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A7250 - Impact of a Mitochondria-Targeted DNA Repair Glycosylase on Pulmonary Vascular Endothelial Barrier Function in a Rodent Model of Lung Donation After Circulatory Determination of Death
Author Block: Y. Tan1, V. Pastukh2, O. Gorodnya2, J. Simmons1, M. Gillespie2; 1Surgery, University of South Alabama, Mobile, AL, United States, 2Pharmacology, University of South Alabama, Mobile, AL, United States.
RATIONALE: The demand for donor lungs greatly exceeds the supply. An underutilized resource that can address the deficiency is lungs derived from donors after circulatory determination of death (DCDD). However, less than 2% of current lung transplants in the USA are from DCDD lungs, in part because of post-mortem metabolic degradation that renders the organs more susceptible to ischemia-reperfusion (IR) lung injury. Mitochondrial (mt) DNA is highly prone to oxidative damage from the reactive oxygen species generated during IR. Because mtDNA damage has been linked to endothelial barrier disruption, we developed a fusion protein construct targeting the DNA repair glycosylase, Ogg1, to mitochondria (mt-Ogg1) and used it to determine if protection from mtDNA damage attenuates endothelial barrier dysfunction after IR injury in a rat model of DCDD.
METHODS: Procurement, cold storage, and ex vivo lung perfusion mimicked protocols used in humans. Briefly, lungs from pentobarbital-euthanized rats were excised immediately or at 60 minutes after death. Lungs were then flushed and subjected to cold-ischemia for 2 hours. Subsequently, they were mounted and gradually re-warmed in a constant flow perfusion apparatus while physiological parameters were monitored. One group served as control, and was perfused with medium only. Two other groups were perfused with medium containing either mt-Ogg1 or a repair-deficient mutant mt-Ogg1. The vascular filtration coefficient and wet to dry lung weight ratio were employed as indices of pulmonary endothelial barrier function. The extent of oxidative damage to lung mtDNA was evaluated using Southern blot analysis, while mtDNA Damage Associated Molecular Patterns (DAMPs) released into perfusate were quantified using RT-PCR of selected mtDNA sequences.
RESULTS: Mitochondria-targeted Ogg1 attenuated the rise in both vascular coefficient and wet to dry lung weight ratio incurred after the one-hour post-mortem period. The increase in mtDNA damage sustained from the post-mortem period was also reduced by mitochondria-targeted OGG1. Addition of OGG1 also reduced the quantity of pro-inflammatory mtDNA fragments in the perfusate. The repair-deficient mt-Ogg1 mutant failed to protect lungs from the adverse effects of DCDD procurement.
CONCLUSION: These findings support the concept that endothelial barrier dysfunction is associated with prolonged post-mortem period and increased mtDNA damage. The finding that mitochondria-targeted OGG1 improved endothelial barrier function in DCDD procured lungs supports that idea that mtDNA damage contributes to physiologic abnormalities after DCDD and suggests that strategies to enhance mtDNA repair could be a useful means of alleviating the shortage of transplantable lungs.
Funded by NIH.