Intratracheal administration of siRNA targeting FAS reduces ischemia-reperfusion-induced lung injury

Abstract

INTRODUCTION. Ischemia-reperfusion injury has been identified as the main cause of primary graft dysfunction after lung transplantation. Fas-mediated apoptosis is one of the most relevant mechanisms involved in the pathogenesis of ischemia-reperfusion injury. Exogenous administration of small interfering RNA (siRNA) is a new strategy of gene therapy able to specifically silence the expression of proteins through blocking the translation of mRNA. OBJECTIVES. The aim of this study was to demonstrate in an ex vivo mouse model of lung ventilation and perfusion that a specific siRNA targeting Fas is able to reduce ischemia- reperfusion injury through the modulation of apoptosis. METHODS. C57BL/6 male mice were randomized to intratracheally receive a specific sequence of siRNA targeting FAS (siRNA-FAS) or a scrambled siRNA 48 h before undergoing 15 h of cold ischemic time (4®C) and 2 h of ex vivo ventilation (peak inspiratory pressure = 7 cmH2O, PEEP = 2 cmH2O, RR = 100 b/min, FiO2 = 100%) and reperfusion (4% bovine serum albumin RPMI medium with 10% fresh blood at 1 ml/min flow rate) in a predisposed humidified chamber at 37®C. At the end of experiment, lung elastance, assessed through tidal volume, total protein and MIP- 2 concentration in the bronchoalveolar lavage (BAL) fluid, and the%of cell apoptosis on lung section (TUNEL) were measured. A separate set of lungs were analysed by Western Blot before undergoing cold ischemia to assess the expression of FAS protein. One-way analysis of variance with Student's t-test for comparison between groups was performed. RESULTS. The intratracheal administration of siRNA-FAS reduced the expression of FAS in the lung by 44% (siRNA-FAS 0.90 ± 0.11 vs scrambled siRNA 1.61 ± 0.18 AU). After the ischemia and reperfusion period, lung elastance, BAL total protein and MIP-2 concentration, and apoptosis were significantly reduced in the siRNA-FAS group as compared to control. (Table presented) CONCLUSIONS. The intratracheal administration of siRNA targeting FAS reduces lung apoptosis and improves alveolar membrane permeability during ischemia reperfusion injury.