Obesity is a pre-disposing condition for chronic obstructive pulmonary disease, asthma, and pulmonary arterial hypertension. Accumulating evidence suggests that metabolic influences during development can determine chronic lung diseases (CLD). We demonstrate that maternal obesity causes early metabolic disorder in the offspring. Here, interleukin-6 induced bronchial and microvascular smooth muscle cell (SMC) hyperproliferation and increased airway and pulmonary vascular resistance. The key anti-proliferative transcription factor FoxO1 was inactivated via nuclear exclusion. These findings were confirmed using primary SMC treated with interleukin-6 and pharmacological FoxO1 inhibition as well as genetic FoxO1 ablation and constitutive activation. In vivo, we reproduced the structural and functional alterations in offspring of obese dams via the SMC-specific ablation of FoxO1. The reconstitution of FoxO1 using IL-6-deficient mice and pharmacological treatment did not protect against metabolic disorder but prevented SMC hyperproliferation. In human observational studies, childhood obesity was associated with reduced forced expiratory volume in 1 s/forced vital capacity ratio Z-score (used as proxy for lung function) and asthma. We conclude that the interleukin-6-FoxO1 pathway in SMC is a molecular mechanism by which perinatal obesity programs the bronchial and vascular structure and function, thereby driving CLD development. Thus, FoxO1 reconstitution provides a potential therapeutic option for preventing this metabolic programming of CLD.

Prematurely born infants often require supplemental oxygen that impairs lung growth and results in arrest of alveolarization and bronchopulmonary dysplasia (BPD). The growth hormone (GH)- and insulin-like growth factor (IGF)1 systems regulate cell homeostasis and organ development. Since IGF1 is decreased in preterm infants, we investigated the GH- and IGF1 signaling (1) in newborn mice with acute and prolonged exposure to hyperoxia as well as after recovery in room air; and (2) in cultured murine lung epithelial cells (MLE-12) and primary neonatal lung fibroblasts (pLFs) after treatment with GH, IGF1, and IGF1-receptor (IGF1-R) inhibitor or silencing of GH-receptor (Ghr) and Igf1r using the siRNA technique. We found that (1) early postnatal hyperoxia caused an arrest of alveolarization that persisted until adulthood. Both short-term and prolonged hyperoxia reduced GH-receptor expression and STAT5 signaling, whereas Igf1 mRNA and pAKT signaling were increased. These findings were related to a loss of epithelial cell markers (SFTPC, AQP5) and proliferation of myofibroblasts (αSMA⁺ cells). After recovery, GH-R-expression and STAT5 signaling were activated, Igf1r mRNA reduced, and SFTPC protein significantly increased. Cell culture studies showed that IGF1 induced expression of mesenchymal (e.g., Col1a1, Col4a4) and alveolar epithelial cell type I (Hopx, Igfbp2) markers, whereas inhibition of IGF1 increased SFTPC and reduced AQP5 in MLE-12. GH increased Il6 mRNA and reduced proliferation of pLFs, whereas IGF1 exhibited the opposite effect. In summary, our data demonstrate an opposite regulation of GH- and IGF1- signaling during short-term/prolonged hyperoxia-induced lung injury and recovery, affecting alveolar epithelial cell differentiation, inflammatory activation of fibroblasts, and a possible uncoupling of the GH-IGF1 axis in lungs after hyperoxia.

Despite improved neonatal intensive care, the risk of premature-born infants developing bronchopulmonary dysplasia (BPD) and encephalopathy of prematurity (EoP) remains high. With hyperoxia being a major underlying factor, both preterm-birth-related complications are suggested to be closely interrelated. However, experimental models are lacking for the assessment of the potentially close interplay between both organs. To establish a model, suitable for the assessment of both affected organs, Wistar rats were exposed to 80% oxygen from postnatal day 2 (P2) for seven days. Brain and lung tissues were analysed via histomorphometry, immunohistochemistry, real-time PCR, and western blot at term P11. In the brain, hyperoxia induced significant hypomyelination accompanied by a reduction in oligodendrocytes and CD68 expression on microglia cells. These changes correlate with arrested alveolarisation and an increased number of macrophages in the lung. Interestingly, in contrast to the reduced formation of pulmonary microvessels, an increased vascular density was detected in the brain. Seven days of hyperoxia induces typical characteristics of BPD and EoP in neonatal rats, thereby linking impaired alveolarisation with disturbed myelination in the brain and providing an experimental model for understanding pathophysiological mechanisms and identifying organ-spanning novel therapeutic interventions targeting both diseases.

Preterm infants often require oxygen supplementation, resulting in high risk for bronchopulmonary dysplasia (BPD) and neurodevelopmental deficits. Despite a growing number of studies, there is still little knowledge about brain injury in BPD models. Therefore, we exposed neonatal C57BL/6 mice to 85% oxygen from birth to postnatal day (P) 14. At P28, two weeks after recovery under normoxic conditions, right hemisphere was used for the analysis of mRNA and the left hemisphere for protein expression of neuronal cells, neuroinflammatory and vascularisation markers, analysed by real-time PCR and Western blot, respectively. Hyperoxia led to an altered expression of markers associated with neuronal and oligodendrocyte maturation and neuroinflammation such as Dcx, Nestin, Il-1β, Il-6, NG2, and YM1/2. These changes were accompanied by an increased expression of genes involved in angiogenesis and vascular remodelling, e.g., Vegf-a, Nrp-1, and Icam-1. Together, 14 days of hyperoxia triggered a phenotypic response, resembling signs of encephalopathy of prematurity (EoP).

Rationale: Supplementary oxygen leads to bronchopulmonary dysplasia (BPD) in preterm infants, a disease characterized by lung growth arrest and matrix remodeling. We demonstrated that hyperoxia reduces Krüppel-like factor 4 (Klf4), a key transcription factor in cell biology. Since myofibroblasts are crucial in lung injury and remodeling, we investigated the functional role of Klf4 in the pathogenesis of BPD and in the homeostasis of lung fibroblasts. Methods: (1) Newborn C57BL/6N mice were exposed to 85% O2 (HYX) or 21% O2 (NOX) until postnatal day 28 (P28). (2) Primary neonatal murine lung fibroblasts (pnF) were transfected with a Klf4 overexpressing plasmid or siRNA technique for overexpression (OE) and knockdown of Klf4 (del), respectively, and exposed to HYX or NOX. (3) BioID (Proximity-dependent Biotin Identification) and CHIP-Seq were performed. Results: (1) Reduced Klf4 expression in lungs after HYX was associated with an activation of TGFβ signaling and an increase of myofibroblasts, promoting thereby matrix remodeling. (2) TGFβ inhibited Klf4 expression in cultured pnF. Klf4del decreased migration, induced proliferation and increased mRNA expression of matrix markers (Ctgf, Col1a1 & Col4a1) and Pdgfra (myofibroblasts). In contrast, Klf4OE increased adhesion, but reduced proliferation, apoptosis, migration and expression of fibrotic genes in pnF. (3) Finally, BioID and CHIP-Seq identified a novel Klf4 interactome that regulates FoxO1 transcriptionally and its activation via acetylation. Conclusion: Our data unveil a novel Klf4-FoxO1 axis in lung fibroblasts that could promote matrix remodeling and lung growth arrest in lungs with BPD.