric and allometric techniques [17, 18]. It ranges from 15.9% in humans, 14.2% in rats, 11.8% in canine, and 7.7% in baboons. The number of AEC2s present in the 8 week old adult mouse lung are most likely to come from fetal and postnatal proliferation because turnover of adult AEC2s is >30 days [9, 19]. Mechanisms controlling proliferation of AEC2s during fetal and postnatal lung development are not known. We recently showed how exposure to 100% oxygen between birth and pnd4 accelerates proliferation of mouse AEC2s [20]. However, these cells were not retained when mice were recovered in room air. Furthermore, adult mice exposed to 100% oxygen as neonates develop fibrotic lung disease when infected with a sublethal dose of HKx31 H3N2 Influenza A Virus (IAV) or administered bleomycin [21, 22]. Because AEC2s maintain alveolar homeostasis, their loss in mice exposed to high oxygen could explain why children born preterm and exposed to high oxygen at birth often exhibit greater respiratory morbidity when infected with viruses [23]. This prompted us to use a combination of BrdU labeling, genetic lineage studies, and targeted expression of the anti-oxidant extracellular superoxide dismutase (ECSOD) to AEC2s to better understand how the oxygen environment at birth influences the proper postnatal expansion of AEC2s and AEC1s. Because airway Scgb1a1+ Club cells can function as adult stem cells for AEC2s [5, 24, 25], we used two lines of transgenic mice (Scgb1a1-Cre and Scgb1a1-CreER) to investigate whether postnatal expanding AEC2s derive from the Scgb1a1+ lineage.. Our results show how a nonlinear response to oxygen promotes expansion of AEC2s through two distinct lineages, which unexpectedly do not contribute to the postnatal expansion of AEC1s.

MATERIALS AND METHODS

Mice

Sftpc-EGFP [26], Scgb1a1-Cre [27], Scgb1a1-CreER [28], Sftpc-CreER [25], Rosa26R-mT/mG [29], and Sftpc-ECSOD [30] mice have been described. The University of Rochester Animal Care and Use Committee approved the use of these mice. To induce recombination between e14.5 and e18.5, pregnant dams containing Scgb1a1-CreER; Rosa26R-mT/mG fetuses were injected four times with tamoxifen (0.05mg/g) in corn oil. To label AEC2s prior to birth, pregnant dams containing Sftpc-CreER; Rosa26R-mT/mG fetuses were injected once on e18.5 with tamoxifen (0.1mg/g). Since tamoxifen induces late fetal abortion, fetuses were removed on e19.5 from dams administered tamoxifen and fostered to tamoxifen-free dams.

Oxygen Exposures

Newborn mice were exposed to room air or hyperoxia (>21%) as described [31], or in hypoxia (<21%) created by mixing medical-grade compressed air with pure nitrogen to a specified concentration. Oxygen levels, humidity of 40–70%, and temperature of 37°C was constantly monitored. Mice were continuously exposed to hypoxia or hyperoxia through pnd10. Some mice were injected i.p. with BrdU and sacrificed two hours later [32]. Wet to dry lung ratios were determined by weighing the freshly excised lung, which was then oven dried at 80°C for 72 hours to obtain a stable dry weight.

Influenza A Virus

Female mice were infected intranasally with 120 hemagglutinating units (HAU) of IAV (strain HKx31, H3N2) in 25 μL saline [22, 31]. Lungs were removed on post-infection day 14. The right lobes were fixed and processed for histology.

Real-time PCR analysis

Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA) treated with DNase I to remove potential contamination of genomic DNA using a TURBO DNA-free™ Kit (Life Technologies, Carlsbad, CA) and reverse transcribed using the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA). cDNA was then amplified with SYBR Green I dye on CFX96™ Touch and CFX384™ Touch Real-Time PCR detection system (Bio-Rad Laboratories, Hercules, CA). PCR products were amplified with sequence-specific primers or 18S rRNA used to normalize equal loading of the template cDNAs (Supplement Table S2).

Histology and Immunohistochemistry

Lung tissues were fixed overnight at 4°C in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained as described [32, 33]. Sections were stained with antibodies against proSP-C and ABCA3 (Seven Hills Bioreagents, Cincinnati, OH), T1α (Developmental Studies Hybridoma Bank at the University of Iowa, Iowa City, IA), Scgb1a1 (EMD Millipore Corp., Billerica, MA), EGFP and BrdU (Abcam, Cambridge, MA), and TTF-1/NKx2.1 (Dako, Carpinteria, CA). Sections were incubated with fluorescently labeled secondary antibody and stained with 4′,6-diamidino-2-phenylindole (DAPI). Slides were visualized with a Nikon E-800 fluorescence microscope (Nikon Instruments, Microvideo Instruments, Avon MA). Images were captured with a SPOT-RT digital camera (Diagnostic Instruments, Sterling Heights, MI). For quantifying positively stained cells, five random images of the right lung lobes were captured at 40X magnification [32, 33]. A minimum of 200 cells per image were counted using Metamorph analysis software (Molecular Devices Corp., Sunnyvale, CA).

Statistical Analysis

Values are expressed as mean ± SEM obtained from at least three separate experiments in each group. All data were analyzed using JMP11 software (SAS Institute, Cary, NC). A 2-way ANOVA was first used to determine overall significance, followed by Tukey-Kramer HSD tests.

RESULTS

Postnatal expansion of the alveolar epithelium is controlled by a non-linear response to oxygen

To evaluate how the alveolar epithelium responds to different levels of oxygen at birth, newborn mice were exposed to 12%, 17%, 21% (room air), 40%, 60%, or 100% oxygen between birth and postnatal day (pnd) 10 (Figure 1A). Quantitative RT-PCR (qRT-PCR) was used to evaluate mRNA expressed by alveolar epithelial and endothelial cells. Relative to room air, exposure to 12% or ≥60% oxygen increased expression of the AEC2-specific Sftpc and Abca3 mRNAs 10–20 fold (Figure 1B). Low (12%) and high (≥60%) oxygen also stimulated mRNA for Lamp1, Lamp2, Muc1, Lyz2, and Ctsh (Figure 1C), which have recently been used to identify different populations of AEC2s [34, 35]. Exposure to low or high oxygen also stimulated mRNA for T1α and Aqp5, genes expressed by AEC1 cells (Figure 1B). This non-linear response to oxygen was specific for the alveolar epithelium because low, but not high oxygen, stimulated mRNA for hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (Vegf), V-cadherin (V-cad) (Figure 1D). High oxygen also inhibited expression of PECAM (CD31).

Figure 1. The oxygen environment at birth exerts different effects on genes expressed by epithelial and endothelial cells.

Figure 1

Open in a new tab

(A) Experimental model showing newborn mice were exposed to a range of oxygen levels between postnatal days 0 (birth) and 10. (B) Fold change in mRNA expression for Sftpc, Abca3, T1α, Aqp5, in mice exposed to varying amounts of oxygen. (C) Fold change in mRNA expression for Lamp1, Lamp2, Muc1, Lyz2, and Ctsh in mice exposed to low (12%), room air (RA), or high (100%) oxygen. (D) Fold change in mRNA expression of Hif1-1α, Vegf, V-Cadherin, and Pecam in mice exposed to low (12%) or high (100%) oxygen graphed relative to room air. Data are presented as mean ± SEM fold change over room air, * P < 0.05, ** P <0.01, ***<0.001, when compared to 21% room air, n =5 per group.

To further analyze the effects of oxygen, lungs were stained with hematoxylin and eosin or with antibodies against proSP-C and T1α. Relative to room air, alveolar simplification was seen in lungs exposed to low (12%) or high (100%) oxygen (Figure 2A). Alveolar septal thickening appeared greater in lungs exposed to high oxygen than in lungs exposed to low oxygen or room air. Exposure to low or high oxygen also increased the number of proSP-C+ cells and the staining intensity of T1α. The thin squamous nature of AEC1s made it difficult to morphometrically quantify T1α+ cells. Instead, the number of AEC2s was determined by staining lungs with antibodies against proSP-C, ABCA3, and TTF-1, and morphometrically quantifying the number of positive cells. Because enhanced green fluorescence protein (EGFP) will be used to map proliferation of AEC2s, we also used transgenic Sftpc-EGFP mice to assess how the oxygen environment at birth influenced the number of EGFP+ AEC2s. Compared to room air, exposure to 12% or ≥60% oxygen clearly increased the number of alveolar cells expressing proSP-C, ABCA3, TTF-1, or EGFP (Figure 2B). Approximately 20% of alveolar cells express proSP-C, ABCA3, EGFP, or TTF-1 (Figures 2C–F). Exposure to low or high oxygen increased the number of positive cells ~2-fold.

Figure 2. The oxygen environment at birth regulates growth of the alveolar epithelium.

Figure 2

Open in a new tab

Newborn mice were exposed to 12%, 21% (room air), 60%, or 100% oxygen between postnatal days 0 and 10. (A) Lungs were stained with hematoxylin and eosin or with antibodies against proSP-C (red), T1α (red), and DAPI (blue). (B) Lungs were stained for proSP-C, ABCA3, or TTF-1 (red), EGFP (green), and DAPI (blue). (C–F) The proportion of proSP-C, ABCA3, EGFP, and TTF-1 positive cells were quantified in 5 images taken from 3 mice and graphed as a percent relative to the total number of DAPI+ cells in the field. * P<0.05, ** P<0.01, *** P<0.001, when compared to 21% room air. Scale bar in (A, B) = 50 μm.

BrdU labeling studies were used to determine when the oxygen environment stimulates proliferation of AEC2s. Because antigen retrieval used to detect BrdU reduced proSP-C immunostaining to background levels, we used EGFP as a surrogate marker of AEC2 cells. Pregnant Sftpc-EGFP mice were injected with BrdU on e14.5, e16.5, e18.5, and pups were injected on postnatal days 0, 4, and 10. Consistent with previous studies in mice and rats showing that epithelial proliferation declines during late fetal lung development [36, 37], BrdU labeled EGFP+ epithelial cells were readily detected on e14.5 and declined by birth (Supplement Figure S1). The very low number of BrdU labeled epithelial cells preceding birth prompted us to investigate whether the oxygen rich transition at birth quickly stimulated proliferation of AEC2s. Newborn Sftpc-EGFP mice were exposed to low (12%), room air (21%), or high (100%) oxygen at birth and injected with BrdU at 6–12 hours of life or on pnd4 (Figure 3A). It was immediately evident that low and high oxygen increased the number of EGFP+ cells in the lung within 6–12 hours of birth. In mice exposed to room air, EGFP was detected in approximately 20% of all alveolar cells, or 1% of EGFP+ AEC2s (Figure B,C). In contrast, exposure to low or high oxygen increased the number of EGFP+ cells by 50–150%. In these mice, BrdU was detected in 3–6% of EGFP+ AEC2s. The effect of oxygen on overall lung growth, including increased proliferation of AEC2s was sufficiently robust to cause a significant change in wet/dry lung ratio (Supplement Table S1).

Figure 3. The oxygen environment at birth stimulates proliferation of AEC2s and airway Club cells.

Figure 3

Open in a new tab

(A) Lungs of newborn Sftpc-EGFP mice exposed to 12%, 21% (RA), or 100% oxygen through pnd1 were stained for EGFP (green), BrdU (red), and DAPI (Blue). (B, C) The proportion of EGFP+ cells present in alveoli or BrdU detected in EGFP+ cells were quantified and graphed. Values represent mean ± SEM of 8 mice, *P < 0.05, **P <0.01, when compared to 21% room air. Scale bar in (A upper row) = 50 μm and (A lower row) = 20 μm.

The oxygen environment at birth stimulates differentiation of airway Scgb1a+ progenitor cells

As exemplified by the lack of BrdU labeling in airway Scgb1a1+ cells, the increased BrdU labeling seen in one-day-old mice exposed to low or high oxygen was restricted to the alveolar space (Figure 4A). However by pnd4, BrdU labeled airway Scgb1a1+ Club cells were detected in mice exposed to low or high oxygen, and occasionally seen in room air. Proliferating Club cells were not considered bronchoalveolar stem cells (BASCs) because they were distributed randomly in the distal airway and did not express proSP-C [38].

Figure 4. The Scgb1a1+ lineage contributes to the oxygen-dependent expansion of AEC2s.

Figure 4

Open in a new tab

(A) Mice were exposed to 12%, 21% (RA), or 100% oxygen between birth and pnd1 or pnd4. Lungs were stained for Scgb1a1 (green), BrdU (red), and DAPI (blue). (B) Scgb1a1-Cre; Rosa26R-mT/mG mice and (C) Scgb1a1-CreER; Rosa26R-dmT/mG mice administered tamoxifen between e16.5 and birth were exposed to 12%, 21% (RA), or 100% oxygen at birth. Lungs were harvested on pnd4 and stained for EGFP (green), Scgb1a1 (red) and DAPI (blue). The number of alveolar EGFP+ cells were quantified in Scgb1a1-Cre (C) and Scgb1a1-CreER (D) mice and graphed relative to the total number of DAPI+ cells in the field. Values represent mean fold change of 5 images taken from 3 mice. (E) Scgb1a1 mRNA expression was determined by qRT-PCR, normalized to 18S ribosomal RNA, and graphed as fold change over room air. ** P<0.01 and *** P<0.001 compared to 21% (RA) values. Scale bar in (A) = 20 mM and (B) = 100 μm.

We used two different Scgb1a1+ reporter mice to determine if the proliferating Scgb1a1 lineage seen on pnd4 contributed to the oxygen-dependent expansion of AEC2s. Scgb1a1-Cre; Rosa26R-mT/mG mice that statically express Cre recombinase in airway Club cells were exposed to 12%, 21% (RA), or 100% oxygen between birth and pnd4 [27]. In mice exposed to room air, EGFP was detected in some airway Club cells and rarely detected in alveolar cells. In contrast, EGFP was readily detected in alveolar cells of mice exposed to low or high oxygen at birth (Figure 4B). Since Cre is expressed under control of the rat Scgb1a1 promoter, which in the rat is normally expressed by AEC2 cells, we used a second line of mice that expresses tamoxifen-responsive CreER under control of the mouse Scgb1a1 promoter [28]. Scgb1a1-CreER; Rosa26R-mT/mG pregnant mice were administered tamoxifen between e14.5 and e18.5, and exposed to 12%, 21% (RA) or 100% oxygen between birth and pnd 4. While EGFP was detected in airway Scgb1a1+ cells of all mice, it was also detected in alveolar cells of mice exposed to low or high oxygen (Figure 4C). Alveolar cells expressing EGFP were considered AEC2 because they also express proSP-C, but not Scgb1a1 or T1α. In both lines of Cre mice, the number of alveolar cells expressing EGFP was less than 1% in mice exposed to room air and increased ~8-fold in mice exposed to low or high oxygen (Figure 4D,E). Increased expression of EGFP in alveolar cells was not attributed to oxygen stimulating expression of the Scgb1a1 gene. In fact and as previously reported [39, 40], exposure to low or high oxygen inhibited Scgb1a1 mRNA expression (Figure 4F).

As further evidence that the oxygen environment at birth stimulates expansion of AEC2s through two lineages, we tested the hypothesis that over-expression of the antioxidant extracellular superoxide dismutase (ECSOD) in AEC2s would inhibit oxygen-dependent self-renewal of existing AEC2s but not differentiation of airway Club cell progenitors. We used Sftpc-ECSOD mice because they are resistant to oxygen-dependent changes in alveolar development and epithelial injury [30, 41]. As expected, oxygen-dependent increase in Sftpc and Abca3 mRNAs was attenuated in Sftpc-ECSOD mice (Figure 5B). To investigate the effects on AEC2 self-renewal, bi-transgenic Sftpc-ECSOD; Sftpc-EGFP mice were exposed to 12%, 21% (RA), or 100% oxygen. Over-expression of ECSOD significantly reduced the oxygen-dependent increase in number of EGFP+ alveolar cells (Figure 5C,D). To investigate effects on the Scgb1a1 lineage, triple transgenic Scgb1a1-Cre; Rosa26R-mT/mG; Sftpc-ECSOD mice were exposed to 12%, 21% (RA), or 100% oxygen. Over-expression of ECSOD in AEC2s did not attenuate oxygen-dependent increase in alveolar EGFP+ cells produced from the Scgb1a1+ lineage (Figure 5E,F).

Figure 5. Over-expression of ECSOD in AEC2s does not inhibit oxygen-dependent expansion of the Scgb1a1+ lineage.

Figure 5

Open in a new tab

(A) Experimental model showing newborn mice exposed to a range of oxygen tensions at birth were analyzed on pnd4 and pnd10. (B) Wildtype and Sftpc-ECSOD mice were exposed to low (12%), 21% (RA), or high (100%) oxygen. Lungs were harvested on pnd10 and mRNA expression of Sftpc and Abca3 were determined by qRT-PCR and graphed. (C) Sftpc-EGFP and bitransgenic Sftpc-EGFP; Sftpc-ECSOD mice were exposed to 12%, 21% (RA), or 100% oxygen. Lungs were harvested on pnd4 and stained for EGFP (green) and DAPI (blue). (D) The proportion of EGFP+ alveolar cells were quantified and graphed. (E) Bi-transgenic Scgb1a1-Cre; Rosa26R-mT/mG and triple transgenic Scgb1a1-Cre; Rosa26R-mT/mG; Sftpc-ECSOD mice were exposed to low (12%), 21% (RA), or high (100%) oxygen. Lungs were harvested on pnd4 and stained for EGFP (green) and DAPI (blue). (F) The proportion of EGFP+ alveolar cells were quantified and graphed. Values represent means ± SEM for 5 mice per treatment. * P < 0.05, ** P <0.01. Scale bar in (C, E) =50μm.

Newborn AEC2s do not contribute to the postnatal expansion of AEC1s

Because low and high oxygen also increased T1α and aquaporin 5 expressed by AEC1s, genetic lineage studies were used to determine whether AEC1s were derived from the self-renewing and/or the Scgb1a1+ lineage of AEC2s. Pregnant dams containing Sftpc-CreER; Rosa26R-mT/mG fetuses were administered tamoxifen on e18.5. Newborn pups were then exposed to 12%, 21% (RA) or 100% oxygen through pnd10. While EGFP was readily detected in cuboidal proSP-C+ AEC2s, we did not observed any EGFP expression in T1α+ squamous AEC1s. Using a similar strategy, EGFP was conditionally activated in Scgb1a1-CreER, Rosa26R-mT/mG mice that were then exposed to 12%, 21% (RA) or 100% oxygen through pnd10. EGFP+ cells expressing proSP-C were occasionally detected in mice exposed to 12% or 100% oxygen but not exposed to room air. Like the self-renewing population of AEC2s, EGFP+ alveolar cells derived from the Scgb1a1 lineage did not express T1α.

To confirm that adult AEC2s have the capacity to differentiate into AEC1s, Sftpc-CreER, Rosa26R-mT/mG mice were administered tamoxifen on e18.5. At 8 weeks of age, EGFP was still restricted to cuboidal proSP-C+ AEC2s (Supplement Figure 2S). Mice were then infected with a sublethal dose of influenza A virus (HKx31, H3N2). On post-infection day 14, EGFP was detected in both cuboidal proSP-C+ AEC2 cells and in some squamous T1α+ AEC1 cells.

DISCUSSION

The concept that AEC2s function as a “reserve” or progenitor cell for the injured alveolus originated nearly 50 years ago when Kapanci and colleagues showed how they replace injured AEC1s in adult monkeys exposed to hyperoxia [6]. While numerous studies since then have confirmed this concept and extended it by showing how AEC2s also produce surfactant and innate immune molecules, it is still unclear how the number of alveolar epithelial cells is determined in the adult lung. Here, we provide evidence that postnatal expansion of alveolar epithelial cells is regulated by a non-linear response to oxygen, wherein birth into 12% or ≥60% oxygen stimulates expansion of AEC2s and AEC1s relative to room air. This expanding population of AEC2s is derived from increased self-renewal of existing AEC2s and differentiation of the airway Scgb1a1+ lineage (Figure 7). Surprisingly, neither lineage contributed to the postnatal expansion of AEC1s. Our findings show how the number of alveolar epithelial cells is influenced by the oxygen environment at birth and suggest the stem-like properties of AEC2s may be restricted to the adult lung.

Figure 7. A proposed model for the oxygen-dependent expansion of alveolar epithelial cells.

Figure 7

Open in a new tab

AEC2s progressively expand in room air through self-renewal until the lung has reached maximal size. Exposure to low or high oxygen at birth promotes expansion of AEC2s by increasing self-renewal and promoting differentiation of the airway Scgb1a1+ lineage. Contrary to their ability to function as adult stem cells, neonatal AEC2s do not contribute to the postnatal expanding population of AEC1s.

Gene expression studies in mice has defined a “time-to-birth” program in which genes specifying lung structure are expressed first, while genes involved in oxygen transport, protection against reactive oxygen species, and host defense are expressed later [42, 43]. Late fetal production of anti-oxidant defenses likely serve to maintain a constant redox state as amniotic fluid containing less than 1% oxygen is replaced with air containing much higher levels of oxygen [44]. As anti-oxidant levels rise in late fetal lung development, proliferation of AEC2s declines perhaps as another way to defend against oxidative stress and damage at birth [45]. AEC2 proliferation resumes between pnd4 and pnd10, and then declines by pnd21 to the slow steady state level seen in adult lung [36, 37]. In the current study, morphometric counting of AEC2s and genetic lineage labeling with static Sftpc-EGFP and conditional Sftpc-CreER; Rosa26R-mT/mG mice revealed how exposure to 12% or ≥60% oxygen at birth stimulated proliferation of existing AEC2s within 6–12 hours of birth. The effects of low or high oxygen on the developing lung were swift and sufficiently robust to increase lung weight by pnd1 and promote alveolar simplification by pnd10. Targeting the anti-oxidant ECSOD to AEC2s blunted oxygen-dependent changes in proliferation, indicating that the redox state of the alveolus is a critical regulator of neonatal AEC2 proliferation. Indeed, a redox balance has been observed in fetal AEC2s wherein hypoxia activates HIF-1α and hyperoxia activates NF-κB [46]. Both of these transcription factors play an important role in alveolar epithelial cell differentiation and survival of newborn mice at birth [47–49].

In addition to stimulating self-renewal of AEC2s, birth into low or high oxygen stimulated BrdU labeling of airway Scgb1a1+ cells by pnd4. Proliferating cells were not bronchoalveolar stem cells (BASCs) because they were detected randomly in the distal airway epithelium and they did not express proSP-C. Genetic lineage studies using two lines of Scgb1a1+ Cre drivers revealed some AEC2s also derive from the airway Scgb1a1 lineage. This supports our earlier study in which Scgb1a1-rtTA; otet-Cre; Rosa26R-mT/mG mice were used to show that exposure to 100% oxygen stimulated expansion of AEC2s [20]. Because leaky Cre expression in AEC2s has been observed in these mice, we could not interpret whether high oxygen expanded existing labeled AEC2s or differentiation of the airway Scgb1a1+ lineage [50]. However, the current study using BrdU labeling along with Scgb1a1-Cre; Rosa26R-mT/mG and Scgb1a1-CreER; Rosa26R-mT/mG mice support the idea that some AEC2s derive from a proliferating Scgb1a1+ lineage. Since adult airway Scgb1a1+ progenitors repopulate AEC2s following bleomycin or influenza virus injury [5, 24], differentiation of these cells in neonatal mice exposed to low or high oxygen may reflect a response to oxidant injury. Decreased expression of Scgb1a1 (Figure 4) and increased 8-oxoguanine staining (data not shown) support this conclusion.

Oxygen-dependent expansion of airway progenitors was not seen in an earlier study where the Scgb1a1-CreER; Rosa26R-mT/mG mice were administered tamoxifen at birth [28]. We reproduced this observation and discovered only a small number of airway progenitor cells labeled with EGFP when tamoxifen was administered once (Supplement Figure S3). The limited number of cells labeled with EGFP may explain why differentiation of airway progenitors was not easily seen until tamoxifen was administered earlier and more often. It is worth noting that over-expression of ECSOD in AEC2s protects against neonatal hyperoxia as defined by preserving alveolar structure during exposure and preventing the loss of excess AEC2s when mice exposed to 100% oxygen were recovered in room air [41, 51]. Since ESCOD does not inhibit expansion of the Scgb1a1 lineage, their differentiation into AEC2s and subsequent loss seems less disruptive to alveolar homeostasis than the effects on AEC2 self-renewal. This is consistent with the genetic fate mapping studies showing how the number of AEC2s derived from the Scgb1a1+ lineage is significantly less than the number derived from self-renewal. Whether the small number of AEC2s derived from airway Scgb1a1+ progenitors express unique genes or functions remains to be determined. In fact, growing evidence suggests subpopulations of AEC2s exist that can be distinguished by relative expression of Lamp1, Lamp2, Muc1, Lyz2, and Ctsh [34, 35]. However, none of these cells appear to be selectively responding to the oxygen environment at birth because expression of these genes responded similarly to different doses of oxygen. Hence, oxygen-dependent proliferation of existing AEC2s and differentiation of Scgb1a1 lineage does not appear to selectively expand a unique subpopulation of AEC2s.

Proliferating AEC2s presumably must also maintain expansion with AEC1s. In newborn rats, the number of AEC1s increased between pnd1-7 and pnd10-21 [37]. An even greater increase occurred between pnd7-10, which coincided with 3H-thymidine labeling of AEC2s. Because adult AEC2s can function as stem cells for AEC1s, increased AEC1s seen between pnd7-10 were thought to come from proliferating AEC2s. Hence, it was not surprising to find that low and high oxygen also increased T1α and Aqp5, genes expressed by AEC1 cells. However, differentiation of AEC1 from AEC2s was not seen with Sftpc-CreER; Rosa26R-mT/mG mice or with Sftpc-CreER driver lines. Our inability to demonstrate differentiation of AEC2s to AEC1s within the first 10 days of life is consistent with a recent study showing neonatal AEC2s are not progenitor cells for AEC1s [35]. Interestingly, our genetic lineage studies reveal the population of AEC2s present at birth can undergo differentiation to AEC1s when adult mice are infected with influenza A virus. This implies their ability to function as stem cells for AEC1s develops postnatally. Our findings therefore suggest AEC1s are not developmentally derived from AEC2s, but their production during alveolar development is coordinated with AEC2s.

Morphometric and allometric studies evaluating lung volume, alveolar surface area, diffusing capacity, and cell number indicate that the relative proportion and size of individual cells is constant between species containing a 200,000 fold difference in body mass [17, 18]. Our morphometric studies counting AEC2s and genetic lineage studies with Sftpc-CreER; Rosa26R-mT/mG mice suggest AEC2s comprise ~20% of alveolar cells at pnd4 and at 8 weeks of age (Figure 4C). To maintain this ratio, increased proliferation of AEC2s between pnd4 and pnd10 should theoretically also coordinate with PDGR-α mesenchymal cells that function as support cells in alveolosphere cultures [5]. However, as the oxygen environment stimulates expansion of AECIIs, it surprisingly suppresses mRNA for both Pdgr-α and Pdgr-β (data not shown). This is consistent with a recent paper showing Pdgr-α levels are suppressed in newborn mice exposed to 75% oxygen and in human BPD samples [52]. Vascular endothelial cells are another population that theoretically should maintain growth with AEC2s. But as defined by changes in expression of V-cadherin and PECAM (CD31), low oxygen stimulates angiogenesis and high oxygen inhibits angiogenesis. An aberrant oxygen environment may therefore uncouple the coordinated growth of different cell populations required to build a functioning alveolus.

Why would the oxygen environment at birth influence growth of the newborn lung? Analysis of iron oxide in rock suggests that the oxygen environment fluctuated between 15% and 40% as the transition from aquatic to terrestrial habitation by tetrapods and the development of a lung breathing air occurred approximately 300 million years ago [53]. These fluctuating oxygen levels may have influenced how the lung evolved so that it is best suited to function in that level of oxygen. As shown here, when exposed outside of this normative range, the lung functionally adapts, in part, by expanding the alveolar epithelium. Hypothetically, exposure to low oxygen stimulates alveolar epithelial and vascular growth so as to maximize ability to capture oxygen in a low oxygen environment. In contrast, exposure to low oxygen stimulates alveolar epithelial growth but inhibits angiogenesis perhaps because the diffusing capacity of oxygen is great in a high oxygen environment. However, functionally adapting to oxygen can also affect other functions of the lung that manifest as airway hyperreactivity and altered host-pathogen responses to respiratory viral infections. Remarkably, a similar non-linear dose response to oxygen has been reported to negatively influence life-span of Drosophila melanogaster [54]. Likewise, too much oxygen used to treat preterm infants in respiratory distress alters lung development and increases severity of lung disease following respiratory viral infections [23]. Conversely, children born into low oxygen at high altitude develop larger lungs and heart, and are also more likely to be re-hospitalized following a respiratory viral infection [55]. Whether respiratory morbidity is related to oxygen-dependent changes in AEC2s or other stem cell niches requires further investigation.

SUMMARY

Despite nearly 50 years of research showing AEC2s can self-renew and differentiate into AEC1s, how the optimal number of alveolar epithelial cells is determined in the adult lung is not known. Our findings show how postnatal expansion of AEC2s in mice is regulated by a nonlinear response to oxygen at birth controlling both self-renewal of existing AEC2s and differentiation of the airway Scgb1a1+ lineage. Surprisingly, neither lineage contributes to the postnatal expansion of AEC1s. The apparent inability of AEC2s to produce AEC1s developmentally, yet still function as adult stem cells following injury questions the concept that regeneration recapitulates developmental processes. A better understanding of how the oxygen environment at birth influences expansion of alveolar epithelial cells and how AEC2s acquire a stem-like phenotype could lead to new opportunities for improving respiratory health.

Supplementary Material

Supp Fig S1
Supp Fig S2
Supp Fig S3
Supp Info

Figure 6. Newborn AEC2s do not function as progenitor cells for AEC1s.

Figure 6

Open in a new tab

Newborn (A) Scgb1a1-Cre X Rosa26R-mT/mG and (B) Scgb1a1-CreER X Rosa26R-mT/mG mice administered tamoxifen between e16.5 and birth were exposed to 12%, 21% (RA) or 100% oxygen. Lungs were harvested on pnd 10 and stained from SP-C and T1α (red), EGFP (green), and counterstained with DAPI (blue). Inset boxes shown in (B) are magnified below the image. Scale bar in (A) =20 μm and (B) = 50 μm.

Acknowledgments

We thank members of the O’Reilly laboratory for critiquing this paper. This work was supported by grants from NIH grants HL091968 and HL097141 (M.A.O), ES017250 and ES023260 (B.P.L), and HL094431 (T.M.). The animal inhalation facility and tissue processing cores were supported by NIEHS Center grant ES01247. We thank John Heath and Brigid Hogan for sharing their advice and transgenic mice with us.

Footnotes

SUPPLEMENTAL INFORMATION

Supplemental information for this article includes 3 figures and 2 Tables.

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

The authors have no potential conflicts of interest to disclose.

Author Contributions:

M.Y.: Concept and Design, Collection and Assembly of Data, Data Analysis and Interpretation, Final Approval of Manuscript; R.G.: Concept and Design, Collection and Assembly of Data, Final Approval of Manuscript; T.J.M.: Provision of Study Material, Data Analysis and Interpretation, Final Approval of Manuscript; B.P.L.: Provision of Study Material, Data Analysis and Interpretation, Final Approval of Manuscript; M.A.O’R.: Concept and Design, Financial Support, Data Analysis and Interpretation, Manuscript Writing, Final Approval of Manuscript

References

  • 1.Carvalho TC, Peters JI, Williams RO., 3rd Influence of particle size on regional lung deposition--what evidence is there? International journal of pharmaceutics. 2011;406:1–10. doi: 10.1016/j.ijpharm.2010.12.040. [DOI] [PubMed] [Google Scholar]
  • 2.Hogan BL, Barkauskas CE, Chapman HA, et al. Repair and regeneration of the respiratory system: complexity, plasticity, and mechanisms of lung stem cell function. Cell stem cell. 2014;15:123–138. doi: 10.1016/j.stem.2014.07.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.McCormack FX, Whitsett JA. The pulmonary collectins, SP-A and SP-D, orchestrate innate immunity in the lung. J Clin Invest. 2002;109:707–712. doi: 10.1172/JCI15293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Fehrenbach H. Alveolar epithelial type II cell: defender of the alveolus revisited. Respir Res. 2001;2:33–46. doi: 10.1186/rr36. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Barkauskas CE, Cronce MJ, Rackley CR, et al. Type 2 alveolar cells are stem cells in adult lung. J Clin Invest. 2013;123:3025–3036. doi: 10.1172/JCI68782. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Kapanci Y, Weibel ER, Kaplan HP, et al. Pathogenesis and reversibility of the pulmonary lesions of oxygen toxicity in monkeys. II. Ultrastructural and morphometric studies. Lab Invest. 1969;20:101–118. [PubMed] [Google Scholar]
  • 7.Crapo JD, Barry BE, Foscue HA, et al. Structural and biochemical changes in rat lungs occurring during exposures to lethal and adaptive doses of oxygen. The American review of respiratory disease. 1980;122:123–143. doi: 10.1164/arrd.1980.122.1.123. [DOI] [PubMed] [Google Scholar]
  • 8.Evans MJ, Cabral LJ, Stephens RJ, et al. Transformation of alveolar type 2 cells to type 1 cells following exposure to NO2. Exp Mol Pathol. 1975;22:142–150. doi: 10.1016/0014-4800(75)90059-3. [DOI] [PubMed] [Google Scholar]
  • 9.Evans MJ, Bils RF. Identification of cells labeled with tritiated thymidine in the pulmonary alveolar walls of the mouse. The American review of respiratory disease. 1969;100:372–378. doi: 10.1164/arrd.1969.100.3.372. [DOI] [PubMed] [Google Scholar]
  • 10.Gonzalez RF, Allen L, Dobbs LG. Rat alveolar type I cells proliferate, express OCT-4, and exhibit phenotypic plasticity in vitro. Am J Physiol Lung Cell Mol Physiol. 2009;297:L1045–1055. doi: 10.1152/ajplung.90389.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Jain R, Barkauskas CE, Takeda N, et al. Plasticity of Hopx(+) type I alveolar cells to regenerate type II cells in the lung. Nature communications. 2015;6:6727. doi: 10.1038/ncomms7727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Tuder RM, Yoshida T, Arap W, et al. State of the art. Cellular and molecular mechanisms of alveolar destruction in emphysema: an evolutionary perspective. Proc Am Thorac Soc. 2006;3:503–510. doi: 10.1513/pats.200603-054MS. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Coward WR, Saini G, Jenkins G. The pathogenesis of idiopathic pulmonary fibrosis. Therapeutic advances in respiratory disease. 2010;4:367–388. doi: 10.1177/1753465810379801. [DOI] [PubMed] [Google Scholar]
  • 14.Otto-Verberne CJ, Ten Have-Opbroek AA, Willems LN, et al. Lack of type II cells and emphysema in human lungs. The European respiratory journal: official journal of the European Society for Clinical Respiratory Physiology. 1991;4:316–323. [PubMed] [Google Scholar]
  • 15.Zoz DF, Lawson WE, Blackwell TS. Idiopathic pulmonary fibrosis: a disorder of epithelial cell dysfunction. The American journal of the medical sciences. 2011;341:435–438. doi: 10.1097/MAJ.0b013e31821a9d8e. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Sisson TH, Mendez M, Choi K, et al. Targeted injury of type II alveolar epithelial cells induces pulmonary fibrosis. Am J Respir Crit Care Med. 2010;181:254–263. doi: 10.1164/rccm.200810-1615OC. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Crapo JD, Young SL, Fram EK, et al. Morphometric characteristics of cells in the alveolar region of mammalian lungs. The American review of respiratory disease. 1983;128:S42–46. doi: 10.1164/arrd.1983.128.2P2.S42. [DOI] [PubMed] [Google Scholar]
  • 18.Stone KC, Mercer RR, Gehr P, et al. Allometric relationships of cell numbers and size in the mammalian lung. Am J Respir Cell Mol Biol. 1992;6:235–243. doi: 10.1165/ajrcmb/6.2.235. [DOI] [PubMed] [Google Scholar]
  • 19.Bowden DH, Davies E, Wyatt JP. Cytodynamics of pulmonary alveolar cells in the mouse. Arch Pathol. 1968;86:667–670. [PubMed] [Google Scholar]
  • 20.Yee M, Buczynski BW, O’Reilly MA. Neonatal hyperoxia stimulates the expansion of alveolar epithelial type II cells. Am J Respir Cell Mol Biol. 2014;50:757–766. doi: 10.1165/rcmb.2013-0207OC. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Yee M, Buczynski BW, Lawrence BP, et al. Neonatal hyperoxia increases sensitivity of adult mice to bleomycin-induced lung fibrosis. Am J Respir Cell Mol Biol. 2013;48:258–266. doi: 10.1165/rcmb.2012-0238OC. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.O’Reilly MA, Marr SH, Yee M, et al. Neonatal hyperoxia enhances the inflammatory response in adult mice infected with influenza A virus. Am J Respir Crit Care Med. 2008;177:1103–1110. doi: 10.1164/rccm.200712-1839OC. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Weisman LE. Populations at risk for developing respiratory syncytial virus and risk factors for respiratory syncytial virus severity: infants with predisposing conditions. The Pediatric infectious disease journal. 2003;22:S33–37. doi: 10.1097/01.inf.0000053883.08663.e5. discussion S37–39. [DOI] [PubMed] [Google Scholar]
  • 24.Zheng D, Limmon GV, Yin L, et al. Regeneration of alveolar type I and II cells from Scgb1a1-expressing cells following severe pulmonary damage induced by bleomycin and influenza. PloS one. 2012;7:e48451. doi: 10.1371/journal.pone.0048451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Rock JR, Barkauskas CE, Cronce MJ, et al. Multiple stromal populations contribute to pulmonary fibrosis without evidence for epithelial to mesenchymal transition. Proc Natl Acad Sci U S A. 2011;108:E1475–1483. doi: 10.1073/pnas.1117988108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Lo B, Hansen S, Evans K, et al. Alveolar epithelial type II cells induce T cell tolerance to specific antigen. J Immunol. 2008;180:881–888. doi: 10.4049/jimmunol.180.2.881. [DOI] [PubMed] [Google Scholar]
  • 27.Simon DM, Arikan MC, Srisuma S, et al. Epithelial cell PPAR[gamma] contributes to normal lung maturation. FASEB J. 2006;20:1507–1509. doi: 10.1096/fj.05-5410fje. [DOI] [PubMed] [Google Scholar]
  • 28.Rawlins EL, Okubo T, Xue Y, et al. The role of Scgb1a1+ Clara cells in the long-term maintenance and repair of lung airway, but not alveolar, epithelium. Cell stem cell. 2009;4:525–534. doi: 10.1016/j.stem.2009.04.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Muzumdar MD, Tasic B, Miyamichi K, et al. A global double-fluorescent Cre reporter mouse. Genesis. 2007;45:593–605. doi: 10.1002/dvg.20335. [DOI] [PubMed] [Google Scholar]
  • 30.Ahmed MN, Suliman HB, Folz RJ, et al. Extracellular superoxide dismutase protects lung development in hyperoxia-exposed newborn mice. Am J Respir Crit Care Med. 2003;167:400–405. doi: 10.1164/rccm.200202-108OC. [DOI] [PubMed] [Google Scholar]
  • 31.Maduekwe ET, Buczynski BW, Yee M, et al. Cumulative neonatal oxygen exposure predicts response of adult mice infected with influenza A virus. Pediatr Pulmonol. 2014 doi: 10.1002/ppul.23063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Yee M, Vitiello PF, Roper JM, et al. Type II epithelial cells are critical target for hyperoxia-mediated impairment of postnatal lung development. Am J Physiol Lung Cell Mol Physiol. 2006;291:L1101–1111. doi: 10.1152/ajplung.00126.2006. [DOI] [PubMed] [Google Scholar]
  • 33.Yee M, Chess PR, McGrath-Morrow SA, et al. Neonatal oxygen adversely affects lung function in adult mice without altering surfactant composition or activity. Am J Physiol Lung Cell Mol Physiol. 2009;297:L641–649. doi: 10.1152/ajplung.00023.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Treutlein B, Brownfield DG, Wu AR, et al. Reconstructing lineage hierarchies of the distal lung epithelium using single-cell RNA-seq. Nature. 2014;509:371–375. doi: 10.1038/nature13173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Desai TJ, Brownfield DG, Krasnow MA. Alveolar progenitor and stem cells in lung development, renewal and cancer. Nature. 2014;507:190–194. doi: 10.1038/nature12930. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Amy RW, Bowes D, Burri PH, et al. Postnatal growth of the mouse lung. J Anat. 1977;124:131–151. [PMC free article] [PubMed] [Google Scholar]
  • 37.Kauffman SL, Burri PH, Weibel ER. The postnatal growth of the rat lung. II. Autoradiography. Anat Rec. 1974;180:63–76. doi: 10.1002/ar.1091800108. [DOI] [PubMed] [Google Scholar]
  • 38.Kim CF, Jackson EL, Woolfenden AE, et al. Identification of bronchioalveolar stem cells in normal lung and lung cancer. Cell. 2005;121:823–835. doi: 10.1016/j.cell.2005.03.032. [DOI] [PubMed] [Google Scholar]
  • 39.Raffay TM, Locy ML, Hill CL, et al. Neonatal hyperoxic exposure persistently alters lung secretoglobins and annexin A1. BioMed research international. 2013;2013:408485. doi: 10.1155/2013/408485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Ramsay PL, Luo Z, Major A, et al. Multiple mechanisms for oxygen-induced regulation of the Clara cell secretory protein gene. FASEB J. 2003;17:2142–2144. doi: 10.1096/fj.03-0048fje. [DOI] [PubMed] [Google Scholar]
  • 41.Buczynski BW, Yee M, Martin KC, et al. Neonatal hyperoxia alters the host response to influenza A virus infection in adult mice through multiple pathways. Am J Physiol Lung Cell Mol Physiol. 2013;305:L282–290. doi: 10.1152/ajplung.00112.2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Kho AT, Bhattacharya S, Mecham BH, et al. Expression profiles of the mouse lung identify a molecular signature of time-to-birth. Am J Respir Cell Mol Biol. 2009;40:47–57. doi: 10.1165/rcmb.2008-0048OC. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Xu Y, Wang Y, Besnard V, et al. Transcriptional programs controlling perinatal lung maturation. PloS one. 2012;7:e37046. doi: 10.1371/journal.pone.0037046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Sjostedt S, Rooth G, Caligara F. The oxygen tension of the amniotic fluid. American journal of obstetrics and gynecology. 1958;76:1226–1230. doi: 10.1016/s0002-9378(16)36937-x. [DOI] [PubMed] [Google Scholar]
  • 45.Kauffman SL. Cell proliferation in the mammalian lung. Int Rev Exp Pathol. 1980;22:131–191. [PubMed] [Google Scholar]
  • 46.Haddad JJ, Olver RE, Land SC. Antioxidant/pro-oxidant equilibrium regulates HIF-1alpha and NF-kappa B redox sensitivity. Evidence for inhibition by glutathione oxidation in alveolar epithelial cells. J Biol Chem. 2000;275:21130–21139. doi: 10.1074/jbc.M000737200. [DOI] [PubMed] [Google Scholar]
  • 47.Saini Y, Harkema JR, LaPres JJ. HIF1alpha is essential for normal intrauterine differentiation of alveolar epithelium and surfactant production in the newborn lung of mice. J Biol Chem. 2008;283:33650–33657. doi: 10.1074/jbc.M805927200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Bridges JP, Lin S, Ikegami M, et al. Conditional hypoxia inducible factor-1alpha induction in embryonic pulmonary epithelium impairs maturation and augments lymphangiogenesis. Developmental biology. 2012;362:24–41. doi: 10.1016/j.ydbio.2011.10.033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Yang G, Abate A, George AG, et al. Maturational differences in lung NF-kappaB activation and their role in tolerance to hyperoxia. J Clin Invest. 2004;114:669–678. doi: 10.1172/JCI19300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Rawlins EL, Perl AK. The a“MAZE”ing world of lung-specific transgenic mice. Am J Respir Cell Mol Biol. 2012;46:269–282. doi: 10.1165/rcmb.2011-0372PS. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Auten RL, O’Reilly MA, Oury TD, et al. Transgenic extracellular superoxide dismutase protects postnatal alveolar epithelial proliferation and development during hyperoxia. Am J Physiol Lung Cell Mol Physiol. 2006;290:L32–40. doi: 10.1152/ajplung.00133.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Popova AP, Bentley JK, Cui TX, et al. Reduced platelet-derived growth factor receptor expression is a primary feature of human bronchopulmonary dysplasia. Am J Physiol Lung Cell Mol Physiol. 2014;307:L231–239. doi: 10.1152/ajplung.00342.2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Berner RA, Vandenbrooks JM, Ward PD. Evolution. Oxygen and evolution. Science. 2007;316:557–558. doi: 10.1126/science.1140273. [DOI] [PubMed] [Google Scholar]
  • 54.Rascon B, Harrison JF. Lifespan and oxidative stress show a non-linear response to atmospheric oxygen in Drosophila. The Journal of experimental biology. 2010;213:3441–3448. doi: 10.1242/jeb.044867. [DOI] [PubMed] [Google Scholar]
  • 55.Choudhuri JA, Ogden LG, Ruttenber AJ, et al. Effect of altitude on hospitalizations for respiratory syncytial virus infection. Pediatrics. 2006;117:349–356. doi: 10.1542/peds.2004-2795. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supp Fig S1
NIHMS758446-supplement-Supp_Fig_S1.eps (2.6MB, eps)
Supp Fig S2
NIHMS758446-supplement-Supp_Fig_S2.eps (2.9MB, eps)
Supp Fig S3
NIHMS758446-supplement-Supp_Fig_S3.eps (1.2MB, eps)
Supp Info
NIHMS758446-supplement-Supp_Info.docx (138.2KB, docx)

RESOURCES