Mechanical ventilation (MV) is necessary to support patients with acute lung injury (ALI);however, it is well recognized that mechanical stretch induced by MV may exacerbate lung injury and contribute to excess mortality, a process called ventilator-associated lung injury (VALI). Previous animal model studies have demonstrated that high tidal volume (HVT 20 ml/kg) ventilation concomitantly activates the enzyme xanthine oxidoreductase (XOR) and promotes apoptosis of alveolar endothelial and epithelial cells resulting in increases in alveolar capillary permeability. We have shown that pharmacologic inhibition of XOR with allopurinol abrogates HVT- induced increase in vascular leakage indicating that signaling through this pathway is necessary for permeability changes in vivo. In addition, we have also demonstrated using our in vitro model of VALI that cyclic stretch (CS) of endothelial cells (EC) at 18% for 20cycles/min but not 5% or static, is sufficient to promote XOR activity and apoptosis. Objective: Allopurinol pre-treatment of EC in vitro prevents CS induced apoptosis;however, the mechanisms by which XOR potentiates mechanical stress induced apoptosis are not well described and are the focus of this study. We will test the hypothesis that XOR's upregulation of endothelial cell apoptosis in the setting of mechanical stress is a result of the downregulation of molecular inhibitors of apoptosis, namely cIAP2. METHODS: Pulmonary microvascular endothelial cells (EC) will be exposed to pathologic cyclic stretch (18%) versus physiologic stretch (5%) or static conditions for increasing time intervals (2, 4, and 6 hours) with the absence or presence of XOR inhibitor (allopurinol, febuxostat, or RNAi) after which the cells were harvested. Endpoints included mRNA analysis, Western blotting, caspase activity, and analysis of nuclear morphology for characteristic apoptotic changes.
ALI and ARDS account for significant morbidity with 200,000 cases a year in the US and an overall mortality ranging from 35 to 60%. Mechanical stress related to alveolar over-distension and cyclical opening and closing from mechanical ventilation leads to pathogenic events that are poorly understood. Dissection of the pathophysiologic mechanisms underlying VALI will provide a critical impetus for the development of new treatments aimed at decreasing mortality.
|Kim, Bo S; Serebreni, Leonid; Fallica, Jonathan et al. (2015) Cyclin-dependent kinase five mediates activation of lung xanthine oxidoreductase in response to hypoxia. PLoS One 10:e0124189|
|Kim, Bo S; Serebreni, Leonid; Hamdan, Omar et al. (2013) Xanthine oxidoreductase is a critical mediator of cigarette smoke-induced endothelial cell DNA damage and apoptosis. Free Radic Biol Med 60:336-46|