Extracellular matrix hyaluronan is a determinant of the endothelial barrier.

We measured the hydraulic conductivity (Lp) of the extracellular matrix (ECM) obtained after detaching bovine pulmonary microvascular endothelial (BPMVEC) and bovine pulmonary arterial endothelial cell (BPAEC) monolayers from the ECM at different days postseeding. From day 1 to day 5 in culture, the total Lp (i.e., of cell monolayer + ECM) decreased from basal values of 17.1 +/- 4.0 to 8.5 +/- 1.6 x 10(-6) cm.s-1.cmH2O-1 in BPAEC (P < 0.05) and 7.6 +/- 1.1 to 3.7 +/- 0.8 in BPMVEC (P < 0.05), respectively, and on day 5 the total Lp values were lower in BPMVEC than in BPAEC (P < 0.05). On the 5th day, ECM Lp was 55.0 +/- 8.3 in BPAEC and 10.7 +/- 0.9 cm.s-1.cmH2O-1 in BPMVEC (P < 0.05), indicating that the contribution of ECM to the total Lp was greater in BPMVEC than in BPAEC. Treatment of [3H]acetate-labeled ECM with Streptomyces hyaluronidase (HAse; 6 U/ml for 10 min) released sixfold greater radioactivity in BPMVEC compared with untreated BPMVEC controls; a similar treatment of BPAEC did not release detectable radioactivity indicative of a higher hyaluronan content in the BPMVEC ECM. HAse treatment reduced the differences in total Lp between BPMVEC and BPAEC at different days postseeding. Moreover, on the 5th day after seeding, the ECM Lp of BPMVEC increased to a greater extent after HAse treatment than the ECM of BPAEC. These data indicate that the hyaluronan component of the ECM is an important determinant of the endothelial liquid-exchange barrier.

Requirement for receptor-bound urokinase in plasmin-dependent cellular conversion of latent TGF-beta to TGF-beta.

The role of receptor-bound urokinase-type plasminogen activator (uPA) in cellular activation of latent transforming growth factor-beta (LTGF-beta) was investigated in a model system of mouse LB6 cells transfected with either a human uPA receptor cDNA (LhuPAR+), a human prouPA cDNA (LhuPA), or a control neomycin-resistance cDNA (Lneo). When LhuPAR+ cells were co-cultured with LhuPA cells, the plasmin-dependent fibrinolytic activity generated was more than that observed in either homotypic cultures with fivefold greater number of LhuPA cells or co-cultures containing LhuPA and Lneo cells instead of the LhuPAR+ cells. The preferential activation of TGF-beta by co-cultures with the greatest plasmin-generation potential, LhuPAR+ and LhuPA cells, was confirmed by three independent bioassays. In the first assay, a 48% decrease in PA activity, a measure of active TGF-beta production, was observed with BAE cells treated with conditioned medium (CM) from co-cultures of LhuPA and LhuPAR+ cells. Inclusion of neutralizing antibodies to TGF-beta abrogated the inhibitory effect of CM on PA activity demonstrating that the inhibitory molecule was TGF-beta. Addition of the amino terminal fragment of uPA (ATF) or omission of plasminogen from co-cultures blocked both the fibrinolytic activity and the generation of TGF-beta activity in the CM. In the second assay, CM from co-cultures of LhuPA and LhuPAR+ cells inhibited the migration of BAE cells in a wound assay. Controls with anti-TGF-beta IgG indicated that the inhibition was due to TGF-beta. In the third assay, proliferation of mink lung epithelial cells was inhibited by CM generated by co-cultures of LhuPA and LhuPAR+ cells as compared to CM from the same cells cultured in the absence of plasminogen or to CM from a co-culture of LhuPA with LhuPAR- cells. Excess mannose-6-phosphate (M6P) blocked the generation of TGF-beta as assayed by both the BAE migration and PA assays, presumably because it interfered with cell-surface localization of LTGF-beta. Additionally, small numbers of LhuPA and LhuPAR+ cells co-cultured with BAE cells inhibited the BAE cell PA activity via the paracrine action of TGF-beta. These results support the conclusion that plasmin-dependent activation LTGF-beta by LB6 cells is promoted by the surface localization of uPA by its receptor.

Urokinase in conditioned medium from phorbol ester-pretreated endothelial cells promotes polymorphonuclear leukocyte migration.

Serum-free conditioned medium (CM) generated by human umbilical vein endothelial cell monolayers following pretreatment with 100 ng/ml of phorbol myristate acetate (PMA) promoted human polymorphonuclear leukocyte (PMNL) migration as assayed in blindwell chambers. Stimulation of PMNL migration in response to CM was dependent on the dose of PMA used to pretreat the endothelial cells as well as the duration of incubation time to generate CM. Phorbol esters have been previously shown to release plasminogen activators from vascular endothelial cells. In the present study, pretreatment of endothelial cells with PMA also increased plasminogen activator activity in CM at a time course similar to the generation of PMNL chemoattractant activity. Treatment of CM with a polyclonal antibody against human urokinase-type plasminogen activator (uPA) not only inhibited uPA activity, but also significantly reduced PMNL chemoattractant activity when compared with untreated CM. In contrast, treatment of CM with an antibody directed against tissue-type plasminogen activator (tPA) did not affect PMNL migratory activity. Furthermore, when CM was passed over an anti-uPA immunoaffinity column, plasminogen activator activity was reduced 90% and chemoattractant activities was reduced 68%. Both plasminogen activator and chemoattractant activities were reconstituted in the eluate from the anti-uPA column. These data demonstrate that uPA present in the CM from PMA-pretreated endothelial cells stimulates PMNL chemoattractant activity and suggests a possible role for endothelial cell-derived uPA in stimulating migration of peripheral blood leukocytes at an inflammatory locus.

Urokinase-type plasminogen activator mediates basic fibroblast growth factor-induced bovine endothelial cell migration independent of its proteolytic activity.

The dependence of urokinase-type plasminogen activator (uPA) induction on endogenous basic fibroblast growth factor (bFGF) activity during endothelial cell migration was investigated utilizing a combination of wounded endothelial cell monolayers and substrate overlay techniques. Purified polyclonal rabbit immunoglobulin G (IgG) against bFGF blocked the appearance of uPA-dependent lytic activity normally observed at the edge of a wounded bovine aortic endothelial (BAE) cell monolayer. Additionally, the migration of cells into the denuded area was inhibited 30-50% by antibodies either to bFGF or to bovine uPA. Incubation of wounded monolayers with either purified bovine uPA or agents able to induce PA activity, such as phorbol myristate acetate (PMA), vanadate, or bFGF, resulted in enhanced migration of cells (28-50%). Anti-bovine uPA IgG blocked a significant fraction (25%) of BAE cell migration induced by exposure to exogenous bFGF. The role of uPA in migration of wounded BAE cells was not dependent on plasmin generation. Furthermore, the amino terminal fragment (ATF) of human recombinant (hr) uPA, which is enzymatically inactive, stimulated endothelial cell movement in the presence of anti-bFGF IgG. These results suggest that BAE cell migration from the edge of a wounded monolayer is dependent upon local increases of uPA mediated by endogenous bFGF. Moreover, the data support the conclusion that migration is stimulated via a signalling mechanism dependent upon occupancy of the uPA receptor but independent of uPA-mediated proteolysis.

Fibronectin fragments released from phorbol ester-stimulated pulmonary artery endothelial cell monolayers promote neutrophil chemotaxis.

We have recently shown that monolayer cultures of calf pulmonary artery endothelial (CPAE) cells pretreated with phorbol myristate acetate (PMA) generate a conditioned medium that is chemotactic for human polymorphonuclear leucocytes (PMNL). Fibronectin (Fn) is a multidomain protein found in the plasma and subendothelial extracellular matrix that induces attachment and migration of a variety of cell types. The present study was designed to evaluate the role of Fn or fragments of Fn present in conditioned medium from phorbol ester-stimulated endothelial cells as potential chemotactic factors for human PMNL. A large number of Fn fragments were revealed by Western immunoblotting of serum-free conditioned medium 4 hr after treatment of CPAE monolayers with PMA. Gelatin-Sepharose affinity chromatography of 4-hr conditioned medium demonstrated chemotactic activity for PMNL in both gelatin-binding and non-gelatin-binding fractions. The addition of bovine Fn antiserum to the conditioned medium inhibited PMNL chemotaxis in a dose-dependent manner while having no effect on PMNL chemotaxis generated by zymosan-activated serum. One site on the Fn molecule known to interact with phagocytic cells is the cell-binding domain containing the Arg-Gly-Asp (RGD) sequence. Pretreatment of PMNL with a RGD-containing peptide (1 mM GRGDSPK) for 10 min completely inhibited the expression of chemotactic activity present in conditioned medium and in the gelatin-binding and non-gelatin-binding fractions. PMNL chemotaxis was not stimulated by either intact Fn or the RGD-containing septapeptide tested over a wide concentration range. However, incubation of PMNL with a purified 120,000-MW fragment of Fn containing the cell-binding domain stimulated chemotaxis in a dose-dependent manner. In contrast, a purified 45,000 MW fragment of Fn containing the gelatin-binding domain was not chemotactic for PMNL. When a monoclonal antibody directed against the cell-binding domain of Fn was incubated with conditioned medium, a significant reduction in PMNL chemotaxis was observed. These results demonstrate that phorbol ester-stimulated pulmonary artery endothelial cells release Fn fragments and suggest an important role for Fn fragments containing the cell-binding domain in stimulating the migration of PMNL.

Generation of neutrophil chemotactic activity by phorbol ester-stimulated calf pulmonary artery endothelial cells.

Chemotactic activity for human polymorphonuclear leukocytes (PMNL) was detected in serum-free conditioned media 1 to 4 hr after monolayers of calf pulmonary artery endothelial cells were pretreated with phorbol myristate acetate (PMA). Chemotactic activity was increased in conditioned media following pretreatment with either PMA or the less lipophilic active phorbol ester, 4-beta-phorbol-12,13-dibutyrate (P(Bu)2) in a dose-dependent manner. Chemotactic activity of conditioned media from PMA-treated endothelial cells was confirmed by checkerboard analysis. The chemotactic activity in conditioned media from PMA-pretreated endothelial cells was completely inhibited by pretreating endothelial cells with either cycloheximide, actinomycin D, or the lipooxygenase inhibitor, diethylcarbamazine. Furthermore, the chemotactic activity was heat-stable, inhibited by trypsin treatment, and present in both aqueous and lipid phases after ether extraction. The data demonstrate that pulmonary artery endothelial cells exposed to active phorbol esters release potent chemotactic factor(s) for PMNL. These findings suggest a role for activators of protein kinase C in mediating endothelial cell release of chemotactic factor(s) that may be important in the directed migration of circulating leukocytes to sites of vascular injury.