Lung ischemia-reperfusion injury (IRI) is an acute lung injury that contributes to morbidity and mortality following lung transplantation (LTX). IRI i mediated in part by the synthesis of oxidative and nitrosative species that in turn induce DNA single strand breakage and triggering of the nuclear DNA repair enzyme, poly(ADP-ribose) polymerase-1 (PARP-1). Hyperactivation of PARP-1 results in the consumption of its substrate NAD, which in turn exhausts intracellular energetics, leading to ATP depletion and tissue necrosis. PARP-1 activation also induces a widespread expression of pro-inflammatory genes that contribute to vascular permeability, lung edema, neutrophil infiltration, pulmonary shunt, and respiratory failure. In models of IRI injury, genetic deletion or pharmacologic inhibition of PARP-1 potently reduces lung damage, but the extent of protection is subtotal. We propose a more thorough abrogation of IRI-induced tissue damage via the deployment of a bifunctional PARP-1 inhibitor, R-503, formed from the covalent linkage of 2 moieties, each with demonstrated tissue protection: 1) a PARP-1 inhibitor moiety, and 2) a thiol-rich dihydrolipoyl (""""""""DHL"""""""") domain that acts as a broad-spectrum redox degradation catalyst.
Aim #1 : Establish the superiority of the bifunctional PARP-1 inhibitor, R-503, relative to its component domains, in a rat model of warm-ischemic lung IRI. Rat lungs are rendered ischemic in situ for 60 min and reperfused for 4 h. Prior to ischemia, rats are treated with IV R-503, DHL, the monofunctional PARP-1 inhibitor INO-1001, or a combination of DHL and INO-1001. Tissue damage is assessed by histology, levels of PMN infiltration, lipid peroxidation, NF-?B, protein nitrosation, PARP-1 activation, apoptosis, and oxygenation (PaO2). Inflammation is assessed by examining BAL markers of inflammation (protein, PMNs, TNF-?, and MIP-1?).
Aim #2 : Establish the efficacy of R-503 in a syngeneic rat model of orthotopic LTX. Using a translational model of LTX, donor rats are treated IV with R-503 or vehicle 10 min before lung removal. After flushing with cold Perfadex"""""""", spiked with R-503 (30 ?M) or vehicle, donor lungs are stored cold for 12 h before left LTX. Right donor lungs are processed for immunohistochemical analysis of PARP-1 activation. Immediately following LTX with left donor lungs, recipients will receive R-503 or vehicle. The dose of R-503 will be guided by the optimal level elucidated in Aim #1. Recipient rats will be evaluated at 3 different reperfusion times (1, 3, 6 h) for wet/dry weight ratio, graft oxygenation, pulmonary vascular resistance, dynamic compliance, and lung F2?-isoprostane, histology, and immunohistochemistry for 3-nitrotyrosine and poly(ADP-ribose). Specific analyses will be carried out at 3 time points: at 1 h post reperfusion for I?B?, phosphorylation of MAP kinases, nuclear p50;at 3 h post reperfusion for RT-PCR to quantify lung mRNA concentrations of TNF-?, MIP-1?, and Bcl-2;and at 6 h post reperfusion for determination of BALF cellularity, protein concentration, TNF-?, MIP-1?, IL6, and IL1-?.
Ischemia-reperfusion injury is a major medical complication following lung transplantation and contributes to the high mortality in this population. At present there are no approved prophylactic measures. We are developing a novel drug that targets the basic mechanisms of this condition and will test this agent in two clinically-relevant animal models.
