Stimulation of plasminogen activator and inhibitor in the lymphatic endothelium

Chromogenic assays, immunoblotting, and Northern blot hybridization methods were employed to assess the effects of various agonists on the production of tissue plasminogen activator (t similar to PA) and plasminogen activator inhibitor type 1 (PAI-1) by the lymphatic endothelium (LEC). Fibrin autogr aphy showed that plasminogen-dependent fibrinolytic activity occurred at M- r of 110 kDa, which represents a complex of tPA with PAI-1, and 65- and 55- kDa bands corresponding to tPA and uPA, respectively. The fractionation of lymph collected from ovine lymphatic vessels also produced a prominent lyti c band of similar to 110 kDa, suggesting the formation of PA/PAI complexes in lymph. The stimulationn of various agonists produced large-scale increas es in tPA mRNA, as shown by Northern blot hybridization analyses. The effec ts of ECGF, histamine, and LPS on the presence of tPA and on enhancing the levels of mRNA reached maximum activity at 4 h and declined to levels below that of controls by 8 h. However, phorbol-treated cells exhibited reduced levels of tPA mRNA at 4 h, but was significantly increased by 8 h. A large- scale increase in PAI-1 mRNA steady-state levels was also stimulated by the agonists used in these studies. Both the 3.4- and 2.4-kb species of PAI-1 mRNA were increased. These observations demonstrated that tPA and PAI-1 are produced and secreted by LEC monolayer cultures and are also present in ly mph. (C) 2000 Academic Press.