Intrapleural activation, processing, efficacy, and duration of protection of single-chain urokinase in evolving tetracycline-induced pleural injury in rabbits

Abstract

Intrapleural fibrinolysins have been used to treat pleural loculations. However, the efficacy of clinically available agents has recently been questioned, providing a rationale for investigation of new interventions. Single-chain urokinase plasminogen activator resists inhibition by serpins, and repeated, daily intrapleural administration of this agent prevents intrapleural loculation more effectively than complexes of this proenzyme with its receptor (Idell S, Mazar A, Cines D, Kuo A, Parry G, Gawlak S, Juarez J, Koenig K, Azghani A, Hadden W, McLarty J, Miller E. Am J Respir Crit Care Med 166: 920-926, 2002). Understanding of the protective mechanism and intrapleural processing remains unclear. We speculated that single-chain urokinase could induce sustained local fibrinolysis and protection by selective administration either before, during, or following loculation after pleural injury induced by tetracycline in rabbits. Enzymography, immunoassays, histology, immunohistochemistry, morphology, and morphometry were used to test the efficacy, duration of protective effect, and processing of single-chain urokinase. Intrapleural single chain urokinase prevented loculation at 72 h after injury (P < 0.01) if given either before or during adhesion formation and was converted to two-chain high-molecular-weight urokinase, which remained active for at least 24 h within pleural fluids. The effect was dose dependent, and established loculations at 72 h after tetracycline-induced injury were reversed at 96 h by single-dose treatment. Single-chain urokinase bound and saturated intrapleural plasminogen activator inhibitory (PAI)-1-like activity and urokinase-related immunoreactivity of the mesothelium was comparable in treatment or vehicle-control groups. Adhesions recurred by 2 wk after treatment with recurrence of excess local PAI activity. Single-chain urokinase induces sustained local fibrinolysis and reversibly prevents pleural loculation for up to 48 h after intrapleural administration after tetracycline-induced injury.

Intrapleural Adenoviral Delivery of Human Plasminogen Activator Inhibitor–1 Exacerbates Tetracycline-Induced Pleural Injury in Rabbits

“…Levels of statistical significance were determined using the MannWhitney rank sum test, as described previously (31,37,38).…”

“…The severity of pleural injury in animals that successfully overexpressed hPAI-1 ( Figure 3C) was clearly apparent versus control animals ( Figures 3A and 3B). The extent of pleural injury was assessed by comparing gross loculation/adhesion injury scores, using a scoring system we previously described (38,41), with minor modifications detailed in the online supplement. The gross loculation scores of pleural injury in rabbits injected with AdPAI-1/AdlacZ and overexpressing hPAI-1 were increased compared with animals intrapleurally injected with PBS (P ¼ 0.029) or AdEV/AdlacZ (P ¼ 0.021; Figure 3D).…”

“…Fibrinolytic Therapy with scuPA or sctPA TCN-induced pleural fibrosis in rabbits was successfully treated with intrapleural fibrinolytic therapy, using effective doses of sctPA (0.145 mg/kg) or scuPA (0.5 mg/kg) (35,37,38). To test the effects of overexpressed hPAI-1 on IPFT, animals injected with AdPAI-1/AdlacZ and then with TCN 3 days after transduction (n ¼ 6) were subjected to IPFT (on Day 5 after transduction) with effective doses of scuPA and sctPA, or with PBS (vehicle control) (n ¼ 2 each).…”

Lipoprotein Receptor–Related Protein 1 Regulates Collagen 1 Expression, Proteolysis, and Migration in Human Pleural Mesothelial Cells

“…To detect cell-associated uPA activity, fibrin gel enzymography was performed as previously described (21). Cells were then washed with glycine buffer (pH 3.0) and incubated in the presence of PBS, ATN-617 (an antibody that blocks binding of uPA to uPAR [22]), or isotypematched IgG-treated cells.…”

“…Cells were then washed with glycine buffer (pH 3.0) and incubated in the presence of PBS, ATN-617 (an antibody that blocks binding of uPA to uPAR [22]), or isotypematched IgG-treated cells. Cells were incubated in the presence or absence of 10 or 20 nM uPA on ice for 20 minutes, and 50 mg of cleared lysate was resolved on a 10% SDS-PAGE and assayed via enzymography, as previously described (21).…”