Binding of type 1 plasminogen activator inhibitor to the extracellular matrix of cultured bovine endothelial cells

The binding of type 1 plasminogen activator inhibitor (PAI-1) to the extracellular matrix (ECM) of cultured bovine aortic endothelial cells was investigated using purified 125I-labeled or L-[35S]methionine-labeled PAI-1 as probes. Little specific binding of latent PAI-1 to ECM previously depleted of endogenous PAI-1 could be demonstrated. In contrast, the guanidine-activated form of PAI-1 bound to ECM in a dose- and time-dependent manner, and binding was saturable. The dissociation constant (Kd) for this interaction was estimated to be 60 nM by Scatchard analysis, and approximately 6 pmol of activated PAI-1 was bound per cm2 of ECM. Binding was relatively specific since unlabeled, activated PAI-1 competed with 35S-labeled PAI-1 for binding to ECM, but latent PAI-1 did not. Moreover, PAI-2, protein C inhibitor (i.e. PAI-3), protease nexin-1, and alpha 2-antiplasmin were not able to compete. Tissue-type plasminogen activator (tPA) also inhibited binding, but diisopropyl fluorophosphate-inactivated tPA did not. Pretreatment of ECM with tPA, urokinase-type PA, or thrombin had no effect on its ability to subsequently bind PAI-1, whereas trypsin, plasmin, and elastase pretreatment greatly reduced its ability to bind PAI-1. Guanidine-activated, radiolabeled PAI-1 resembled active endogenous PAI-1 since it was unstable in solution but stable when bound to ECM. In addition, it formed complexes with tPA that had a relatively low affinity for ECM. These data suggest that ECM of bovine aortic endothelial cells contains a protease-sensitive structure that binds active PAI-1 tightly and relatively selectively and that this association stabilizes PAI-1 against the spontaneous loss of activity that occurs in solution.     

Regulation of type I plasminogen activator inhibitor synthesis by protein kinase C and cAMP in bovine aortic endothelial cells.

The second messengers and protein kinases involved in the induction of type I plasminogen activator inhibitor (PAI-1) synthesis by various agents were evaluated in cultured bovine aortic endothelial cells. Phorbol myristate acetate (PMA) induced PAI-1 in these cells implicating the protein kinase C (PK-C) pathway. However, bradykinin, which also activates PK-C in bovine aortic endothelial cells, did not induce PAI-1. Moreover, when PK-C was down-regulated by PMA pretreatment, subsequent induction of PAI-1 by transforming growth factor beta (TGF beta) and tumor necrosis factor alpha (TNF alpha) was unaltered, and induction by lipopolysaccharide (LPS) was decreased by only 50%. LPS increased phospholipid second messengers which can activate PK-C but TGF beta and TNF alpha did not. Agents which increase cAMP, (e.g., forskolin and isobutylmethylxanthine) blocked the induction of PAI-1 synthesis by PMA, LPS, TGF beta and TNF alpha suggesting that induction may occur by lowering cAMP. This possibility seems unlikely since cAMP levels did not change in response to any of these agents. Moreover, somatostatin lowered cAMP but did not induce PAI-1. PAI-1 was not induced by treating the cells with cGMP, Na+/H+ ionophore and calcium ionophore or arachidonic acid.     

Plasminogen activator activity by cultured bovine aortic endothelial cells

This communication reports the observations that bovine aortic endothelial cells (EC) in culture during their log phase of growth secrete plasminogen activator. Hydrocortisone, dibutyryl cAMP, theophylline, colchicine and cycloheximide, dependent upon concentration, inhibit plasminogen activator activity. Several substances associated with inflammation and thrombosis, such as thrombin, serotonin,catecholomines, histamine, vasopressin, endotoxin, and indomethacin, at the concentrations tested, did not significantly alter plasminogen activator activity when compared with controls.     

The majority of type 1 plasminogen activator inhibitor associated with cultured human endothelial cells is located under the cells and is accessible to solution-phase tissue-type plasminogen activator

The interactions between exogenously added tissue-type plasminogen activator (t-PA) and the active form of type 1 plasminogen activator inhibitor (PAI-1) produced by and present in cultured human umbilical vein endothelial cells (HUVECs) were investigated. Immunoblotting analysis of the conditioned media obtained from monolayers of HUVECs treated with increasing concentrations of t-PA (less than or equal to 10 micrograms/ml) revealed a dose-dependent formation of both t-PA/PAI-1 complexes, and of a 42,000-Mr cleaved or modified form of the inhibitor. Immunoradiometric assays indicated that t-PA treatment resulted in a fourfold increase in PAI-1 antigen present in the conditioned media. This increase did not result from the release of PAI-1 from intracellular stores, but rather reflected a t-PA-dependent decrease in the PAI-1 content of the Triton X-100 insoluble extracellular matrix (ECM). Although the rate of t-PA-mediated release of PAI-1 was increased by the removal of the monolayer, similar quantities of PAI-1 were removed in the presence or absence of the cells. These results suggest that the cells only represent a semipermeable barrier between ECM-associated PAI-1 and exogenous t-PA. Treatment of HUVECs with t-PA (1 microgram/ml, 2 h) to deplete the ECM of PAI-1 did not affect the subsequent rate of PAI-1 production and deposition into the ECM. Immunogold electron microscopy of HUVECs not only confirmed the location of PAI-1 primarily in the region between the culture substratum and ventral cell surface but failed to demonstrate significant (less than 1%) PAI-1 on the cell surface. Thus, the majority of PAI-1 associated with cultured HUVEC monolayers is present under the cells in the ECM and is accessible to solution-phase t-PA.     

Fibrinolytic system of cultured endothelial cells: regulation by plasminogen activator inhibitor

Cultured bovine aortic endothelial cells have a relatively complex fibrinolytic system that is responsive to both the physiological state of the cell itself and to a variety of agents added to the culture medium. The fibrinolytic activity of these cells results from the production of both urokinase-type and tissue-type plasminogen activators and is regulated by an inhibitor capable of neutralizing their activities. The properties of these fibrinolytic components will be reviewed, and their respective roles in initiating and regulating the fibrinolytic activity of the cells will be summarized. A cDNA coding for the inhibitor has been isolated, and its sequence will be compared to that of other serine proteinase inhibitors.     

Interaction of tissue-type plasminogen activator and plasminogen activator inhibitor 1 on the surface of endothelial cells

The site of the reaction between plasminogen activators and plasminogen activator inhibitor 1 (PAI-1) was investigated in cultures of human umbilical vein endothelial cells. In conditioned medium from endothelial cells, two forms of a plasminogen activator-specific inhibitor can be demonstrated: an active form that readily binds to and inhibits plasminogen activators and an immunologically related quiescent form which has no anti-activator activity but which can be activated by denaturation. In conditioned medium, only a few percent of PAI-1 is the active form. However, the addition of increasing concentrations of tissue-type plasminogen activator (t-PA) or urokinase to confluent endothelial cells produced a saturable (3.0 pmol/5 x 10(5) cells), dose-dependent increase of the activator-PAI-1 complex in the conditioned medium even in the presence of actinomycin D or cycloheximide. This resulted also in a dose-dependent decrease of the residual PAI activity measured by reverse fibrin autography both in the conditioned medium and cell extracts. Short-time exposure of endothelial cells to a large amount of t-PA caused almost complete depletion of all cell-associated PAI activity. Although there was no detectable PAI activity even after activation of PAI by denaturants or antigen in the culture medium at 4 degrees C without the addition of t-PA, the addition of t-PA at 4 degrees C not only resulted in the formation of 70% of the amount of the t-PA.PAI complex in conditioned medium at 37 degrees C, but also induced PAI-1 antigen in a time and dose-dependent manner in the conditioned medium. Moreover, 125I-labeled t-PA immobilized on Sepharose added directly to endothelial cells formed a complex with PAI-1 in a dose-dependent manner. On the other hand, no detectable complex was formed with PAI-1 when Sepharose-immobilized 125I-labeled t-PA was added to endothelial cells under conditions in which the added t-PA could not contact the cells directly but other proteins could pass freely by the use of a Transwell. All these results suggest that a "storage pool" on the surface of endothelial cells or the extracellular matrix produced by endothelial cells contains almost all the active PAI-1, and reaction between PA and PAI-1 mainly occurs on the endothelial cell membranes, resulting in a decrease of the conversion of active PAI-1 to the quiescent form.     

Vascular origin determines plasminogen activator expression in human endothelial cells. Renal endothelial cells produce large amounts of single chain urokinase type plasminogen activator

Positioned at the boundary between intra- and extravascular compartments, endothelial cells may influence many processes through their production of plasminogen activators (PA). Available data have shown that tissue-type plasminogen activator (t-PA) is the major form produced by human endothelial cells. We have compared the molecular forms of PA produced by human endothelial cells from different microvascular and large vessel sources including two different sites within the circulation of the kidney. Using combined immunoactivity assays specific for u-PA and t-PA activity and antigen, we found that both human renal microvascular and renal artery endothelial cells produced high levels of u-PA antigen (60.48 ng/10(5) cells/24 h and 50.42 ng/10(5) cells/24 h, respectively) and corresponding levels of u-PA activity after activation with plasmin. Activity was not evident before plasmin activation, showing that the u-PA produced is almost exclusively as single chain form U-PA. In contrast, human omental microvascular endothelial cells and human umbilical vein endothelial cells produced exclusively t-PA (8.80 ng/10(5) cells/24 h and 2.17 ng/10(5) cells/24 h, respectively). Neither endothelial cell type from human kidney produced plasminogen activator inhibitor, as determined by reverse fibrin autography and titration assays. Agents including phorbol ester, thrombin, and dexamethasone were shown to regulate the renal endothelial cell production and mRNA expression of both u-PA and t-PA. Among the macro- and microvascular endothelial cells tested, only those from the renal circulation produced high levels of single chain form U-PA, suggesting the vascular bed of origin determines the expression of plasminogen activators.     

Endotoxin induction of an inhibitor of plasminogen activator in bovine pulmonary artery endothelial cells

We have examined the effects of bacterial lipopolysaccharide (endotoxin) on the fibrinolytic activity of bovine pulmonary artery endothelial cells. Endotoxin suppressed the net fibrinolytic activity of cell extracts and conditioned media in a dose-dependent manner (threshold dose, 0.1 ng/ml; maximal dose, 10-100 ng/ml). The effects of endotoxin required at least 6 h for expression. Cell extracts and conditioned media contained a 44-kDa urokinase-like plasminogen activator. Media also contained multiple plasminogen activators with molecular masses of 65-75 and 80-100 kDa. Plasminogen activators in extracts and media were unchanged by treatment of cells with endotoxin. Diisopropyl fluorophosphate (DFP) abolished fibrinolytic activity of extracts and conditioned media. DFP-treated samples from endotoxin-treated but not untreated cells inhibited urokinase and tissue plasminogen activator, but not plasmin. Inhibitory activity was lost by incubation at pH 3 or heating to 56 degrees C for 10 min. These treatments did not affect inhibitory activity of fetal bovine serum. Incubation of 125I-urokinase with DFP-treated medium from endotoxin-treated cells produced an inactive complex with an apparent molecular mass of 80-85 kDa. The complex could be detected by chromatography on Sephadex G-100, but not by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These findings suggest that low doses of endotoxin suppress fibrinolytic activity in endothelial cells by stimulating the production or expression of a fast-acting, relatively labile inhibitor of plasminogen activator.     

Immunoelectron microscopic localization of type 1 plasminogen activator inhibitor on the surface of activated endothelial cells

Immunogold EM was employed to compare the distribution of type 1 plasminogen activator inhibitor (PAI-1) on the surface of agonist-activated human umbilical vein endothelial cells (HUVECs) with that of control, unactivated cells. As previously observed, (Schleef, R.R., T.J. Podor, E. Dunne, J. Mimuro, and D.J. Loskutoff. J. Cell Biol. 110:155-163), analysis of cross-sections of nonpermeabilized control HUVEC monolayers stained first with affinity-purified rabbit antibodies to PAI-1 and then with gold-conjugated goat anti-rabbit IgG, revealed the presence of relatively few gold particles (less than 1-2% of the total) on the apical cell surface. The majority of gold particles were detected primarily in the extracellular matrix between the culture substratum and the cell membrane. In contrast, treatment of HUVECs with tumor necrosis factor alpha (TNF alpha; 200 U/ml, 24 h) or with lipopolysaccharide (LPS; 10 micrograms/ml, 24 h) resulted in an increased staining of PAI-1 not only in the extracellular matrix, but also on the apical cell surface (10-fold increase). Immunoabsorption of the rabbit anti-PAI-1 with purified PAI-1, or treatment of HUVECs with tissue-type plasminogen activator (2.5 micrograms/ml, 2 h, 4 degrees C) reduced the amount of staining both on the apical surface and in the extracellular matrix of agonist-activated HUVECs by 80-95%. The topographical location of PAI-1 on the cell surface was examined further by coupling immunogold staining with high resolution surface replication. Transmission EM of surface replicas from TNF alpha- or LPS-activated HUVECs revealed a general increase in PAI-1 staining both on planar regions and within indentations of the apical cell surface. Nonactivated HUVECs revealed PAI-1-specific immunogold particles only in areas of exposed extracellular matrix between the cells and occasionally at regions of cell-cell contacts. Analysis of activated bovine aortic endothelial cells by immuno-electron microscopy, immunologic assays, and flow cytometry revealed similar increases in surface PAI-1. These increases in surface PAI-1 could be detected by 3 h and continued over a 24-h period. The expression of PAI-1 on the luminal surface of endothelial cells during immune or inflammatory reactions could reduce endothelial fibrinolytic activity, thus, promoting the localized, pathologic formation of intravascular thrombi.     

Regulation of tissue-type plasminogen activator and plasminogen activator inhibitor type-1 in cultured rat Sertoli and Leydig cells

New data are provided to show that (i) rat Sertoli cells produce two types of plasminogen activators, tissue type (tPA) and urokinase type (uPA), and a plasminogen activator inhibitor type-1 (PAI-1); (ii) both tPA (but not uPA) and PAI-1 secretion in the culture are modified by FSH, forskolin, dbcAMP, GnRH, PMA and growth factors (EGF and FGF), but not by hCG and androstenedione (△4); (iii) in vitro secretion of tPA and PA-PAI-1 complexes of Sertoli cells are greatly enhanced by presence of Leydig cells which produce negligible tPA but measurable PAI-1 activity;(iv) combination culture of Sertoli and Leydig cells remarkably increases FSH-induced PAI-1 activity and decreases hCG- and forskolin-induced inhibitor activity as compared with that of two cell types cultured alone. These data suggest that rat Sertoli cells, similar to ovarian granulosa cells, are capable of secreting both tPA and uPA, as well as PAI-1. The interaction of Sertoli cells and Leydig cells is essential for the cells to response to     

Binding of tissue plasminogen activator to human aortic endothelial cells.

The experiments described in this paper were designed to examine the specific binding of tissue plasminogen activator (tPA) to cultured human aortic endothelial (HAE) cells. When 125I-labelled tPA was incubated with the cells at 4 degrees C, binding was found to plateau within 90 min after incubations were begun. Binding was saturable and the bound enzyme dissociated from the sites with a half-time of approx. 48 min. Scatchard analyses were performed using tPA molecules isolated from human melanoma and colon cells as well as from C127 and Chinese hamster ovary cells that had been transfected with the human tPA gene. These enzymes showed very similar binding characteristics in spite of the fact that they differ substantially in the types of sugars which comprise their side chains. Neither the chainedness of the molecules (one-chain or two-chain) nor the sites at which they are glycosylated (type I or type II) appear to affect their ability to interact with binding sites. The tPA molecules were found to have an average equilibrium dissociation constant of (1.15 +/- 0.10) x 10(-9) M and HAE cells appeared to have a single, homogeneous population of independent binding sites present at a concentration of (1.57 +/- 0.13) x 10(6) sites per cell. Lowering the pH of the binding buffer from 7.4 to 6.5 resulted in a reversible increase in specific binding of between 2-fold and 7-fold depending upon the particular preparation of cells. Preincubation of tPA with plasminogen activator inhibitor 1 (PAI-1) was found to have little effect on binding, suggesting that tPA interacts at sites distinct from surface-bound PAI-1. No evidence for either internalization or degradation of tPA was observed in assays run at 37 degrees C. This suggests that, like urokinase, tPA remains on cell surfaces for an extended period of time.