In the clinical setting of sepsis related ARDS, the presence of liver failure markedly increases mortality and studies of endotoxin induced lung injury in animals implicate effects on the liver as important for the full expression of the injury. The sequence of biochemical events that dictates the physiologic response to endotoxin may be activation of critical transcription factors, especially nuclear factor kappa B (NFkappaB) and CCAAT enhancer binding protein beta (C/EBPbeta, a.k.a. NF-IL6) in both the lungs and the liver, leading to generation of pro-inflammatory cytokines. Our preliminary data as well as data of others implicate eicosanoids as modulators of these endotoxin effects and induction of the cyclooxygenase (COX-the proximal enzyme in prostanoid synthesis) also involves NFkappaB and C/EBPbeta activation. To elucidate relationships among the physiologic, biochemical, molecular and pathophysiologic events related to liver-lung interactions in the endotoxin response we propose a series of studies in an in situ perfused swine model in which the lungs can be perfused with or without the liver in the perfusion circuit. We will: 1) determine effects of endotoxemia on pulmonary vascular resistance, lung vascular permeability, lung water content, tissue, perfusate and bronchoalveolar lavage fluid (BALF) concentrations of eicosanoids and cytokines, BALF total and differential cell counts, expression of TNFalpha, COX-1, COX- 2 and 5-lipoxygenase genes, activation of nuclear factor kappa B (NFkappaB) and C/EBPbeta in the lungs (and liver) with and without inclusion of the liver in the perfusion circuit; 2) manipulate concentrations of prostanoids in lung or liver by local controlled infusion of PGE2 into either organ, targeted delivery of a COX inhibitor to liver or lungs or transfecting the organ(s) with a construct expressing the COX gene and determine effects of these interactions on generation of eicosanoids and on endotoxin responses; 3) increase p20, the inhibitor isoform of C/EBPbeta, in the liver or the lungs by partial hepatic resection, delivery of a p20 membrane translocation signal fusion protein or transfection of the liver or lungs with a p20 gene and determine effects of these interventions on the endotoxin response; 4) inhibit activation of NFkappaB in liver or lungs by transfection of either organ with a gene encoding a transdominant inhibitor of NFkappaB activation or administration of a cell-permeable NFkappaB inhibitory fusion protein and determine effects on the endotoxin response. These studies will link pathophysiology to pathogenesis of the endotoxin response, providing a basis for identifying new potentially therapeutic interventions.
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