Oxygen therapy in neonatal chronic lung disease – acute and sustained effects and their indicators

Gonzalez, Erika (2022): Oxygen therapy in neonatal chronic lung disease – acute and sustained effects and their indicators. Dissertation, LMU München: Medizinische Fakultät

Abstract

Premature neonates often require life-saving oxygen therapy, which increases their risk of developing bronchopulmonary dysplasia (BPD). Although survival improves with oxygen supplementation, morbidity increases with higher oxygen concentration and longer exposures. The induction of lung injury by short-term postnatal hyperoxia has been suggested by clinical observations and was in part supported by experimental studies. However, underlying mechanisms of lasting effects provoked by clinically relevant oxygen levels remain poorly understood. Despite this, most of the experimental models of BPD employ very high oxygen concentrations (FiO2 > 0.8) for several days or weeks. Thus, our knowledge of the effects of oxygen therapy in the developing lung derives from models of severity that most likely do not reflect the clinical setting. Even though the effects of treatment with severe hyperoxia concentrations have been extensively characterized, there is still a poor understanding of the maladaptive responses that lead to BPD. More importantly, a real gap of knowledge remains when it comes to clinically relevant hyperoxia concentrations i.e., FiO2 ≤ 0.4. Thus, it is critical to identify and understand the early mechanisms involved in the injury response to hyperoxia treatment in order to harness this knowledge towards therapeutics that restore normal lung function. Therefore, my studies addressed significant and functionally relevant changes in the three major lung cell types, i.e., ATII, EC, and MFB, and confirmed their lasting effects. This study presents previously unidentified immediate and sustained effects of the early postnatal exposure to clinically relevant hyperoxia concentrations (FiO2 = 0.4). One of the main findings of this study was that treatment with O2 (FiO2 = 0.4) arrests the cell cycle potentially through the downregulation of the pre-replication complex, which is critical for cell cycle progression. The predominant downregulation of genes following hyperoxia exposure revealed a significant overlap between the MFB and ATII cell transcriptome pattern, partially shared by the lung EC. The significant overlap in the transcriptome profile between MFBs and ATII cells potentially indicates similar effects (and coping strategies) in response to oxidative stress. In Mcm2 knockdown experiments in vitro, I established a causal link between cell-cycle arrest in G1 and changes in developmentally relevant gene expression 8 and cell function in MFB and AT2 cells. Furthermore, Mcm2 knockdown cells presented with aberrations in DNA damage response and repair genes. In vivo, we were able to recapitulate our main in vitro findings. A model employing FiO2 = 0.4 for 8 hours revealed indications of cell cycle arrest at the proteomic level in the absence of classical apoptosis. Longer O2 treatment revealed a similar signature of cell cycle arrest and the regulation of other critical proteins involved in the DDR such as Parp1. Moreover, 24 hours of O2 did not result in classical apoptosis. Interestingly, 8 hours of O2 treatment led to the upregulation of VEGFA but no other changes in developmentally relevant proteins were observed. Remarkably, studies in 18-month-old mice that received 8 hours of O2 as newborn pups revealed Pdgf-Rα and VE-cadherin to be significantly downregulated while eNOS, another vascular marker, was significantly increased. These results paint a picture of aberrant signaling that although not evident immediately after treatment, appears to be long-lasting. To study deeper the potential consequences of short-term O2 treatment, we treated mice with O2 for 24 hours and allow them to recover after treatment. Three and seven weeks after treatment, the lungs were harvested. We observed that at the transcriptional level, the downregulation of genes like Pdgf-Rα can already be seen seven weeks after treatment. This model also revealed alterations in Wnt5a mRNA expression three and seven weeks after treatment. Remarkably, we elucidated aberrations in DDR genes i.e., Chk1 and Cdkn1a, and in BER pathway genes three weeks after treatment. Several of these changes did not return to baseline by the seven-week post-treatment. Additionally, with a double-hit injury model, we delineated the changes in DNA damage response and the BER pathway that arise from O2-lung priming. For several of the target genes evaluated, previous treatment with O2 modulated the response to the second-hit injury, perhaps making the lung more vulnerable. Finally, we translated these results to the bedside by analyzing samples collected from pre-mature babies that received oxygen therapy. Remarkably, in plasma proteome analysis from blood collected during the first week of life, protein levels of Apex1, a key BER gene, were positively correlated with O2 days mirroring our observations in vivo. In summary, the results reported in this dissertation delineate the short, long-term effects and potential epigenetic regulation in the developing lung arising from treatment with clinically relevant hyperoxia levels. Furthermore, here it is extensively established that cell cycle arrest works as an initial mechanism by which the cells respond to DNA damage arising from oxidative stress. Moreover, my findings demonstrate that priming the developing lung with clinical hyperoxia levels for short periods of time alters the response to a second-hit injury as well as leaving long-term aberrations in DNA repair. These findings have profound clinical implications beyond neonatal chronic lung disease.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Neonatal lung disease, hyperoxia, cell cycle regulation, DNA repair
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 26. September 2022
• 1. Bericht­erstatter:in: Hilgendorff, Anne