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A6355 - The Heme-Oxygenase 1 Induction Prevents Senescence in COPD Lung Fibroblasts by Protecting Against Mitochondrial Dysfunction
Author Block: B. Even1, S. Fayad-Kobeissi2, J. Gagliolo1, R. Motterlini2, J. Boczkowski1, R. Foresti2, M. Dagouassat1; 1Equipe 04, IMRB INSERM U 955, Créteil, France, 2Equipe 12, IMRB INSERM U 955, Créteil, France.
Introduction: Chronic obstructive pulmonary disease (COPD) is associated with lung fibroblast senescence, a process characterized by an irreversible proliferation arrest associated with secretion of inflammatory mediators. Reactive oxygene species (ROS) production, known to induce senescence, is increased in COPD fibroblasts. Other mechanisms, including mitochondrial dysfunction and defective mitophagy (protective mechanism which minimizes the proportion of dysfunctional mitochondria), may also contribute to ROS-mediated senescence in COPD. Among the battery of cellular responses against oxidative stress damage, heme-oxygenase (HO)-1, an antioxidant enzyme catalyzing heme degradation, plays a critical role in defending the lung against oxidative stress and inflammation but is also known to improve mitochondrial biogenesis. Therefore, we investigated if pharmacological induction of HO-1 by chronic hemin treatment prevents senescence and protects cells against defective mitophagy. Methods: Fibroblasts from smoker controls (S-C) (n=13) and smokers with moderate COPD (n=16) were isolated from lung biopsies. Groups were similar in age and smoking history. Fibroblasts were long-term cultured in the presence or absence of hemin (HO-1 inducer), and/or ZnPP or QC-15 (HO-1 inhibitors). We analyzed at non-senescent and senescent stages (passages 3 and 7 respectively): cells proliferative capacity, senescence markers (p21 and p16 mRNA levels, β-galactosidase staining), inflammatory mediators (IL6, IL8 mRNA levels), and parameters related to mitochondria biology: genes involved in mitochondria biogenesis (NRF1, PGC-1α), total and mitochondrial ROS production (using DCFH-DA and mitosox probes respectively), and mitochondrial activity (respiration, glycolysis and ATP levels, by using Seahorse technology). Furthermore, we analyzed mitophagy by analyzing the number of mitochondria (nonyl-acridine orange probe), and the expression level of PINK1 and Parkin by western blot. Results: Lung fibroblasts from S-C and COPD patients displayed a senescent phenotype at passage 7, characterized by a reduced proliferative capacity and an increase in senescence and inflammatory markers. These parameters were significantly higher in senescent COPD versus S-C fibroblasts, and were accompanied by a decreased mitochondrial activity, elevated mitochondrial ROS production and an impaired mitophagy in the former as compared to the latter group. Exposure to hemin increased HO-1 enzymatic activity and diminished ROS levels, senescence, the inflammatory profile by preventing mitochondria dysfunction and restoring mitophagy in COPD cells. These effects were abolished by a co-treatment with ZnPP or QC-15. Conclusion: HO-1 prevents senescence in COPD fibroblasts by protecting, at least in part, against mitochondria dysfunction and restoring mitophagy. Moreover, these results set up the basis for the development of new therapeutic strategies in COPD aimed to restore defective mitophagy.