Organismal adaptation to low oxygen levels is primarily regulated at the transcriptional level by hypoxia-inducible factors (HIFs). HIFs are heterodimeric transcription factors comprised of an oxygen-sensitive alpha subunit (1a, 2a or 3a) and a constitutively expressed beta subunit (1b, also referred to as aryl hydrocarbon receptor nuclear translocator (ARNT)). All three alpha subunits, as well as ARNT, are critical modulators of embryonic mouse development as demonstrated in gene ablation studies (Maltepe et al., 1997; Iyer et al., 1998; Ryan et al., 1998; Compernolle et al., 2002; Scortegagna et al., 2003; Yamashita et al., 2008). HIF1a activity is largely regulated at the level of protein stability, governed by a family of prolyl hydroxylase domain (PHD)-containing enzymes, PHD1, 2 and 3 (Bruick and McKnight, 2001; Epstein et al., 2001; Ivan et al., 2002). Under normoxic conditions, the PHDs hydroxylate two conserved proline residues within the oxygen-dependent degradation domain (ODD) of human HIF1a, P402 and P564, in a process that requires O₂, Fe²⁺ and 2-oxoglutarate. Hydroxylated HIF1a is then recognized by the E3 ubiquitin complex von-Hippel-Lindau (pVHL), tagged with polyubiquitin and degraded by the 26S proteasome (see (Schofield and Ratcliffe, 2004) for review). In hypoxia, PHD-mediated hydroxylation is inhibited and HIF1a escapes proteasomal degradation. Stable HIF1a subunits then accumulate in the cytoplasm and translocate into the nucleus where they interact with ARNT. Functional HIF1a/ARNT dimers bind a conserved DNA binding motif known as the hypoxia responsive element (HRE) and activate a large number of target genes that regulate oxygen delivery, energy maintenance and survival (Wenger et al., 2005). In addition to regulation at the protein stability level, HIF1a transcriptional activity is also controlled via hydroxylation by the asparaginyl hydroxylase factor inhibiting HIF (FIH) (Mahon et al., 2001). Hydroxylation of HIF1a occurs at asparagine 803 (N803) and prevents interaction with the potent co-activator p300/CBP, thereby dampening the transcriptional activation of HIF1a under normoxic conditions (Ema et al., 1999; Lando et al., 2002b).

HIF1a protein is present at low levels in normoxia-cultured primary and transformed pulmonary cell types derived from multiple species and is highly induced under hypoxic conditions (Yu et al., 1998). Endogenous HIF1a and ARNT are primarily restricted to epithelial cells of the first trimester human fetal lung (Groenman et al., 2007), suggesting a role for epithelial HIF1a signaling in pulmonary development and/or maturation. In agreement, Cre-mediated deletion of HIF1a from the developing pulmonary epithelium using the Sftpc-rtTA/tetO7-Cre system has been reported to result in neonatal respiratory distress associated with decreased maturation of distal epithelial cells (Saini et al., 2008). In extremely premature infants, respiratory distress is a common morbidity resulting from insufficient maturation of the lung. This perturbation of maturation, coupled with mechanical ventilatory support, is thought to underlie the susceptibility of this patient population to developing a form of chronic lung disease known as bronchopulmonary dysplasia (BPD) (Baraldi and Filippone, 2007). Studies performed in large animal models of BPD have shown that HIF1a protein is decreased in the premature lung, associated with decreased vascularization and diminished levels of a pivotal HIF1a target gene, Vegfa (Asikainen et al., 2005; Asikainen et al., 2006b; Grover et al., 2007); chemical stabilization of HIF1a in the premature baboon model dramatically improved the pathophysiological pulmonary abnormalities (Asikainen et al., 2006a). Collectively, these data demonstrate that HIF1a signaling is required for the proper maturation and function of alveolar type II cells during fetal lung development.

Previous work from our lab has shown that the threshold level of SatPC required for perinatal lung function in mice lies between 4.3 and 5.6nmol/mg tissue weight, amounts that represent 46.0–60.0% of wild-type levels (Bridges et al., 2010). Based on these data, it is unlikely that the 21% decrease in SatPC levels seen in HIF1a ΔODD,N803A mice solely underlies the respiratory distress and neonatal lethal phenotype. However, this deficiency in SatPC production, combined with substantial decreases in mature SFTPB and ABCA3 protein expression, both of which are required for the transition to air breathing (Clark et al., 1995; Ban et al., 2007; Fitzgerald et al., 2007; Hammel et al., 2007), may have been sufficient to induce respiratory distress at birth. We do not believe that the HIF1a-mediated inhibition of SFTPB and ABCA3 expression is direct, since HIF1a TPM expression did not suppress baseline expression of the proximal Sftpb and Abca3 promoters in luciferase reporter assays (data not shown). An alternative explanation for the observed phenotype is that the HIF1a ΔODD,N803A protein inhibited endogenous HIF1a function in the developing respiratory epithelium of HIF1a ΔODD,N803A transgenic mice. This possibility stems from our observation that transient HIF1a ΔODD,N803A expression in HeLa cells decreased the hypoxic induction of endogenous HIF1a protein (Figure 3A). In support of this hypothesis, genetic deletion of HIF1a in the developing respiratory epithelium resulted in phenotypic similarities seen in HIF1a ΔODD,N803A mice, including decreased production of surfactant proteins SFTPB and SFTPC and the phospholipid transporter ABCA3 in distal epithelial cells without defects in branching morphogenesis (Saini et al., 2008).

Epithelial HIF1a TPM expression profoundly affected branching morphogenesis, characterized by hypoplasia and the presence of a cystic distal epithelium that was initially detected at E14.5 and persisted throughout lung organogenesis. One possible explanation for this morphogenetic defect is decreased proliferation of HIF1a TPM-expressing epithelial cells (Figure 7A and 7B). During early embryonic lung development, the rate of cell proliferation is highest in the epithelial cells located at the branch points where it drives both growth and patterning of the developing airways (Hogan and Yingling, 1998; Warburton et al., 2000). In vitro studies have shown that prolonged hypoxic exposure induces HIF1a-dependent cell cycle arrest in a variety of cell types (Gardner et al., 2001; Green et al., 2001; Goda et al., 2003; Koshiji et al., 2004). Arrest occurs at the G1/S transition and is driven by increased expression of the cell cycle regulators Cdkn1a, via HIF1a-mediated displacement of the C-Myc repressor from the Cdkn1a promoter, and direct transcriptional activation of Cdkn1b (Gardner et al., 2001; Goda et al., 2003). In the HIF1a TPM transgenic mice, Cdkn1a was induced in the epithelium whereas Cdkn1b levels were unchanged (Figure 7C and 7D). Importantly, both Cdkn1a and Cdkn1b levels were not induced in HIF1a ΔODD,N803A mice (Supplementary Figure 2), in which branching proceeded normally, suggesting that the differential transcriptional induction of Cdkn1a in HIF1a TPM mice underlies the branching defect. In addition to the induction of Cdkn1a, it is likely that the decreased mitochondrial content and metabolic shunting toward glycolysis also contributed to decreased epithelial proliferation by limiting the amount of ATP required for this energy expensive process. Interestingly, genetic deletion of another Myc family member, N-Myc, resulted in a similar cystic phenotype that was associated with decreased epithelial cell proliferation (Moens et al., 1992; Moens et al., 1993; Sawai et al., 1993; Okubo et al., 2005). Whether the decreased proliferation observed in the HIF1a TPM respiratory epithelium is also due, in part, to an N-Myc-dependent mechanism remains to be determined.

The blood vascular defects in HIF1a TPM lungs did not appear to be due to hypervascularity or gross alterations in patterning of the vascular plexus. One possible explanation for the subpleural hemorrhaging is the HIF1a-mediated induction of endogenous VEGFA protein from the epithelial cells, resulting in increased permeability of the blood vasculature. This hypothesis is consistent with previous reports demonstrating an accumulation of interstitial fluid in transgenic mice that overexpressed the mouse VEGFA₁₆₄ isoform in developing (Zeng et al., 1998) or postnatal (Le Cras et al., 2004) pulmonary epithelial cells, and in adult lungs in which human VEGF-165 was ectopically expressed by adenoviral infection (Kaner et al., 2000). In addition, HIF1a ΔODD,N803A mice, in which blood vascular integrity was normal, did not have elevated VEGFA levels (Supplemental Figure 1A). Gain of HIF1a function in the epidermis, using two distinct HIF1a transgenic constructs and epidermal promoters, increased endogenous VEGFA levels and was associated with a vascular phenotype that was distinct from our model, characterized by hypervascularity in the absence of baseline microvascular leak (Elson et al., 2001; Oladipupo et al., 2011). Many mechanistic differences could be responsible for the discrepancy between the HIF1a-mediated phenotype in the pulmonary and epidermal vascular tissue beds including: 1) potency of HIF1a transgenic constructs; 2) differential VEGF receptor expression on endothelial cells in the developing lung compared to the epidermis; 3) differences in VEGFA isoform expression between the models or 4) dysregulation of alternative pathways that modulate vascular permeability independent of VEGFA. Determining the mechanistic basis underlying the sensitivity of the pulmonary microvasculature to HIF1a-mediated permeability requires further investigation.

The increased lymphangiogenesis in the HIF1a TPM mice was a surprising observation. Previous data had shown that conditional induction of the VEGFA₁₆₄ isoform in the pulmonary epithelium during the pseudoglandular stage of development was sufficient to induce a 3-fold increase in lymphangiogenesis (Mallory et al., 2006). In our model, HIF1a TPM induced expression of VEGFA₁₆₄, but also increased expression of the VEGFA₁₈₈ isoform and another VEGF family member, VEGFC, a bona fide lymphangiogenic growth factor (Karkkainen et al., 2004; Lohela et al., 2008). Furthermore, we observed activation of Notch1 signaling in the lung mesenchyme and induction of several Notch pathway components that are known modulators of blood and lymphatic endothelial cell specification (Figure 10F&G, see (Rocha and Adams, 2009) for review). Ex vivo experiments using epithelium-free fetal lung mesenchyme demonstrated that recombinant VEGFA-165 and VEGFC were independently sufficient to induce the Notch effectors Hey1 and Heyl, and the lymphatic markers Foxc2 and Sox18, but not Prox1 (Figure 11B). In addition, induction of these lymphatic markers by VEGFA or VEGFC was prevented by co-treatment of the mesenchyme with the Notch inhibitor DAPT. Collectively, these data suggest a model whereby HIF1a TPM-mediated lymphangiogenesis is mediated by increased VEGFA and/or VEGFC secretion by the epithelium, engaging the KDR and/or FLT4 receptors on endothelial precursor cells in the fetal lung mesenchyme. KDR and/or FLT4 engagement then activates Notch signaling in an adjacent endothelial precursor cell to govern it toward a lymphatic endothelial cell fate (Figure 11C).

Fetal lung development occurs in a relatively hypoxic environment (Gao and Raj, 2010), suggesting that low oxygen tension promotes organogenesis. Consistent with this concept, ex vivo studies performed with rodent lung explants demonstrate that both airway branching and vascular morphogenesis are increased when cultured in a hypoxic environment (Gebb and Jones, 2003; Gebb et al., 2005; van Tuyl et al., 2005). Using an oligodeoxynucleotide knockdown approach, Van Tuyl et. al. demonstrated that the hypoxia-mediated increase in branching and vascular morphogenesis observed in their model were mediated by HIF1a (van Tuyl et al., 2005). These data, in conjunction with the epithelial localization of HIF1a protein in the developing mouse and human lung (Figure 1E and (Groenman et al., 2007)), led us to hypothesize that the hypoxia-induced phenotype seen ex vivo was driven by increased expression of HIF1a in the epithelium. However, in our transgenic model, epithelial HIF1a TPM expression resulted in the opposite phenotype, characterized by decreased airway branching and vascular leak. Several possibilities exist for which to explain these phenotypic differences. First, the level of epithelial HIF1a expression in the HIF1a TPM mice may have been higher than that observed in hypoxia-cultured lungs, thereby activating alternative molecular pathways or amplifying overlapping pathways that result in distinct phenotypes. Second, although the expression pattern of HIF1a in hypoxia-cultured lungs was not reported at the cellular level (van Tuyl et al., 2005), one would predict that all cells within the lung would stabilize HIF1a protein since HIF1a mRNA is ubiquitously expressed. Therefore, an alternative explanation is that hypoxia-induced HIF1a expression in the mesenchymal cells of the explant model promotes branching and hypervascularity. Third, it is also possible that HIF1a-independent hypoxia effects, including stabilization of EPAS1, activation of the unfolded protein response and downstream target genes (Koritzinsky et al., 2006) and/or increased translation of selective mRNAs including VEGFA (Young et al., 2008), contributed to the hypoxia-mediated increase in airway branching and vascular morphogenesis ex vivo that was not observed in our model. Lastly, although the HIF1a TPM protein is more stable and transcriptionally active that wild type HIF1a (Figure 3), its structure and/or function may differ from that of endogenous HIF1a, contributing to the observed phenotype.

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