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A5739 - Krüppel-Like Factor 4 (Klf4) Is a Novel Regulator of Neonatal Lung Fibroblast Homeostasis and Reduced in Hyperoxia-Induced Lung Injury
Author Block: D. Hirani1, K. Dinger1, J. Mohr1, C. Vohlen1, C. Klaudt1, J. Dötsch2, M. Alejandre Alcazar1; 1Department of Pediatrics, Translational Experimental Pediatrics - Experimental Pulmonology, University Hospital Cologne, Cologne, Germany, 2Department of Pediatrics, University Hospital Cologne, Cologne, Germany.
Rationale: Bronchopulmonary dysplasia (BPD) is a neonatal chronic lung disease, which typically evolves in extremely preterm infants whose immature lungs are exposed to prolonged increased oxygen (O2). Failed alveolar and microvascular formation coupled with perturbed matrix remodeling are characteristics of BPD. Alveolar myofibroblasts are thought to be crucial in secondary septation during alveolarization and fibrotic processes. The transcription factor Krüppel-like factor 4 (Klf4) is important in cell homeostasis and has been shown to regulate fibroblast function. Impaired secondary septation and fibrotic changes in murine models of BPD let us hypothesize that reduced alveolar formation after hyperoxia is mechanistically linked to dysregulated Klf4 in lung fibroblasts.
Methods: (1) Newborn C57Bl/6N mice were exposed to 85% O2 (hyperoxia) or 21% O2 (normoxia) from postnatal day 1 (P1) to P28. Lungs were excised and either snap-frozen or pressure-fixed with paraformaldehyde and paraffin embedded. (2) Primary neonatal murine lung fibroblasts (PnF) were isolated and exposed to hyperoxia or normoxia for up to 48 hours. Klf4-siRNA or vehicle was used to assess effects on proliferation (MTT assay), migration (Boyden chamber assay) and gene expression of PnF.
Results: (1) Klf4 mRNA and protein are highly expressed in lungs during alveolarization. Exposure of newborn mice to hyperoxia markedly reduced Klf4 mRNA and protein at P7 and P28 in lungs when compared to control. Gene and protein expression of αSMA as an indicator of myofibroblasts was increased after hyperoxia. Dual immunofluorescent staining showed a co-localization of Klf4 in myofibroblasts (αSMA-positive cells). Moreover, septal thickness and expression of matrix components, such as CTGF, elastin, collagen Iα1, and collagen IIIα1 were significantly increased, whereas alveolar formation was reduced in lungs after hyperoxia. (2) Exposure of cultured PnF to hyperoxia decreased Klf4 protein. Knockdown of Klf4 in PnF using siRNA did not affect proliferation or gene expression of apoptosis-regulators, such as bax or bcl-2. The Boyden chamber assay, however, showed that loss of Klf4 significantly reduced migration of PnF. Moreover, we found that knockdown of Klf4 using siRNA increased gene expression of CTGF, collagen Iα1, collagen IIIα3, and PDGFRα, indicating a transdifferentiation of neonatal lung fibroblast to myofibroblasts.
Conclusion: Our study demonstrates that loss of Klf4 is intimately linked to myofibroblast activation, fibrotic processes and reduced alveolarization. Moreover, we identify Klf4 as a novel regulator of neonatal lung fibroblast migration and differentiation to myofibroblasts. Induction of Klf4 in lungs exposed to hyperoxia could partially protect from hyperoxia-induced lung injury.