Extracellular matrix produced by cultured corneal and aortic endothelial cells contains active tissue‐type and urokinase‐type plasminogen activators

Abstract

Incubation of plasminogen with the subendothelial extracellular matrix (ECM) synthesized by cultured bovine corneal and aortic endothelial cells resulted in generation of fibrinolytic activity, indicated by proteolysis of 125I-fibrin in a time- and dose-dependent manner. Both tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) were identified in the ECM by fibrin zymography, immunoblotting, and inhibition of plasminogen activation by anti-u-PA and anti-t-PA antibodies. Most of the ECM-resident plasminogen activator (PA) activity did not originate from intracellular PA release occurring when the endothelial cells were lyzed and the ECM exposed, since a comparable amount of PA was associated with the ECM when the cells were lyzed with Triton X-100 or removed intact by treatment with 2 M urea. Active u-PA and t-PA were released from ECM by treatment with heparanase (endo-beta-D-glucuronidase), indicating that some of the ECM-resident PA activity is sequestered by heparan sulfate side chains. These results indicate that both u-PA and t-PA produced by endothelial cells are firmly sequestered in an active form by the subendothelial ECM. It is suggested that ECM-resident plasminogen activators participate in sequential matrix degradation during cell invasion and tumor metastasis. PA activity may also function in release of ECM-bound growth factors (i.e., basic fibroblast growth factor) and activation of proenzymes (i.e., prothrombin), resulting in modulation of the ECM growth-promoting and thrombogenic properties.

The extracellular matrix produced by bovine corneal endothelial cells contains progelatinase A

“…The presence of progelatinase A in the ECM may be compared to that of active tissue-type and urokinase-type plasminogen activators [5]. ECM-bound plasminogen, for example, was found to be a better substrate for tissue plasminogen activator (t-PA) than soluble plasminogen and it was protected from inhibition by plasmin inhibitor [8].…”

“…The subendothelial ECM was exposed by dissolving (5 min, room temperature) the cell layer with sterile PBS containing 0.5% Triton X-100 and 20 mM NH4OH, followed by four washes in PBS. The ECM remained intact, free of cellular debris and firmly attached to the entire area of the tissue culture dish [5,21,22]. ECM extracts for zymography were prepared by scrapping the ECMcoated culture dishes (35 mm) with the aid of a rubber policeman in 250 /11 of Laemmli sample gel buffer (10% SDS, 30% glycerol, 0.25 M Tris-HCl, pH 6.8, 0.1% Bromophenol blue) [23] either solubilized ECM, serum-free conditioned media or purified enzymes were subjected to gelatin-SDS-PAGE without heating or reduction.…”

“…bFGF, TGFfl) [3,4], enzymes (e.g. tPA, uPA) [5,6], enzyme inhibitors (e.g. PAll) [7] and plasma proteins (e.g.…”

Minimal and inducible regulation of tissue factor pathway inhibitor-2 in human gliomas

“…Most (70-90%) of this protease inhibitor is associated with the extra cellular matrix (ECM) (Iino et al, 1998), a complex mixture of proteoglycans that participates in cell adhesion, migration, growth and dierentiation (Hascall et al, 1991). ECM also serves as a reservoir for several growth factors (Vlodavsky et al, 1987;Vukicevic et al, 1992), proteases (McGuire and Seeds, 1989;Korner et al, 1993;Nagase and Woessner, 1999) and protease inhibitors (Mimuro et al, 1987;Chen et al, 1992;Gomez et al, 1997) that are important for homeostasis. However, the functional signi®cance of the association of TFPI-2 with the ECM, as well as the identity of the matrix components that interact with TFPI-2, is largely unknown.…”

Urokinase and Tissue-type Plasminogen Activator Are Required for the Mitogenic and Chemotactic Effects of Bovine Fibroblast Growth Factor and Platelet-derived Growth Factor-BB for Vascular Smooth Muscle Cells

“…Although the general contribution of thrombosis to the development of atherosclerosis has been acknowledged for a long time, recent investigations suggested that the fibrinolytic system may also play an important role in the process of cell proliferation or migration (3,4). Indeed, several authors described both a mitogenic and a chemotactic effect of two types of plasminogen activators: t-PA and u-PA for several cell types including vascular smooth muscle cells (5-8) 2 and a recent study (10) showed that both u-PA and t-PA produced by endothelial cells were sequestered in an active form by the subendothelial extracellular matrix suggesting that they may participate in sequential matrix degradation during cell invasion but also function in the release of extracellular matrixbound growth factor-like bFGF, that will stimulate SMC growth, a crucial step in atherogenesis or post-percutaneous transluminal coronary angioplasty restenosis.…”