TNF-alpha -induced matrix Fn disruption and decreased endothelial integrity are independent of Fn proteolysis

Theresa M. Curtis, Robert F. Rotundo, Peter A. Vincent, Paula J. McKeown-Longo, and Thomas M. Saba

Department of Physiology and Cell Biology, Albany Medical College, Albany, New York 12208

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Exposure of confluent pulmonary arterial endothelial monolayers to tumor necrosis factor (TNF)-alpha causes both a reorganization and/or disruption of fibronectin (Fn) in the extracellular matrix and an increase in transendothelial protein permeability. However, the factors initiating this response to TNF-alpha have not been defined. Because TNF-alpha can induce proteinase expression in endothelial cells, we determined whether proteinases cause both the alteration of the Fn matrix and the permeability increase as is often speculated. Incubation of calf pulmonary arterial endothelial monolayers with TNF-alpha (200 U/ml) for 18 h caused a disruption of the Fn matrix and an increase in transendothelial protein permeability. A reduced colocalization of cell-surface alpha 5beta 1-Fn integrins with the Fn fibers in focal contacts was also observed. TNF-alpha treatment of endothelial monolayers with matrices prelabeled with 125I-human Fn (hFn) did not cause the release of Fn fragments or alter the content of Fn antigen in the medium as analyzed by SDS-PAGE coupled with autoradiography. Both the content and fragmentation pattern of Fn within the cell layer and the insoluble Fn matrix also appeared unchanged after TNF-alpha exposure as confirmed by Western immunoblot. Fn-substrate zymography revealed that TNF-alpha increased the expression of two proteinases within the conditioned medium in which activity could be blocked by aprotinin but not by EDTA, 1,10-phenanthroline, leupeptin, or pepstatin. However, inhibition of the Fn proteolytic activity of these two serine proteinases did not prevent either the TNF-alpha -induced disruption of the Fn matrix or the increase in permeability. Thus the reorganization and/or disruption of the Fn matrix and the temporally associated increase in endothelial permeability caused by TNF-alpha appear not to be due to proteolytic degradation of Fn within the extracellular matrix. In contrast, decreased alpha 5beta 1-Fn integrin interaction with Fn fibers in the matrix may be important in the response to TNF-alpha exposure.

lung vascular permeability; fibronectin; tumor necrosis factor-alpha ; proteinases

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

PULMONARY ENDOTHELIAL PROTEIN permeability is increased during inflammatory-induced septic lung injury, an event associated with the release of cytokines such as tumor necrosis factor (TNF)-alpha from activated monocytes and macrophages sequestered in the lung (19, 29, 30). This increase in lung vascular permeability contributes to the etiology of pulmonary edema and acute respiratory distress syndrome (ARDS) in septic surgical and trauma patients (1, 23). Under both in vivo and in vitro conditions, TNF-alpha has been shown to decrease the integrity of the endothelial barrier (5, 7, 9, 19, 21), but the mechanism of this alteration in endothelial barrier permeability is unclear.

Fibronectin (Fn) in the subendothelial matrix influences cell adhesion to the substratum, a process that can affect endothelial cell shape and cytoskeletal conformation (11, 25) and, therefore, the integrity of the endothelial barrier. Exposure of calf pulmonary arterial endothelial (CPAE) monolayers to TNF-alpha causes both an increase in protein permeability and a temporally associated reorganization and/or disruption of the fibrillar organization pattern of Fn in the extracellular matrix (ECM) (5, 37). Indeed, the insoluble pool of Fn normally incorporated within the lung matrix is disturbed by many of the same agents that can also increase lung vascular permeability in vivo (6, 17, 20, 33), suggesting a potential functional relationship between stability of the ECM and integrity of the lung vascular barrier. The addition of purified soluble human plasma Fn (hFn) to the medium of TNF-alpha -treated endothelial monolayers can prevent as well as reverse the TNF-alpha -induced increase in protein permeability, and this protective effect appears to require incorporation of the added soluble Fn into the ECM (5, 35). Such findings suggest that the increase in protein permeability caused by TNF-alpha may be due to disruption of the Fn matrix, leading to altered endothelial adhesion and barrier integrity.

Exposure of isolated endothelial cells to TNF-alpha causes the release of the serine proteinase urokinase-type plasminogen activator (uPA) as well as of two matrix metalloproteinases, i.e., gelatinase B (MMP-9), and stromelysin-1 (MMP-3) (8, 14, 16, 32). Accordingly, these proteinases could potentially mediate the altered integrity of the ECM and thus destabilize a confluent endothelial cell monolayer. However, TNF-alpha exposure can also increase the release of proteinase inhibitors such as plasminogen activator inhibitor-1 from endothelial cells (31). From this perspective, the proteolytic activity of a proteinase secreted by endothelial cells may, in theory, be regulated or attenuated by endogenous inhibitors also secreted by the same cells. Thus although the addition of TNF-alpha to the culture medium of CPAE monolayers does cause the release of proteinases, their speculated role in both the TNF-alpha -induced disruption of Fn in the ECM and the increase in protein permeability has yet to be validated.

The present study was designed to determine whether the reorganization and/or disruption of the Fn matrix as well as the temporally associated increase in transendothelial protein permeability (5, 37, 38) observed in CPAE monolayers after exposure to TNF-alpha was dependent on proteolysis of Fn in the subendothelial ECM. Our study, which is the first to analyze the content, molecular weight, and deposition pattern of Fn within the subendothelial matrix of endothelial monolayers after TNF-alpha exposure, suggests that both the reorganization of Fn in the ECM and the increase in monolayer protein permeability are not dependent on the proteolytic degradation of matrix localized Fn. In contrast, these TNF-alpha -induced changes in endothelial permeability and ECM organization may be associated with a reduced colocalization of alpha 5beta 1-Fn integrins with fine Fn fibers within the ECM.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Endothelial Cell Monolayer

CPAE cells (American Type Culture Collection CCL-209, Rockville, MD) were grown in MEM (GIBCO BRL, Grand Island, NY) supplemented with 20% fetal bovine serum (FBS; Hyclone), penicillin (100 U/ml), and streptomycin (100 µg/ml). Experiments were performed between passages 17 and 23. When the CPAE monolayers were treated with TNF-alpha , MEM supplemented with 5% FBS, penicillin, and streptomycin was used.

TNF-alpha , Leukocyte Elastase, and Proteinase Inhibitors

Purified recombinant human TNF-alpha (Cellular Products, Buffalo, NY) had a specific activity of 24 × 106 U/mg protein. Human leukocyte elastase (Sigma, St. Louis, MO) had a specific activity of 50 U/mg protein. The three proteinase inhibitors, i.e., aprotinin, leupeptin, and pepstatin, were purchased from Boehringer Mannheim (Indianapolis, IN). EDTA and 1,10-phenathroline were purchased from Sigma.

Antibodies

The following antibodies were purchased: rabbit anti-bovine Fn (bFn; Calbiochem, La Jolla, CA), FITC-conjugated goat F(ab')2 fragment to hFn (Cappel, Durham, NC), rabbit anti-bovine collagen IV (Biodesign International, Kennebunk, ME), and mouse monoclonal antibody to human alpha 5beta 1-integrin (MAb 1999, Chemicon, Temecula, CA). The secondary antibodies, rhodamine isothiocyanate (RITC)-conjugated goat anti-rabbit IgG (Cappel) and RITC-conjugated goat anti-mouse IgG (Boehringer Mannheim), were purchased commercially.

Immunofluorescence

CPAE cells were seeded (280,000 cells/well) on glass coverslips in 12-well tissue culture dishes and grown to confluence (3 days). The cell monolayers were then treated with medium alone or medium containing TNF-alpha (200 U/ml) for 18 h. The cells were then rinsed three times with PBS (pH 7.4) and fixed in 3.0% formaldehyde in PBS for 15 min. After fixation, endothelial cell monolayers were rinsed three times with PBS, and the monolayers were permeabilized with a HEPES solution containing 0.5% Triton X-100 on ice for 5 min followed by three rinses with PBS. The cell monolayers were first blocked with 2% BSA-50 mM glycine-0.2% Tween 20 in PBS for 1 h and then incubated with various primary and secondary antibodies (see Immunofluorescence Protocols). Control studies using no primary antibodies were negative. The coverslips were again washed three times in PBS and mounted in 2% N-propylgallate in 25% glycerol. The slides were kept in the dark until they were viewed and photographed with a fluorescent microscope (Nikon Microphot SA). All immunofluorescent experiments were performed at least in triplicate.

Immunofluorescence Protocols

Detection of bFn. After the TNF-alpha treatment interval was over, the monolayers were prepared for indirect immunofluorescence. The primary antibody used was rabbit anti-bFn (1:100), and the secondary antibody was a RITC-conjugated goat anti-rabbit IgG (1:50).

Simultaneous detection of bFn and hFn. After the TNF-alpha treatment interval was over, the monolayers were prepared for analysis by dual-label immunofluorescence. The primary antibody used to detect bFn was a rabbit anti-bFn (1:100), and the primary antibody used to detect hFn was a FITC-conjugated goat F(ab')2 fragment to hFn (1:50). The secondary antibody, RITC-conjugated goat anti-rabbit IgG (1:50), was then added to visualize bFn. To ensure specificity of the antibodies and avoid cross-reactivity, the antibodies to either bFn or hFn were subjected to affinity chromatography against the opposite species antigen with either hFn- or bFn-Sepharose columns, respectively.

Simultaneous detection of hFn and bovine collagen IV. Similar to the tracer experiments, we seeded CPAE cells in the presence of medium containing FBS (deficient in bFn) plus exogenous hFn. Soluble plasma hFn added to the culture medium before confluence will incorporate into the ECM and colocalize with the endogenous bFn. Moreover, after treatment with TNF-alpha , both the endogenous bFn and the added hFn will show similar reorganization and/or disruption. The monolayers were treated with TNF-alpha for 18 h and then prepared for dual-label immunofluorescence. The hFn was detected with an FITC-conjugated goat anti-hFn (1:100), and collagen IV was detected with a rabbit anti-bovine collagen IV IgG and an RITC-conjugated goat anti-rabbit IgG (1:100). Such a dual-label approach to detect collagen IV and bFn in the matrix of the same monolayer avoided a potential antibody cross-reactivity with rabbit anti-bFn and rabbit anti-bovine collagen IV.

Simultaneous detection of hFn and alpha 5beta 1-Fn integrins. An important assumption with regard to the endothelial monolayer on a Fn-rich ECM is that cell-surface integrins, especially alpha 5beta 1-Fn integrins, located primarily on the basal surface of such adherent cells, are bound or colocalized with Arg-Gly-Asp (RGD) sites in Fn fibers in the matrix. Such colocalization should take place within focal contact regions (3). Whether there would be reduced colocalization after TNF-alpha exposure is not known. In the present study, the cells were pulsed before confluence with hFn, the rapid covalent incorporation of which into the ECM allowed for the simultaneous staining of Fn within the matrix and cell-surface alpha 5beta 1-integrins. The monolayers were again treated with TNF-alpha for 18 h and then prepared for dual-label immunofluorescence. On the same monolayers, hFn was detected with an FITC-conjugated goat anti-hFn (1:100), and cell-surface alpha 5beta 1-integrins were detected with both the mouse monoclonal antibody to human alpha 5beta 1-integrin (1:500) and a secondary antibody that was an RITC-conjugated goat anti-mouse IgG (1:100).

hFn preparation. Purified hFn was provided to our laboratory in a pasteurized and lyophilized form (RHCG, USV/Armour Pharmaceuticals). This hFn preparation has been previously used for both in vitro and in vivo studies, including a phase II clinical study in septic and nonseptic surgical patients (22, 24). It enhances the phagocytosis of gelatin-coated particles by macrophages (22, 24) and retains its ability to be rapidly incorporated into the ECM of the lung and other tissues after intravenous injection (22, 24). It will also incorporate into the subendothelial matrix of substrate-attached lung endothelial cells in culture (37) as well as into the ECM of tissue slices (18).

Iodination of hFn. hFn was iodinated with the chloramine T method. Fn was iodinated by mixing 400 µl of Fn (1 mg/ml in 0.2 M phosphate buffer, pH 7.4) with 1 mCi of Na125I and 35 µl of chloramine T (4 mM in 0.4 M phosphate buffer, pH 7.4). After a 1-min reaction time, 35 µl of sodium metabisulfite (8 mM in PBS) were added to stop the reaction. The mixture was then applied over a G-25 gel-filtration column (PD-10, Pharmacia, Sweden) to remove free Na125I. The peak fractions contained radiolabeled Fn that was intact as verified by SDS-PAGE and autoradiography. The 125I-Fn was then dialyzed against PBS and stored at -80°C until use.

Labeling the Fn matrix. The experimental protocol for developing a prelabeled Fn matrix is shown in Fig. 1. In essence, 125I-hFn was added to the culture medium of endothelial cells, which then incorporated Fn into their matrix. In this protocol, CPAE cells were seeded at a concentration of 140,000 cells/well in a 24-well plate in the presence of 1-2 µg of 125I-Fn (specific activity ~1 × 106 counts · min-1 · µg-1). The cells were seeded, and the tracer was added in MEM containing 20% FBS that was Fn deficient. Fn in the FBS was removed by affinity chromatography with gelatin-Sepharose. After 48 h, the wells were washed extensively with PBS to remove unbound labeled Fn. We then added MEM containing normal 20% FBS (containing ~5 µg bFn/well) to the wells for 24 h to chase all of the labeled soluble Fn into the deoxycholate (DOC)-insoluble pool (pool II), which reflects Fn incorporated into the ECM. After the chase period, ~90% of the radiolabeled Fn was in pool II. Medium alone or medium containing TNF-alpha (200 U/ml) was then added to the washed and chased endothelial monolayers for 18 h.


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Fig. 1.   Protocol to prelabel fibronectin (Fn) in matrix of calf pulmonary arterial epithelial (CPAE) monolayers. CPAE cells were seeded in presence of 1-2 µg of soluble 125I-Fn and incubated for 48 h to allow incorporation of 125I-Fn into their extracellular matrices. Cell layers were then washed, and unlabeled Fn was added for 24 h to chase residual-bound but not incorporated 125I-Fn into pool II. Thereafter, cell layers were exposed to either medium or medium containing tumor necrosis factor (TNF)-alpha (200 U/ml) for 18 h. Conditioned medium and cell layer-matrix from both control and TNF-alpha -treated monolayers were then analyzed. DOC, deoxycholate.

After the incubation, the content of Fn in the medium as well as the potential presence of Fn fragments was analyzed by first counting all medium samples for 125I activity and then analyzing them with SDS-PAGE and autoradiography. The medium was also analyzed for free 125I after TCA precipitation. The cell layers were then washed three times with ice-cold PBS and solubilized in boiling SDS-sample buffer (reduced and nonreduced) and also analyzed by SDS-PAGE and autoradiography. To allow for comparison among the wells, a constant volume was loaded on the gel from each well. All gels were performed at least in triplicate, with one representative gel shown. The cell layers were also evaluated for 125I-Fn content in both pool I and pool II (as defined in Pool I and pool II determination). Radioactivity was measured with a TM Analytic 1193 Gamma Trac gamma counter.

Pool I and pool II determination. Washed endothelial monolayers were extracted for 15 min with 1% DOC in 0.02 M Tris (pH 8.3) containing 2 mM phenylmethylsulfonyl fluoride, 2 mM EDTA, 2 mM N-ethylmaleimide, and 2 mM iodoacetic acid. They were then scraped with a rubber policeman and centrifuged (35,000 g) at 4°C for 30 min. Both the supernatant and pellet were evaluated for radioactivity. In accordance with previous studies (12, 18), the soluble 125I-Fn in the culture medium as well as the 125I-Fn bound to the cell layer will be the DOC-soluble fraction (pool I), whereas 125I-Fn actually covalently incorporated into the ECM is contained within pool II.

Gel electrophoresis and immunoblotting. SDS-PAGE was performed according to Laemmli (10) with 4-15% gradient gels. Samples were diluted 1:1 in gel buffer (4% SDS and 20% glycerol in 0.125 M Tris, pH 6.8) either with or without 5% beta -mercaptoethanol. For immunoblotting, proteins were transferred to nitrocellulose with a Bio-Rad transblot apparatus. Blots were then incubated with a blocking buffer (3% BSA and 0.5% Tween 20 in Tris-buffered saline, pH 7.4) for 1 h and then immunoblotted with the primary antibody (rabbit anti-bFn, 1:100,000). The blots were washed three times with Tris-buffered saline and 0.5% Tween 20 followed by a 1:100,000 dilution of goat anti-rabbit peroxidase-linked secondary antibody. The immunoblots were developed with enhanced chemiluminescence (Super Signal Ultra Substrate, Pierce, Rockford, IL), and one representative immunoblot (out of three) is shown.

Zymography. To determine the presence of proteinases capable of degrading Fn, we used Fn-substrate zymography to analyze both the conditioned medium and the cell layer or ECM from the endothelial monolayers treated with either medium alone or medium containing TNF-alpha . Fn was added to the standard Laemmli (10) acrylamide polymerization mixture at a final concentration of 1 mg/ml. Nondiluted culture medium or the residual cell layer and ECM were mixed 1:1 with gel sample buffer (0.125 M Tris-4% SDS-20% glycerol, pH 6.8) and electrophoresed through 10% polyacrylamide gels. The gels were then soaked in 2.5% Triton X-100 with gentle shaking for 90 min at room temperature, with two changes of the detergent solution. The gels were incubated overnight (16 h) at 37°C in the substrate buffer (50 mM Tris · HCl buffer, pH 7.4, and 10 mM CaCl2) with or without the addition of proteinase inhibitors to block the potential Fn degradation process. The gels were stained with Coomassie blue to allow for visualization of areas of Fn degradation. The Fn zymograms were also performed at least in triplicate.

Protein permeability with the dual-chamber monolayer system. The dual-chamber monolayer technique was used to evaluate endothelial protein permeability as previously described (4, 38). This technique allows for measurement of transendothelial albumin flux in the absence of hydrostatic or oncotic pressure gradients (4). Briefly, CPAE cells were seeded (75,000 cells/well) on a tissue culture-ready filter (6.5-mm diameter, 0.4-µm pore size; Nuclepore Polycarbonate Membrane, Costar, Cambridge, MA) in the upper luminal chamber. To both the luminal and abluminal chambers, BSA containing MEM solution was added, but the 125I-albumin tracer was added only to the top luminal chamber. To measure albumin clearance, 400-µl aliquots of medium were collected from the abluminal chamber every 5 min over a 60-min interval and assayed for 125I radioactivity with a gamma counter. The clearance, expressed in microliters per minute, was determined by weighted least squares nonlinear regression for all 5-min experimental time intervals over the 50- to 60-min test period.

Radiolabeled albumin. Bovine albumin (Sigma) was iodinated with Na125I with the chloramine T method. Five millicuries of 125I were combined with 100 mg of albumin. The 125I-albumin was maintained in dialysis against PBS (pH 7.4) until used. The ratio of free to protein-bound 125I was determined by using Centricon 30 microconcentrator filters (Amicon), and only preparations that had <0.5% free 125I were used.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

The data in Fig. 2, obtained with the use of monoclonal antibodies to the alpha 5beta 1-complex, document the existence of alpha 5beta 1-Fn integrins on the pulmonary endothelial cells in culture in focal adhesion structures (Fig. 2A) as well as the presence of a fibrillar Fn subendothelial matrix (Fig. 2B). An 18-h exposure of CPAE monolayers to TNF-alpha (200 U/ml) caused both a reorganization of the fibrillar Fn matrix (Fig. 2E) and a marked reduction in the immunofluorescent detection (red staining), indicative of alpha 5beta 1-integrins within focal contact-like structures on many of the cells (Fig. 2D). Figure 2, C and F, are the computer-generated overlapping images of both fields, confirming extensive colocalization of the alpha 5beta 1-integrins with the fine Fn fibers in the matrix (yellow staining) of normal monolayers (Fig. 2C) but reduced association after TNF-alpha (Fig. 2F). Such fluorescent images, which were observed repeatedly in separate studies, are consistent with the possibility that TNF-alpha may disturb the interaction of endothelial cell-surface alpha 5beta 1-Fn integrins with Fn fibers in the matrix.


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Fig. 2.   Effect of TNF-alpha treatment on organization of both Fn in matrix and alpha 5beta 1-Fn-binding integrin. Confluent endothelial cells were treated with either medium (A-C) or medium containing TNF-alpha (200 U/ml; D-F) for 18 h. Cell layers were fixed, permeabilized, and processed for dual-label immunofluorescence to detect localization of both Fn and alpha 5beta 1-Fn-binding integrin in the same cell monolayers. A and D: alpha 5beta 1-integrin immunofluorescence. B and E: Fn matrix immunofluorescence. C and F: computer overlay of alpha 5beta 1-integrin and Fn matrix immunofluorescence. TNF-alpha exposure (F) caused a reorganization of matrix Fn and loss of alpha 5beta 1-Fn-binding integrin from focal contact-like structures compared with control (C). Diffuse red punctate staining of a less dense pattern (D and F) reflects presence of endothelial cells with either a diffuse distribution of integrins on their surface or increased staining of those integrins that had been endocytosed.

To determine whether the rearrangement of Fn in the matrix of CPAE monolayers was specific to Fn, we examined the organization of another major matrix protein found in the ECM of pulmonary endothelial cell matrices, i.e., collagen IV (Fig. 3). Confluent endothelial monolayers were treated with medium alone or medium containing TNF-alpha (200 U/ml) for 18 h, after which time the cell layers were fixed, permeabilized, and processed for dual-label immunofluorescence to examine both Fn and collagen IV in the ECM in the same cells. As shown in Fig. 3, after treatment with TNF-alpha , there was a dramatic reorganization and/or disruption of Fn in the matrix, whereas collagen IV appeared to be organized in large bands both before and after TNF-alpha treatment.


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Fig. 3.   Effect of TNF-alpha treatment on organization of Fn and collagen IV in extracellular matrix. Confluent endothelial cells were treated with either medium (A and B) or medium containing TNF-alpha (200 U/ml; C and D) for 18 h. Cell layers were fixed, permeabilized, and processed for dual-label immunofluorescence to detect localization of both Fn (A and C) and collagen IV (B and D) in the same cell monolayers. Cell layers were viewed by immunofluorescence microscopy to detect Fn or collagen IV in matrix.

To determine whether the reorganization of matrix Fn after TNF-alpha was due to proteolysis of the matrix-localized Fn, we used CPAE monolayers with subendothelial matrices preloaded with small tracer doses (1-2 µg) of soluble 125I-hFn (Fig. 4). To initially verify the model, we first documented that the radiolabeled soluble hFn added to the medium as a tracer would be incorporated into the ECM and that this newly incorporated hFn would also be rearranged after TNF-alpha exposure similar to endogenous bFn. Accordingly, dual-label immunofluorescence was utilized to examine the organization of both endogenous bFn and preloaded hFn after TNF-alpha exposure. As shown in Fig. 4, the soluble hFn tracer added to the culture medium of the CPAE monolayer did incorporate into the ECM (Fig. 4B) where it colocalized in a fibrillar pattern with endogenous bFn (Fig. 4A). Moreover, as predicted, both endogenous bFn (Fig. 4A) and the newly added hFn already incorporated into the ECM (Fig. 4B) became rearranged in the disrupted pattern after exposure to TNF-alpha (Fig. 4, C and D), validating that the hFn tracer behaved in a manner similar to endogenous bFn.


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Fig. 4.   Colocalization and parallel reorganization of 125I-human Fn (hFn) with endogenous bovine Fn (bFn) after TNF-alpha exposure. CPAE cells were seeded in presence of 1-2 µg of soluble 125I-hFn (tracer) similar to protocol in Fig. 1. Confluent CPAE monolayers were then treated with either medium or medium containing TNF-alpha (200 U/ml) for 18 h. Cell layers were fixed, permeabilized, and processed for dual-label immunofluorescence to detect localization of both endogenous bFn (A and C) and tracer hFn (B and D) in the same cell monolayers in the same field. A and B: Fn matrix of control monolayers. C and D: Fn matrix of TNF-alpha -treated monolayers. TNF-alpha exposure causes an identical reorganization and/or disruption of both bFn and hFn in subendothelial matrix.

It should be noted that in the above experiment (Fig. 4), only tracer (1-2 µg) doses of 125I-hFn were used, not the typical 300- to 600-µg hFn dose used to attenuate or normalize the protein permeability after TNF-alpha exposure (5, 37, 38). If the larger treatment hFn dose is used, then the deposition pattern of the added plasma Fn within the ECM appears, in part, dependent on when hFn is added to the culture medium in relationship to the TNF-alpha challenge. If soluble hFn is added at -18 h (with the TNF-alpha ), then much of the added hFn also is reorganized in a disrupted pattern by 12-18 h, like endogenous bFn (5), because it is rapidly incorporated into the ECM, even within 2-3 h. Under these circumstances, it appears difficult to visualize by immunofluorescence the smaller portion of hFn incorporated within the ECM in a fibrillar pattern because of the intense background of a disrupted Fn matrix. However, we observed that if hFn is added 12 h after the addition of TNF-alpha , when endogenous bFn is already reorganized or disrupted, then 6 h later when the permeability is lowered, the added hFn can be readily visualized (37) in a fibrillar deposition in the ECM.

CPAE monolayers with a prelabeled Fn matrix were then exposed to TNF-alpha (200 U/ml) for 18 h, and both radiolabeled Fn released into the medium and the presence of either intact or fragmented Fn in the medium were analyzed with SDS-PAGE and autoradiography. The amount of 125I-Fn in the medium of both control and TNF-alpha -treated monolayers was not significantly different (Fig. 5A). Also, analysis of medium samples diluted with 12% TCA to allow for separation of 125I-bound Fn from free 125I indicated similar amounts of free 125I in the control and TNF-alpha -treated monolayers (control, 12.0 ± 0.46%; TNF-alpha , 8.6 ± 0.38%). In addition, SDS-PAGE and autoradiographic analysis (Fig. 5B) showed that Fn present in the medium from both control and TNF-alpha -treated monolayers was not different. Thus exposure of the monolayer to TNF-alpha did not cause any significant release of Fn fragments into the culture medium.


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Fig. 5.   Analysis of 125I-Fn recovered from medium after TNF-alpha exposure. CPAE monolayers were treated with either medium or medium containing TNF-alpha (200 U/ml) for 18 h. Medium was removed, and its 125I-Fn content was determined isotopically (A). Values are means ± SE. Medium was also analyzed by SDS-PAGE and autoradiography (B). A constant volume of medium was taken from control (C) and TNF-alpha -treated (T) cells and run under nonreducing (NR) or reducing (R) conditions. Amount of 125I-Fn released into medium and level of Fn fragments in medium were both unaffected by TNF-alpha exposure for 18 h. CPM, counts/min.

We next analyzed the relative amount of 125I-Fn associated with either the cell layer (pool I) or incorporated in the subendothelial matrix (pool II) after TNF-alpha exposure (18 h). To our surprise, after an 18-h exposure to TNF-alpha (200 U/ml), there was no significant decrease in either the amount of 125I-Fn bound to the cell layer (pool I) or the 125I-Fn incorporated into the ECM (pool II; Fig. 6A). Also, SDS-PAGE and autoradiographic analysis showed that TNF-alpha exposure caused no Fn fragments either in the cell layer or within the ECM (Fig. 6B). To verify that our autoradiographic technique was sensitive enough to detect Fn fragments if they existed, parallel experiments were done after leukocyte elastase, a proteinase known to degrade Fn, was added to the endothelial monolayers. Leukocyte elastase (2 × 10-4 U/ml) added for 18 h caused both the release of Fn fragments and a loss of Fn from the cell layer-matrix as expected (Curtis and Saba, unpublished data). These findings indicated that TNF-alpha exposure caused a reorganization of Fn in the matrix without an actual loss of Fn from the matrix or any obvious proteolysis of Fn incorporated within the ECM.


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Fig. 6.   Analysis of 125I-Fn incorporated in subendothelial matrix or associated with cell layer after TNF-alpha exposure. CPAE monolayers were treated with either medium alone or medium containing TNF-alpha (200 U/ml) for 18 h. Amount of 125I-Fn in pool I and pool II was quantitated (A). Values are means ± SE. Cell layer and matrix after TNF-alpha treatment was also analyzed by SDS-PAGE and autoradiography under NR and R conditions (B). As a reference control, an aliquot of starting material (SM) that was used to prelabel Fn matrix was shown to be intact. After an 18-h TNF-alpha exposure, there was no difference in amount of 125I-Fn either associated with cell layer or in matrix compared with control cells. Level of Fn fragments associated with cell layer or in matrix was similar in both control and TNF-alpha exposed cells. Exposure to leukocyte elastase (2 × 10-4 U/ml) for 18 h caused both release of Fn fragments and a reduced Fn content in cell layer, especially ECM pool II.

SDS-PAGE and immunoblotting were also used to examine the endogenous bFn after treatment with TNF-alpha . Confluent monolayers were treated with either medium alone (control) or medium containing 200 U/ml of TNF-alpha for 18 h. Then, either the medium (Fig. 7A) or the resulting cell layer and matrix (Fig. 7B) were analyzed by SDS-PAGE and immunoblotting with an antibody specific to bFn. As predicted, high-molecular-weight Fn multimers located in the stacker portion of the gel, as previously shown (Fig. 6), could not be transferred and therefore visualized during immunoblotting. TNF-alpha did not cause any change in the amount of Fn detected within the medium or cell layer-matrix (Fig. 7). Thus exposure of the CPAE monolayer to 200 U/ml of TNF-alpha for 18 h does not appear to cause any major release of Fn fragments from the matrix into the culture medium or significant proteolysis of Fn already incorporated in the ECM.


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Fig. 7.   Molecular mass of endogenous bFn detected in either conditioned medium (A) or cell layer-matrix (B) after treatment with medium (control) or medium containing TNF-alpha (200 U/ml) for 18 h. Both medium and resulting cell layer-matrix (constant volume) were analyzed by SDS-PAGE under either NR or R conditions. Gels were transferred to nitrocellulose and immunoblotted with an anti-bFn antibody. There was no difference in amount of Fn found in medium or cell layer-matrix of control or TNF-alpha -treated monolayers. Also, no additional fragments were found in medium or cell layer-matrix after treatment with TNF-alpha .

We then employed sensitive Fn-substrate zymography to determine whether Fn-degrading proteinases were increased in both the medium and the associated cell layer-ECM after TNF-alpha . Confluent CPAE cells were first treated with either medium alone (control) or medium containing 200 U/ml of TNF-alpha , and after 18 h, an equal volume of medium or cell layer-matrix was analyzed by Fn-substrate zymography. As shown in Fig. 8A, TNF-alpha treatment caused an increased expression in proteinase bands in the medium, with two distinct molecular weights. These two proteinase bands were also present in the cell layer-matrix, but their activity was not changed with TNF-alpha (Fig. 8B). They were likely serine proteinases because their degradative activity was completely inhibited by aprotinin (4 µg/ml) but not by EDTA (20 mM), 1,10-phenanthroline (0.1 mM), leupeptin (8 µg/ml), or pepstatin (0.4-1.2 µg/ml), which have inhibitory activities toward metalloproteinases (EDTA and 1,10 phenanthroline), trypsin-like and cysteine proteinases (leupeptin), and aspartic proteinases (pepstatin; Fig. 9).


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Fig. 8.   Fn proteolytic activity in medium (A) or associated with cell layer-matrix (B) as detected by Fn-substrate zymography. Confluent endothelial monolayers were treated with medium alone (control) or medium containing TNF-alpha (200 U/ml) for 18 h. A: after incubation interval was complete, either 5, 10, 15, or 20 µl of medium were analyzed with Fn-substrate zymography. B: 40 µl of cell layer-matrix were analyzed with Fn-substrate zymography under either control or TNF-alpha treatment conditions. TNF-alpha exposure causes an increase in Fn proteolytic activity found in medium.


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Fig. 9.   Effect of various proteinase inhibitors on proteolytic activity associated with medium or cell layer-matrix as analyzed by Fn-substrate zymography. Either 20 µl of medium (A) or 40 µl of cell layer-matrix (B) from TNF-alpha -treated cell monolayers were analyzed by Fn-substrate zymography in presence of various proteinase inhibitors. Activation of proteinases in the gel was done by a 16-h incubation at 37°C in a Tris buffer containing CaCl2. Gels were incubated with either protease activation mixture alone (TNF-alpha ) or activation mixture in presence of various proteinase inhibitors such as aprotinin (serine protease inhibitor), EDTA and 1,10-phenanthroline (metalloproteinase inhibitors), leupeptin (trypsin-like and cysteine proteinase inhibitor), and pepstatin (aspartic proteinase inhibitor). Serine proteinase inhibitor aprotinin inhibited both proteolytic bands detected in Fn-substrate gels. Fn zymograms were also performed at least in triplicate, and 1 representative zymogram is shown.

Because increased protease activity obviously existed, we then determined whether the TNF-alpha -induced rearrangement of the Fn matrix could be blocked by the serine proteinase inhibitor aprotinin. To our surprise, the TNF-alpha -induced Fn matrix reorganization was identical (Fig. 10) in the absence and presence of aprotinin (4 µg/ml), further supporting the conclusion that the reorganization of the Fn matrix was likely not dependent on Fn proteolysis. Actually, a dose-response study was performed with aprotinin at concentrations as high as 12 µg/ml, but, even at this higher dose, aprotinin had no ability to block the TNF-alpha -induced matrix reorganization (micrographs not shown). We also observed that the TNF-alpha -induced increase in endothelial protein permeability as measured by 125I-albumin clearance was also not blocked by the serine proteinase inhibitor aprotinin (Fig. 11). Indeed, the typical 300-400% increase in protein permeability in response to TNF-alpha was seen (Fig. 11) in both the absence and presence of aprotinin (4 µg/ml), suggesting that the permeability increase was also being mediated by a process independent of the proteolysis of Fn incorporated with the ECM.


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Fig. 10.   Effect of proteinase inhibitor aprotinin on TNF-alpha -induced Fn matrix reorganization. Confluent monolayers were incubated for 18 h with medium alone (-Aprotinin; A), aprotinin (+Aprotinin; 4 µg/ml; B), TNF-alpha (200 U/ml; C), or TNF-alpha +aprotinin (D). Cells were fixed and permeabilized, and bFn matrix was stained with rabbit antiserum to bFn and rhodamine isothiocyanate-conjugated goat anti-rabbit IgG. TNF-alpha -induced disruption of Fn matrix was not prevented by aprotinin.


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Fig. 11.   Effect of proteinase inhibitor aprotinin on TNF-alpha -induced increase in endothelial monolayer protein permeability. Confluent endothelial monolayers were treated for 18 h with medium alone (control), aprotinin (4 µg/ml), TNF-alpha (200 U/ml), or TNF-alpha +aprotinin. Values for protein clearance are means ± SE with 12-24 wells/group. TNF-alpha -induced increase in protein permeability was not prevented by addition of aprotinin to culture medium.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

The inflammatory cytokine TNF-alpha has been shown to cause an increase in the protein permeability of lung endothelial monolayers and a dramatic disruption of the fibrillar Fn in the ECM (5, 37). Also, the addition of soluble RGD peptides [but not Arg-Gly-Glu (RGE) peptides] or antibodies to alpha 5beta 1-integrins to the culture medium of endothelial monolayers can increase their protein permeability and rearrange the Fn in the ECM similar to the pattern observed with TNF-alpha (5), suggesting that the TNF-alpha -induced decrease in endothelial mono-layer integrity and rearrangement of the Fn-rich matrix may be mediated by a change in the ability of the cells to adhere to Fn in the matrix. The effect of these agents on the protein permeability of lung endothelial monolayers could be initiated by proteinase modification of the matrix Fn because TNF-alpha causes the release of both uPA and the metalloproteinases gelatinase B and stromelysin-1 from endothelial cells (8, 14, 16, 32).

RGD peptides themselves as well as anti-alpha 5beta 1-integrins have the ability to potentially stimulate protease secretion from the endothelial cells. Werb et al. (36) demonstrated that the adhesion of fibroblasts to immobilized Fn peptides or to immobilized antibody against the alpha 5beta 1-integrin induced both collagenase and stromelysin gene expression, whereas adhesion of fibroblasts to intact Fn did not induce this response. They suggested that intact Fn may signal the cell differently than either an antibody to the alpha 5beta 1-receptor or RGD peptides. In the present study, we could not detect evidence of Fn proteolysis after TNF-alpha treatment, suggesting that the TNF-alpha -induced increase in endothelial protein permeability may be due to a change in the ability of cell-surface integrins to bind to matrix Fn. This alternate mechanism is supported by our observed change in the surface distribution of the alpha 5beta 1-integrin after TNF-alpha exposure (Fig. 2), indicative of reduced interaction or actual dissociation of many of the cell-surface alpha 5beta 1-Fn integrins with Fn fibers located in the subendothelial matrix.

In blood vessels, the ECM underlying the endothelium supports cell attachment, spreading, migration, and proliferation. After injury, it may be the composition and organization of the ECM that is directing the responses of the endothelial cells (11). In response to binding an immobilized ligand, such as Fn within the ECM, many integrins will cluster and become immobilized within focal adhesion complexes due to binding interactions with actin-associated cytoskeletal proteins (2). The structural link among ECM, integrins, and the actin cytoskeleton stabilizes cell adhesion and also provides the mechanical basis by which integrins can contribute to endothelial shape and associated endothelial barrier integrity. Changes in the ability of the cells to bind to the ECM results in cytoskeletal reorganization and global changes in cell shape (13, 25). Some insight into the mechanism by which changes in adherence to the ECM promotes coordinated changes in cell shape is apparent from the findings of Sims et al. (25). They showed that changes in cell shape can be caused by cytoskeletal tension generated by an actomyosin filament sliding mechanism, which is physically resisted or opposed by the binding of cell-surface integrins to immobilized adhesion sites within the ECM. These studies suggest that the ability of the integrins, such as alpha 5beta 1-Fn integrin, to bind to attachment sites with the ECM may be what controls cell shape by physically resisting the resident or ongoing cytoskeletal tension.

Fn exists in two general forms: soluble Fn in the plasma or lymph fluid and insoluble Fn (plasma or cellular) found in the ECM (22, 23). Plasma Fn can incorporate into the tissue ECM and codistribute with locally produced cellular Fn (22, 23). In the lung, Fn has been localized to the subendothelial matrix, under epithelial cells, and in the interstitial matrix (28). The release of Fn from the lung ECM has been associated with lung vascular injury (6, 17, 20, 33). For example, Peters et al. (17) documented the release of intact cellular Fn from the matrix of isolated perfused rabbit lungs after oxidant- or leukocyte-induced vascular injury. Release of intact extra domain (ED)-rich cellular Fn from the perfused rabbit lung after oxidant-induced vascular injury is not prevented by prior inhibition of protein synthesis in the lung (33). Moreover, postoperative gram-negative bacterial infusion in sheep, which elicits an increase in lung protein permeability, also causes the rapid release of matrix-localized ED1-containing Fn from the lung interstitial ECM into the postnodal lymph (20). In essentially all of these in vivo experiments, very few Fn fragments could be detected. This suggests that the loss of lung barrier integrity in vivo coupled with the large water flux across the endothelial barrier may have facilitated the washout of intact Fn from the disrupted interstitial matrix by a process unrelated to Fn proteolysis, or, because soluble Fn can be rapidly assembled and/or incorporated into the ECM (5, 12, 18), perhaps there is a rapid matrix Fn disassembly process activated by TNF-alpha .

The exposure of lung endothelial monolayers to TNF-alpha for 18 h did not change the content or molecular weight of the Fn recovered from the conditioned medium or the cell layer-ECM. It should be noted that Stolpen et al. (27), using human umbilical vein endothelial cells, showed a loss of Fn from the matrix by immunofluorescent techniques, but this loss of stainable Fn was not apparent until 72-96 h after TNF-alpha treatment, a time course much greater than that used in the present study. Partridge and colleagues (15, 16) used SDS-PAGE analysis (under reducing conditions) and reported loss of a 220-kDa band (Fn monomer) after treatment of microvessel endothelial cells with TNF-alpha , but loss of the Fn monomer did not correspond with any increase in Fn fragments. Additionally, because the high-molecular-weight multimers of matrix Fn, which are known to be retarded within the stacking portion of the gel, were not shown, the loss of Fn monomers after TNF-alpha may simply be due to an increase in high-molecular-weight Fn multimers resistant to reducing agents.

Niedbala and Picarella (14) showed that TNF-alpha treatment of human umbilical vein endothelial cells (low passage) caused proteolysis of a [3H]glucosamine-labeled ECM as measured by the release of radioactivity into the medium over time. This TNF-alpha -mediated ECM degradation was plasminogen dependent and could be inhibited by an anticatalytic uPA monoclonal antibody. Plasmin can degrade multiple ECM components, so the relationship of this finding to any potential proteolysis of matrix Fn can only be speculated. The present study, which is the first extensive analysis of the content, molecular weight, and organization of Fn in the subendothelial matrix after treatment with the cytokine TNF-alpha , suggests that TNF-alpha alters the organization of Fn in the matrix and increases the protein permeability of the endothelial cell monolayers independent of Fn proteolysis.

Proteinase activity against Fn was readily detected in both the medium and cell layer-matrix, and such proteolytic activity was increased in the medium after TNF-alpha treatment. The proteinase activity detected in the Fn zymogram was likely plasmin because the bands detected had a molecular mass of ~90 kDa, and their Fn-degrading activity was inhibited by aprotinin. However, because of the numerous inhibitors in the serum, it is unlikely that any additional plasmin released into the culture medium would have had any significant degradative activity against the Fn found in the subendothelial matrix. It is possible that the aprotinin we added did not have access to cell-associated plasmin, but we added a large concentration of aprotinin over a long time period. Because aprotinin is 6.5 kDa, we did not have reason to believe that it would be limited in gaining access to the basolateral cell surface even in control conditions. In the TNF-alpha treatment conditions, the cells lose their cell-cell contacts so aprotinin would have full access. Using the same confluent endothelial monolayers, we have already shown that the addition of antibodies to alpha 5beta 1-integrins can increase endothelial protein permeability in 18 h. The fact that the antibody to alpha 5beta 1-integrin, which is very large compared with aprotinin, has access to the basolateral surface is proof to us that aprotinin (6.5 kDa) can get to these sites, especially after TNF-alpha . We also measured plasmin activity after either TNF-alpha alone or TNF-alpha coupled with aprotinin. We observed that aprotinin decreased the plasmin activity exhibited after TNF-alpha (Curtis and Saba, unpublished data), consistent with previous findings (26) that aprotinin inhibits both fluid-phase and cell-surface plasmin activity. However, aprotinin was unable to block the TNF-alpha effect on the monolayer. Collectively, such data suggest that an aprotinin-sensitive proteinase did not cause the TNF-alpha -induced Fn matrix disruption or the increase in protein permeability that we observed.

Disruption of the subendothelial Fn matrix after TNF-alpha exposure as reflected by the condensation of the Fn matrix into thicker bands or aggregates of fibers was repeatedly observed. TNF-alpha treatment of endothelial cells did not appear to cause any major reorganization of collagen IV, suggesting that Fn matrix reorganization and/or disruption was specific. Although this response appears to be independent of Fn proteolysis, it could potentially be explained by an altered mechanical balance between the ECM and the cytoskeleton in accordance with the concept of Wang and colleagues (34, 35).

Although disruption of the Fn matrix after TNF-alpha exposure appears not to be due to a proteolytic attack of Fn already incorporated within the ECM, the functional relationship of the matrix Fn disruption to the observed increase in transendothelial protein permeability remains to be clarified. Both RGD peptides and antibodies to alpha 5beta 1-integrin can each increase the protein permeability of CPAE monolayers as well as cause a disruption of the Fn matrix identical to those seen after TNF-alpha exposure (5, 37, 38). Moreover, the soluble plasma Fn added to the culture medium of lung endothelial monolayers can incorporate into the ECM and attenuate the increase in protein permeability observed with all three agents. This suggests that the changes caused by RGD peptides, alpha 5beta 1-integrin antibodies, or TNF-alpha may be mediated by a similar mechanism. Because the ECM can indirectly modulate endothelial barrier function by influencing endothelial cell shape, cell spreading, and cell-matrix adhesion due, in part, to the binding of integrins to matrix molecules, it would appear that reduced interaction of alpha 5beta 1-Fn integrins with Fn fibers in the matrix may be the basis for the response we observed. The previous findings of Curtis et al. (5) of enhanced cell spreading and a decrease in endothelial protein permeability after matrix incorporation of added soluble Fn coupled with the present data indicating that proteolysis is not mediating the response support this conclusion.

    ACKNOWLEDGEMENTS

The secretarial assistance of Debbie Moran is much appreciated.

    FOOTNOTES

This study was supported primarily by National Institute of General Medical Sciences Grant GM-21447 and in part by National Cancer Institute Grant CA-69612.

T. M. Curtis was a predoctoral trainee in the Department of Physiology and Cell Biology (Albany Medical College, Albany, NY), was supported by National Heart, Lung, and Blood Institute Training Grant T32-HL-07194, and is currently a postdoctoral fellow at the University of Virginia (Charlottesville). R. F. Rotundo was a postdoctoral fellow in the Department of Physiology and Cell Biology during these studies, was supported by National Heart, Lung and Blood Institute Training Grant T32-HL-07529, and is currently a Research Associate at Albany Medical College.

Address for reprint requests: T. M. Saba, Dept. of Physiology and Cell Biology (A-134), Albany Medical College, 47 New Scotland Ave., Albany, NY 12208.

Received 21 November 1997; accepted in final form 7 April 1998.

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Discussion
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