Elevated concentrations of plasminogen activator inhibitor-1 (PAI-1) are associated with pleural injury, but its effects on pleural organization remain unclear. A method of adenovirus-mediated delivery of genes of interest (expressed under a cytomegalovirus promoter) to rabbit pleura was developed and used with lacZ and human (h) PAI-1. Histology, b-galactosidase staining, Western blotting, enzymatic and immunohistochemical analyses of pleural fluids (PFs), lavages, and pleural mesothelial cells were used to evaluate the efficiency and effects of transduction. Transduction was selective and limited to the pleural mesothelial monolayer. The intrapleural expression of both genes was transient, with their peak expression at 4 to 5 days. On Day 5, hPAI-1 (40-80 and 200-400 nM of active and total hPAI-1 in lavages, respectively) caused no overt pleural injury, effusions, or fibrosis. The adenovirus-mediated delivery of hPAI-1 with subsequent tetracycline-induced pleural injury resulted in a significant exacerbation of the pleural fibrosis observed on Day 5 (P ¼ 0.029 and P ¼ 0.021 versus vehicle and adenoviral control samples, respectively). Intrapleural fibrinolytic therapy (IPFT) with plasminogen activators was effective in both animals overexpressing hPAI-1 and control animals with tetracycline injury alone. An increase in intrapleural active PAI-1 (from 10-15 nM in control animals to 20-40 nM in hPAI-1-overexpressing animals) resulted in the increased formation of PAI-1/plasminogen activator complexes in vivo. The decrease in intrapleural plasminogen-activating activity observed at 10 to 40 minutes after IPFT correlates linearly with the initial concentration of active PAI-1. Therefore, active PAI-1 in PFs affects the outcome of IPFT, and may be both a biomarker of pleural injury and a molecular target for its treatment.Keywords: pleural injury; plasminogen activator inhibitor-1; intrapleural fibrinolytic therapyThe incidence of complicated pleural infection and empyema, a serious infection of the pleural space often associated with pneumonia, is increasing in the United States (1) and other countries, in both adult and pediatric populations (2-5). The exact cause of this increase is unknown, although the increased prevalence of antibiotic-resistant bacteria, changes in empyema management, and changes in causative bacterial agents have been implicated (1,3,6). Pleural infections, empyema, or complicated parapneumonic effusions develop in approximately 80,000 patients in the United States and the United Kingdom annually (7). In the United Kingdom, a 20% mortality rate was reported for patients with empyema, and 20% of patients require surgical intervention after developing a pleural infection (7). When pleural effusions occur in association with high-grade inflammation, they can organize with the development of loculation, where an effusion becomes trapped behind partly fused visceral and parietal pleura, with pleural thickening (8-10). Persistent pleural loculation and fibrosis increase morbidity and mortality an...