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A2214 - Contribution of the Mitochondrial Intrinsic Pathway to the Apoptosis Resistance Exhibited by Fibroblasts from Idiopathic Pulmonary Fibrosis
Author Block: E. R. Luis-García1, C. Becerril1, J. Pedraza-Chaverri2, M. Orozco-Ibarra3, E. Aparicio-Trejo2, C. Mendoza-Milla1, A. Salgado-Aguayo1, M. Maldonado4, A. Pardo4, M. Selman1; 1Instituto Nacional de Enfermedades Respiratorias, Mexico, Mexico, 2Facultad de Quimica, Universidad Nacional Autonoma de Mexico, Mexico, Mexico, 3Instituto Nacional de Neurologia y Neurocirugia, Mexico, Mexico, 4Facultad de Ciencias, Universidad Nacional Autonoma de Mexico, Mexico, Mexico.
RATIONALE: Idiopathic Pulmonary Fibrosis (IPF) is an aggressive, progressive and frequently lethal disease of unknown etiology, characterized by the expansion of the fibroblast population and destruction of lung parenchyma. Previous reports have shown that IPF fibroblasts are resistant to Fas-mediated apoptosis (extrinsic pathway); however, it is not known whether the apoptotic intrinsic pathway is also involved in this resistance. The inhibition of the mitochondrial permeability transition pore (mPTP) has been proposed as a mechanism involved in the resistance to apoptosis. Then, our aim is to analyze the role of the mitochondria on the apoptosis resistance of fibroblasts from IPF and whether the mPTP contributes to it. METHODS: Primary human lung control and IPF fibroblasts matched by age were used. Cells were studied under basal conditions and after mitomycin C-induced apoptosis. Early apoptosis was analyzed by flow cytometry with Annexin V/IP, cytochrome c release in the cytosolic fraction was evaluated by HPLC and Western blot, while mPTP opening was assessed by the quenching of calcein-AM fluorescence by cobalt chloride through flow cytometry and confocal microscopy. Moreover, mitochondrial respiration was measured as the oxygen consumption in permeabilized and non-permeabilized cells using a type Clark electrode or an Oroboros Instrument, respectively. RESULTS: We found that IPF fibroblasts are resistant to mitomycin C-induced apoptosis and showed less cytochrome c release than control fibroblasts. The co-localization between calcein-AM and cobalt chloride was found increased in IPF fibroblasts indicating a closer mPTP. Likewise, the mPTP of IPF fibroblasts was found less sensitive to ionomycin-induced opening. Regarding oxygen consumption, IPF fibroblasts showed decreased: a) maximum respiratory velocity, b) respiratory control, and c) basal respiration compared to control fibroblasts. Furthermore, tests with permeabilized cells suggested decrease complex I function. CONCLUSIONS: Our results suggest that the mPTP contributes to the apoptosis resistance exhibited by IPF fibroblasts. Most probably, this event is related to the failure in mitochondrial respiration. We are studying the activity and expression of complex I as well as mitochondrial membrane potential and ATP production.