Adenovirus-mediated transfer of the 39 kD receptor-associated protein increases fibrinolytic capacity

Background. The mesothelium has an important role in maintaining an adequat e fibrinolytic capacity in the peritoneal cavity and thus in preventing the formation of fibrinous peritoneal adhesions by secreting the fibrinolytic enzyme tissue-type plasminogen activator (t-PA). The fibrinolytic activity of human mesothelial cells (HMCs) is counteracted by rapid uptake of t-PA v ia the low-density lipoprotein receptor-related protein (LRP). The 39 kD re ceptor-associated protein (RAP) is an inhibitor of binding of I-PA to LRP, but RAP itself is also rapidly degraded via LRP. Methods. Adenovirus-mediated RAP gene transfer technology was used to evalu ate the effect of prolonged overexpression of RAP on L-PA accumulation in c onditioned medium of HMCs under basal and inflammatory conditions. Results. Infection of HMCs with a recombinant adenovirus carrying the RAP c DNA resulted within one day in t-PA levels that were maximally twofold to t hreefold increased as compared with noninfected or adenovirus-beta -galacto sidase-infected cells. Whereas upon prolonged incubation, t-PA levels in th e conditioned medium of uninfected cells leveled off because of rapid uptak e and degradation via LRP, I-PA concentrations in the medium of adenovirus- RAP-infected cells continued to increase, reaching fivefold control levels after 72 hours. The increased t-PA accumulation persisted for seven days an d then slowly returned to control values over the next few weeks. In contra st, the production of a specific inhibitor of t-PA, plasminogen activator i nhibitor-1 (PAI-1), was not affected by adenoviral RAP gene transfer. North ern blotting analysis showed that t-PA, PAI-1and LRP mRNA concentrations we re not changed after adenoviral infection, underlining that the elevated t- PA levels are the result of RAP-blocked uptake and degradation of t-PA rath er than increased t-PA synthesis. RAP gene transfer also restored diminishe d fibrinolytic activity of cytokine-treated mesothelial cells. Conclusions. Adenovirus-mediated transfer of the RAP gene provides an effic ient way of transiently increasing the fibrinolytic capacity of mesothelial cells.