Extracellular matrix of cultured bovine aortic endothelial cells contains functionally active type 1 plasminogen activator inhibitor

Abstract

The extracellular matrix (ECM) of cultured bovine aortic endothelial cells (BAEs) was analyzed by immunoblotting and reverse fibrin autography and shown to contain type 1 plasminogen activator inhibitor (PAI-1). Most PAI-1 in the ECM formed complexes with exogenously added tissue-type plasminogen activator (tPA), demonstrating that this PAI-1 was functionally active. The resulting tPA/PAI-1 complexes were recovered in the reaction solution, indicating that the PAI-1 in such complexes no longer bound to ECM. The PAI-1 could not be removed by incubating ECM in high salt (2 mol/L NaCl), sugars (1 mol/L galactose, 1 mol/L mannose), glycosaminoglycans (10 mmol/L heparin, 10 mmol/L dermatan sulfate), or epsilon-aminocaproic acid (0.1 mol/L). However, PAI-1 could be extracted from ECM by treatment with either arginine (0.5 mol/L) or potassium thiocyanate (2 mol/L), or by incubation under acidic conditions (pH 2.5). ECM depleted of PAI-1 by acid extraction was able to bind both the active and latent forms of PAI-1. In this instance, most of the bound PAI-1 did not form complexes with tPA, indicating that the latent form was not activated as a consequence of binding to ECM. Although the PAI-1 activity in conditioned medium decayed with a half-life (t 1/2) of less than 3 hours, the t 1/2 of ECM- associated PAI-1 was greater than 24 hours. These data suggest that PAI- 1 is produced by cultured BAEs in an active form and is then either released into the medium where it is rapidly inactivated or into the subendothelium where it binds to ECM. The specific binding of PAI-1 to ECM protects it from this inactivation.

Inhibitory Role of Plasminogen Activator Inhibitor‐1 in Invasion and Proliferation of HLE Hepatocellular Carcinoma Cells

“…Forced inhibition of PAI-1 expression decreased cellular PAI-1 antigen and increased uPA activity, whereas secreted PAI-1 and uPA activity did not change, and the level of secreted uPA in PAI-1 − cells is similar to that in control cells. These results may suggest that PAI-1 produced in cancer cells leaves the cells and inactivates secreted uPA protein first, and then free PAI-1 returns to the cancer cell surface and inactivates cell-bound uPA, supporting the theory proposed by Mimuro et al 23) that PAI-1 has a high affinity for some constituents of the ECM and once bound to the ECM, has a longer half life. Moreover, our results may also suggest that the induction of high levels of PAI-1 in cancer cells is necessary to inhibit cell-bound uPA and consequent cellular uPA activity and degradation of the ECM around cancer cells.…”

“…The role of PAI-1 in tumor progression, especially in invasion and metastasis, is still a subject of controversy, and it has been concluded that PAI-1 can be effective in controlling metastasis, depending on the type and location of the primary tumor. Although PAI-1 suppresses uPA activities and ECM degradation in colon cancer, prostate cancer and melanoma, 16,17,23) the role of PAI-1 in invasion and proliferation of HCC cells has not been reported. Our data showed that forced inhibition of PAI-1 increased the invasion index of HCC cells and suggested that the imbalance between uPA and PAI-1 expression and the consequent uPA activity in cancer cells may be important for the invasion of HCC.…”

Plasminogen activator regulation in osteoblasts: Parathyroid hormone inhibition of type‐1 plasminogen activator inhibitor and its mRNA

“…These results are relevant to other work that emphasizes the importance in the regulation of PA activity of the specific inhibitor, PAI-1 (Gelehrter et al, 1987;Seifert and Gelehrter, 1978;Andreasen et al, 1986Andreasen et al, , 1990Gelehrter, 1989, 1990). PAI-1 is bound to vitronectin in the extracellular matrix and retains its functional activity there (Mimuro et al, 1987;Sakata et al, 1988;Salonen et al, 1989), thereby limiting extracellular proteolysis.…”

“…It has been proposed that because of the presence of a specific receptor for uPA (Roldan et al, 19901, the action of uPA would be confined to the limited space around the uPA receptor after release of the inactive precursor single-chain uPA. The regulated production of PAI-1 would inhibit activities of both tPA and uPA, in the latter case possibly by means of PAI-1 located in the extracellular matrix in which the uPA target cells are lying (Mimuro et a]., 1987;Sakata et al, 1988;Salonen et al, 1989;Ciambrone and McKeown-Longo, 1990).…”

Ambulatory blood pressure profiles and plasminogen activator inhibitor (PAI‐1) activity in lean women with and without the polycystic ovary syndrome

“…This relation is independent of body mass (Juhan- Vague et al, 1989) and may reflect insulin stimulated hepatic PAI-1 production (Kooistra et al, 1989). PAI-1 and the plasminogen system is of particular interest in patients with PCOS, a condition characterized by anovulation and ovarian connective tissue abnormalities, as this system is intimately involved in ovarian follicle development and connective tissue remodelling (Mimuro et al, 1987).…”