Cell Biology – Immunology – Pathology
Kidney International (2001) 60, 117–125; doi:10.1046/j.1523-1755.2001.00778.x
Adenovirus-mediated transfer of the 39 kD receptor-associated protein increases fibrinolytic capacity
Martin F Goedde, Jos M Grimbergen, Karin H Toet, Thomas Sitter, Paul H Quax and Teake Kooistra
Gaubius Laboratory, TNO Prevention and Health, Leiden, The Netherlands, and Medizinische Klinik, Klinikum Innenstadt, Ludwig-Maximilians-Universität, Munich, Germany
Correspondence: Teake Kooistra, Ph.D., Gaubius Laboratory, TNO-Prevention and Health, P.O. Box 2215, 2301 CE Leiden, The Netherlands. E-mail: t.kooistra@pg.tno.nl
Received 30 October 2000; Revised 23 January 2001; Accepted 26 January 2001.
Abstract
Adenovirus-mediated transfer of the 39 kD receptor-associated protein increases fibrinolytic capacity.
Background
The mesothelium has an important role in maintaining an adequate 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 via the low-density lipoprotein receptor-related protein (LRP). The 39 kD receptor-associated protein (RAP) is an inhibitor of binding of t-PA to LRP, but RAP itself is also rapidly degraded via LRP.
Methods
Adenovirus-mediated RAP gene transfer technology was used to evaluate the effect of prolonged overexpression of RAP on t-PA accumulation in conditioned medium of HMCs under basal and inflammatory conditions.
Results
Infection of HMCs with a recombinant adenovirus carrying the RAP cDNA resulted within one day in t-PA levels that were maximally twofold to threefold increased as compared with noninfected or adenovirus-
-galactosidase–infected cells. Whereas upon prolonged incubation, t-PA levels in the conditioned medium of uninfected cells leveled off because of rapid uptake and degradation via LRP, t-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 and then slowly returned to control values over the next few weeks. In contrast, the production of a specific inhibitor of t-PA, plasminogen activator inhibitor-1 (PAI-1), was not affected by adenoviral RAP gene transfer. Northern blotting analysis showed that t-PA, PAI-1, and LRP mRNA concentrations were not changed after adenoviral infection, underlining that the elevated t-PA levels are the result of RAP-blocked uptake and degradation of t-PA rather than increased t-PA synthesis. RAP gene transfer also restored diminished fibrinolytic activity of cytokine-treated mesothelial cells.
Conclusions
Adenovirus-mediated transfer of the RAP gene provides an efficient way of transiently increasing the fibrinolytic capacity of mesothelial cells.
Keywords:
tissue-type plasminogen activator, mesothelial cells, peritoneal cavity, CAPD, host defense, adhesion prevention, fibrin
Abbreviations:
Ad--Gal, adenoviral vectors expressing the -galactosidase gene; Ad-RAP, adenoviral vectors expressing the receptor-associated protein gene; BSA, bovine serum albumin; CAPD, continuous ambulatory peritoneal dialysis; CMV, cytomegalovirus; GST, glutathione S-transferase; HMCs, human mesothelial cells; HSA, human serum albumin; HUVEC, human umbilical vein endothelial cell; IL, interleukin; LRP, low-density lipoprotein–receptor-related protein; MTT, 3-4,5dimethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide; PAI-1, plasminogen activator inhibitor-1; PFU, plaque-forming units; RAP, receptor-associated protein; TNF-
, tumor necrosis factor-; t-PA, tissue-type plasminogen activator; u-PA, urokinase-type plasminogen activator
