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A2669 - Pharmacologically Enhanced Mitochondrial DNA Repair Improves Performance of Ex Vivo Perfused Lungs from Brain-Dead Pigs
Author Block: D. Hall1, T. N. Machuca1, D. C. Holden2, J. M. Pickering1, Y. B. Tan3, J. D. Simmons4, V. M. Pastukh3, O. M. Gorodnya3, M. N. Gillespie3; 1Division of Thoracic and Cardiovascular Surgery, University of Florida, Gainesville, FL, United States, 2Laboratory of Inflammation Biology and Surgical Science, University of Florida, Gainesville, FL, United States, 3Department of Pharmacology and Center for Lung Biology, University of South Alabama College of Medicine, Mobile, AL, United States, 4Department of Surgery, University of South Alabama College of Medicine, Mobile, AL, United States.
Rationale: The availability of lungs for transplant and the risk of primary graft dysfunction can be improved by ex vivo lung perfusion (EVLP) prior to implantation. Nevertheless, a pharmacologic means to enhance the quality of marginally suitable lungs has the potential to further increase the number of transplants and reduce the incidence of primary graft dysfunction. Because mitochondrial (mt) DNA damage triggers cellular dysfunction in non-pulmonary organs after ischemia-reperfusion injuries, we used a porcine model of brain death followed by cold ischemia and EVLP to determine if enhancement of mtDNA repair using a fusion protein targeting the DNA glycosylase Ogg1 to mitochondria (mt-Ogg1) increased conversion of lungs to transplantable status.
Methods: Brain death, confirmed by clinical examination and cessation of cerebral blood, was induced in anesthetized and mechanically-ventilated pigs by inflation of a foley catheter introduced in the epidural space. After 6h, lungs were excised, flushed with Perfadex and stored at 4oC for 16h. Subsequently, they were mounted in a perfusion apparatus and subjected to 4h of EVLP with Steen solution during which a battery of physiologic parameters and perfusate concentrations of mtDNA Damage Associated Molecular Patterns (DAMPs) were assessed hourly. At termination of perfusion, lung tissue mtDNA damage was determined by quantitative Southern blot analyses. Two groups of lungs were examined; one (n=8) served as control while the other was treated with 20 ug/ml mt-Ogg1 administered via the perfusate (n=8).
Results: Following clinically determined parameters, the proportion of lungs converted to transplant suitability after EVLP doubled with mt-Ogg1 treatment relative to controls. Mitochondrial DNA DAMPs also increased in perfusion medium as a function of EVLP duration, with coincident accumulation of mtDNA DAMPs harboring oxidized base lesions. While the abundance of perfusuate mtDNA DAMPs tended to be decreased by mt-Ogg1, oxidized mtDNA fragments were significantly reduced. The density of oxidative base damage, apurinic/apyrimidinic sites and strand breaks in lung tissue mtDNA increased after EVLP; these lesions were reduced significantly in mt-Ogg1-treated lungs.
Conclusions: These findings indicate that mtDNA damage accumulates in lung tissue derived from brain dead pigs during EVLP. Because pharmacologically enhanced mtDNA repair prevented both mtDNA damage and physiologic deterioration, these observations also suggest that persistent mtDNA damage may be responsible for the failure of EVLP to restore physiologic viability and limit conversion to transplant suitability.