Department of Physiology and Cell Biology, Albany Medical College, Albany, New York 12208
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ABSTRACT |
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Exposure of lung
endothelial monolayers to tumor necrosis factor (TNF)- causes a
rearrangement of the fibrillar fibronectin (FN) extracellular matrix
and an increase in protein permeability. Using calf pulmonary artery
endothelial cell layers, we determined whether these changes were
mediated by FN multimerization due to enhanced transglutaminase
activity after TNF-
(200 U/ml) for 18 h. Western blot analysis
indicated that TNF-
decreased the amount of monomeric FN detected
under reducing conditions. Analysis of 125I-FN
incorporation into the extracellular matrix confirmed a twofold increase in high molecular mass (HMW) FN multimers stable under reducing conditions (P < 0.05). Enhanced formation of
such HMW FN multimers was associated with increased cell surface
transglutaminase activity (P < 0.05). Calf pulmonary
artery endothelial cells pretreated with TNF-
also formed
nonreducible HMW multimers of FN when layered on surfaces precoated
with FN. Inhibitors of transglutaminase blocked the TNF-
-induced
formation of nonreducible HMW multimers of FN but did not prevent
either disruption of the FN matrix or the increase in monolayer
permeability. Thus increased cell surface transglutaminase after
TNF-
exposure initiates the enhanced formation of nonreducible HMW
FN multimers but did not cause either the disruption of the FN matrix
or the increase in endothelial monolayer permeability.
tumor necrosis factor-; endothelial cells
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INTRODUCTION |
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TUMOR NECROSIS
FACTOR (TNF)- is a major inflammatory cytokine released from
macrophages and monocytes activated during inflammation and/or
gram-negative infection, especially after trauma, burns, and major
surgery (10, 49). The endothelium is one of
the primary targets for such inflammatory cytokines (31),
and high levels of TNF-
are believed to contribute to altered
integrity of the lung endothelial barrier, resulting in excessive
transendothelial leakage of plasma proteins into the interstitium and
interstitial pulmonary edema (16, 17,
46, 47). Purified TNF-
added to the
culture medium of confluent calf pulmonary artery endothelial (CPAE)
monolayers increases their protein permeability (6, 50, 51); and the intravenous infusion of
TNF-
into postsurgical sheep can also increase lung endothelial
protein permeability (39), very similar to that observed
in postsurgical sheep after Pseudomonas or endotoxin
challenge (7, 8, 11,
14). Such observations have suggested that high
concentrations of TNF-
in the lung microcirculation during pulmonary
inflammation and/or bacterial sepsis after surgery or trauma may
contribute to the loss of lung endothelial integrity
(42).
Studies (16, 50) on the TNF--induced
increase in transendothelial protein permeability with
immunofluorescence microscopy have confirmed a dramatic disruption
and/or reorganization of the fine fibrillar fibronectin (FN) network in
the subendothelial extracellular matrix (ECM) of lung endothelial cell
monolayers exposed to TNF-
. This includes an apparent aggregation of
FN in the matrix temporally associated with both an increase in protein permeability and the formation of gaps between cells in previously confluent monolayers (16, 17,
51). A very similar disruption of the FN matrix is seen
after the addition of either monoclonal antibodies to the
5
1-integrin complex or soluble
Arg-Gly-Asp (RGD)-containing peptides to the culture medium
(16, 17).
Addition of endotoxin-free and highly purified human plasma FN to the
culture medium can both attenuate and reverse the TNF--induced increase in endothelial monolayer protein permeability, a protective response that appears to be dependent on the incorporation of the added
soluble FN into the ECM (16, 17,
51). Similarly, the intravenous infusion of such
commercially purified human FN into bacteremic sheep after surgical
trauma to significantly elevate the circulating plasma FN concentration
(by 20-40%) can also attenuate the increase in lung protein
permeability once the infused human FN becomes incorporated into the
lung interstitial ECM (11, 14,
40). Although disruption of the FN subendothelial matrix after TNF-
exposure is now well documented, the mechanisms mediating this change in the fibrillar FN matrix are not known. However, an
alteration of the FN matrix has the potential to either reduce endothelial cell adhesion or spreading, interrupt integrin-mediated ECM
signaling to the cell cytoskeleton, or perhaps modify the integrity of
the vascular barrier, including the exclusion properties of the matrix.
The organization of FN in the ECM of lung endothelial cell layers is
influenced by several processes that include its assembly into fine FN
fibrils within the ECM, its turnover potentially mediated by
proteolysis, and the biochemical structuring of the FN lattice within
the ECM. Structuring of FN in the normal ECM is influenced by disulfide
exchange in the amino-terminal domain of FN and perhaps by
transglutaminase-mediated cross-linking of FN based on experiments
primarily with fibroblasts (3, 4). We
determined whether TNF- could induce changes in the properties of FN
within the ECM to explain its influence on both the fine fibrillar
structure of the FN matrix and the protein permeability of previously
confluent endothelial monolayers. In this regard, we tested the concept
that TNF-
may have enhanced extracellular transglutaminase activity,
resulting in FN multimerization leading to disruption of the FN matrix
and increased protein permeability of endothelial monolayers. Our
observations indicate that the total amount of FN in the matrix as well
as the FN mRNA levels were unchanged after TNF-
exposure. Moreover,
we were unable to detect the release of FN fragments from the cell
layer after TNF-
exposure. In contrast, there was a marked increase
in the amount of nonreducible high molecular mass (HMW) multimers of FN
detected in the ECM of the lung endothelial cell monolayers after
TNF-
exposure that was associated with enhanced extracellular endothelial cell surface transglutaminase activity. Blocking such enhanced transglutaminase activity attenuated the TNF-
-enhanced multimerization of the FN matrix in endothelial monolayers but did not
prevent either the rearrangement of the fibrillar FN matrix as analyzed
by immunofluorescence microscopy or the increase in protein
permeability as quantified by transendothelial 125I-albumin clearance.
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MATERIALS AND METHODS |
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Fibroblast and endothelial cell cultures.
CPAE cells (American Type Culture Collection, Manassas, VA)
were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal bovine serum (FBS; HyClone, Logan, UT), 100 U/ml of
penicillin, and 100 µg/ml of streptomycin. Before TNF- treatment, the culture medium was replaced with DMEM supplemented with 5% FBS
plus the antibiotics. For analysis of the effect on TNF-
on the
release of FN from the endothelial cell layers, the CPAE monolayers
were cultured to confluence in 3-4 days and then washed four times
with phosphate-buffered saline (PBS) before the addition of DMEM
containing FN-deficient FBS prepared by gelatin-Sepharose affinity
chromatography (20). A1-F human foreskin fibroblasts (originally obtained from Dr. Lynn Allen-Hoffman, University of Wisconsin, Madison) were cultured in DMEM supplemented with 10% FBS,
100 U/ml of penicillin, and 100 µg/ml of streptomycin. Both fibroblast and endothelial cell layers were exposed to recombinant human TNF-
at a concentration of 200 U/ml medium for 18 h
before analysis.
Phase-contrast and immunofluorescent microscopy.
Endothelial cells cultured on coverslips were treated with and without
200 U/ml of TNF- for 18 h. The cells were examined and
photographed with an inverted microscope (Olympus IX50).
Immunofluorescent staining for FN in the cell layers was performed as
described by Curtis and colleagues (16, 17)
with an Olympus BX60 microscope. CPAE cells cultured on coverslips were
fixed, permeabilized, and stained with a rabbit polyclonal antibody to
bovine FN (Chemicon International, Temecula, CA) and a fluorescein goat
anti-rabbit IgG secondary antibody (Molecular Probes, Eugene, OR).
Enzyme-linked immunosorbent assay and immunoblot analysis. Standard enzyme-linked immunosorbent assay (ELISA), dot blot, and Western blot analysis of FN were done with a rabbit polyclonal antibody to bovine FN (Calbiochem, San Diego, CA) and a secondary antibody of horseradish peroxidase-conjugated goat anti-rabbit IgG (Calbiochem). For ELISA, FN was detected with the 3,3',5,5'-tetramethylbenzidine microwell peroxidase substrate system (Kirkegaard & Perry Laboratories, Gaithersburg, MD). Dot blot and Western blot analyses were performed with a SuperSignal ULTRA chemiluminescent substrate (Pierce, Rockford, IL) with subsequent exposure to Fuji X-ray films (Fuji Medical Systems USA, Stamford, CT).
RNA isolation and Northern blot analysis.
Poly(A)+ RNA was isolated directly from cell extracts of
CPAE cells with the FastTrack kit (Invitrogen, San Diego, CA). RNA samples (1 µg) were electrophoresed on a 1.3% agarose gel containing 0.66 M formaldehyde and transferred to nitrocellulose as described by
Davis et al. (18). DNA probes were radiolabeled with
[-32P]dCTP with the Random Primer Plus Extension
Labeling System (New Life Science Products, Boston, MA), and
hybridization was performed as previously described (19).
Equal loading of mRNA samples on Northern blots was verified by
simultaneous probing with a cDNA specific to mouse
-actin.
Detection of HMW multimers of 125I-FN incorporated in
the matrix of endothelial cells.
FN purified from fresh human plasma by gelatin-Sepharose affinity
chromatography (20, 38) was iodinated as
described by Rebres et al. (38). The iodinated FN was
confirmed to be intact (440 kDa) by SDS-PAGE and autoradiography. At
48 h after CPAE cells were seeded at a density of 10,000 cells/cm2, the medium of the preconfluent cells was changed
to FN-deficient medium supplemented with 3 µg/ml of
125I-FN. After an 18-h incubation with and without TNF-,
the cells were washed four times with PBS and solubilized in SDS-PAGE
sample buffer [0.125 M Tris (pH 6.8), 4% SDS, and 10% glycerol].
Samples with equal radioactivity were analyzed by SDS-PAGE and
autoradiography. Polyacrylamide gradient gels (4-15%) with a 3%
stacking gel were used for PAGE analysis. When analyzed under reducing
conditions, the concentration of
-mercaptoethanol in the sample
buffer was 2.5% (vol/vol). The gels were then dried and exposed to
Kodak Bio-Max films with an intensifying screen. When the effect of transglutaminase inhibitors on the formation of nonreducible HMW FN was
examined, monodansylcadaverine (MDC; Sigma) at 0.1 mM or cystamine
(Cys; Sigma) was added during TNF-
treatment. Both MDC and Cys are
primary amine substrates that have been routinely used to inhibit
transglutaminase activity (24, 41,
44).
Assay of transglutaminase activity in cell lysate. Transglutaminase activity was determined on the basis of [3H]putrescine incorporation into dimethylated casein as previously described by Korner et al. (29). Cells were washed twice with cold PBS, once with Tris-EDTA-dithiothreitol (TED) buffer (50 mM Tris · HCl, pH 7.4, 1 mM EDTA, and 1 mM dithiothreitol) supplemented with 150 mM NaCl (TED-buffered saline), and then scraped into 1 ml TED-buffered saline/dish. The cells were collected by centrifugation; resuspended in 200 µl of TED buffer containing 0.5 mM phenylmethylsulfonyl fluoride, 5 µg/ml of pepstatin, and 5 µg/ml of leupeptin; and then lysed by three cycles of freezing and thawing. The final reaction mixture (120 µl) consisted of 50 mM Tris · HCl, pH 7.4, 10 mM CaCl2, 15 mM dithiothreitol, 0.5 mM (1 µCi) putrescine, 50 mM NaCl, and 0.8 mg of dimethylated casein, and the cell lysates that contained 80-130 µg of total protein. After incubation at 37°C for 9-15 min, the proteins were precipitated with TCA, and free putrescine was removed by passage through a filter paper as described by Korner et al. Each sample had a negative control in which CaCl2 in the reaction mixture was replaced with 50 mM EDTA. Putrescine incorporation, used as a measure of transglutaminase activity (29), was quantified by scintillation counting.
Assay of cell surface transglutaminase activity.
Cell surface transglutaminase activity was specifically determined by
measuring the incorporation of biotinylated cadaverine (Pierce) into FN
on precoated 96-well plates as described by Verderio et al.
(48). Briefly, the plates were first coated with purified human FN at 10 µg/ml. Then endothelial cells that were either not
exposed to TNF- (control) or pretreated with TNF-
(200 U/ml) were
seeded at various densities from 10,000 to 50,000 cells/well and
incubated at 37°C for 3 h in the presence of 0.5 mM biotinylated cadaverine. The reaction was first stopped by rinsing the wells with 2 mM EDTA in PBS, and then the cell layers were removed by incubation for
20 min with 0.1% sodium deoxycholate in PBS at room temperature.
Cadaverine incorporation was quantified with horseradish
peroxidase-conjugated streptavidin (Amersham, Piscataway, NJ) and the
3,3',5,5'-tetramethylbenzidine color-developing kit (Kirkegaard & Perry Laboratories).
Preparation of preformed endothelial cell matrices and assay of
cell adhesion and spreading.
Confluent endothelial monolayers cultured on glass coverslips in
12-well plates were treated for 18 h with either TNF- (200 U/ml) alone or TNF-
in conjunction with the transglutaminase inhibitor Cys (0.05 mM) to obtain two preformed matrices: one in which
the fine fibrillar FN matrix was disrupted or reorganized with the
parallel formation of the HMW FN multimers inhibited (TNF-
and
cystamine treated), and the other in which the FN matrix was disrupted
in parallel with the TNF-
-induced formation of nonreducible HMW FN
multimers (TNF-
treated).
Radiolabeling of albumin and assay of transendothelial protein permeability. Transendothelial protein permeability was assessed by measuring the diffusive protein (125I-albumin) permeability across the endothelial monolayer with a dual-chamber monolayer system as previously described (15-17, 50, 51). This technique allows for the measurement of transendothelial albumin flux in the absence of a hydrostatic or oncotic pressure gradient (15). This in vitro model system consists of a luminal chamber containing the "tissue culture ready" filter that is covered with a confluent monolayer of CPAE cells. A styrofoam collar around the luminal chamber allows it to float in a larger abluminal chamber. This allows the fluid height in both chambers to be maintained at an identical level to eliminate the convective flux of albumin across the monolayers. During each experiment, the fluid in the abluminal chamber was stirred constantly, and both chambers were kept at 37°C with a thermostatically controlled water bath.
Bovine albumin (Sigma) was iodinated with Na125I with the chloramine T method (50, 51). Five millicuries of 125I were combined with 100 mg of albumin. 125I-albumin was maintained in dialysis against PBS at pH 7.4 until used. The ratio of free to protein-bound 125I was periodically analyzed to ensure that the preparations of albumin used had <0.5% free 125I. Calculation of 125I -albumin clearance (in µl/min) was used to measure the changes in the diffusive permeability of albumin across the lung endothelial monolayers as described by Cooper et al. (15). To conduct this procedure, 25 ml of low-glucose DMEM supplemented with 0.5% BSA were added to the abluminal chamber. Medium from the luminal chamber was first changed after exposure of the endothelial monolayer to TNF- ![]() |
RESULTS |
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TNF- causes a decrease in monomeric FN detectable under reducing
conditions.
Previous immunofluorescent studies (16, 50)
revealed a dramatic reorganization of the FN matrix under the
endothelial cell monolayers after TNF-
exposure. The matrix FN
changed from a fibrillar meshwork of fine FN fibers into a matrix with
predominantly thick bundles of FN after TNF-
exposure. To determine
whether this reorganization of the FN matrix was related to a change in the quality and/or quantity of FN in the matrix after TNF-
exposure, we analyzed the FN in cell lysates by Western blotting under reducing conditions. Unexpectedly, with limited film exposure (Fig.
1A), we observed that the
lysate of TNF-
-treated endothelial cells contained almost no
monomeric FN detectable at 220 kDa. More extensive exposure (Fig.
1B) revealed the presence of monomeric FN that was still
much less after TNF-
exposure than in control cells. We initially
speculated that the reduction in FN monomers could be due to a drastic
detachment of cells, a pronounced increase in FN degradation, and/or a
decrease in FN synthesis. We therefore examined the endothelial cell
monolayers by both phase-contrast and immunofluorescent microscopy
using an antibody to FN. Figure 2 shows
that after an 18-h exposure to TNF-
(200 U/ml), the endothelial cell
layer was still intact, although intercellular gaps in the previously
confluent monolayer became very noticeable (compare Fig. 2,
A and B), and the fine FN subendothelial matrix
was disrupted, with thick bundles of FN apparent (Fig. 2, C
and D). Thus the negligible detection of monomeric FN under
reducing conditions was not due to the loss of CPAE cells from the
monolayer after TNF-
exposure.
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TNF- caused an increase in nonreducible HMW FN multimers in
endothelial cell layers.
Because the total FN content in the cell lysate was also unchanged
after TNF-
(Fig. 3C), we hypothesized that the reduced monomeric FN in TNF-
-treated cells could mean that much of the FN
may have existed as nonreducible HMW complexes, which were perhaps not
transferred onto the nitrocellulose membrane and therefore not
detectable by subsequent antibody probing.
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The increase in nonreducible HMW FN multimers is associated with an
increase in transglutaminase activity in TNF--treated endothelial
cells.
FN can be processed into HMW complexes by a transglutaminase-mediated
cross-linking mechanism (29, 32). Thus one
possible explanation for the observed increase in nonreducible HMW FN
complexes detected after exposure of the endothelial cell layers to
TNF-
is an increase in transglutaminase activity, although this has not been previously documented. Accordingly, we measured
transglutaminase activity in whole endothelial cell lysates by
quantifying the incorporation of [3H]putrescine into
dimethylated casein (29). Transglutaminase activity
increased in the endothelial cell layers in a dose-dependent manner
after TNF-
exposure (P < 0.05; Fig.
5B) in association with the
appearance of nonreducible HMW FN multimers (Fig. 5A), suggesting that the larger amounts of nonreducible HMW FN complexes detected in TNF-
-treated endothelial cells may be due to an
unexpected elevation in transglutaminase activity.
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The amount of nonreducible FN multimers corresponds to the level of
transglutaminase activity in both endothelial cells and fibroblasts.
If a TNF--induced increase in transglutaminase activity was actually
the basis for the significant increase in nonreducible HMW complexes of
FN detected in the endothelial matrix, we predicted that there should
be very little formation of such HMW FN complexes with fibroblast cell
layers because fibroblasts have limited transglutaminase activity. In a
side-by-side comparative analysis, we observed that nearly all of the
FN in the fibroblast ECM existed as HMW species under nonreducing
condition (data not shown). More importantly, the basal level of
transglutaminase activity in control fibroblast cell layers was only
~20% of that detected in endothelial cell layers (Fig.
7). Furthermore, the transglutaminase
activity in the fibroblast cell layers remained low even after
treatment with TNF-
for 18 h, whereas with endothelial cell
layers, transglutaminase activity increased in a dose-dependent manner
in response to TNF-
(P < 0.05; Fig. 7). This
finding was consistent with two additional observations; first, most of
the HMW FN multimers in the fibroblast cultures were readily reducible
to FN monomers (Fig. 8A), and second, TNF-
was unable to further increase the formation of nonreducible HMW FN multimers in the ECM of the fibroblasts, although it could readily do so with endothelial cells (Fig. 8B).
These results suggest a correlation between the formation of HMW
nonreducible complexes of FN in the ECM and the level of
transglutaminase activity. These findings also support the conclusion
that a TNF-
-induced increase in transglutaminase activity in the
CPAE cell layers may have contributed to the increased formation of
such nonreducible HMW FN complexes in the matrix.
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TNF--induced, transglutaminase-mediated FN
cross-linking activity is located on the surface of the
endothelial cell.
If the enhanced formation of nonreducible HMW complexes of FN in the
ECM was indeed caused by TNF-
-induced transglutaminase activity, we
would expect to see an increase in extracellular FN cross-linking
activity on the surface of the endothelial cells and/or in the culture
medium. Thus we examined the ability of both the endothelial cells and
their conditioned medium to cross-link 125I-FN that was
precoated on a cultured surface. Lung endothelial cells were first
preincubated with and without TNF-
(200 U/ml) for 18 h, gently
detached by mild trypsinization, washed in FN-deficient medium, and
then plated onto the 125I-FN coated wells and allowed to
adhere. After 3 h, the cells and their 125I-labeled
matrices were dissolved and analyzed for the presence of HMW
125I-FN complexes.
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Effect of the inhibitors of transglutaminase on TNF--induced
reorganization of the FN matrix, cell adhesion and spreading on the
matrix, and endothelial protein permeability.
Tissue transglutaminase activity has been found in many cell types
including endothelial cells (13, 26).
Regulated expression of transglutaminase may also influence FN
incorporation and play an important role in the regulation of cell
attachment and apoptosis (48). Because exposure of the
endothelial monolayers to TNF-
can induce both a disruption of the
fine FN fibers in the matrix and an increase in endothelial protein
permeability, we determined whether these events were dependent on the
increase in transglutaminase activity and the enhanced formation of
nonreducible HMW FN complexes in the ECM. Accordingly, we treated the
lung endothelial cell layers with TNF-
(200 U/ml) in the absence and
presence of the transglutaminase inhibitor Cys to attenuate the
formation of nonreducible HMW FN multimers in the ECM of
TNF-
-treated cells. Then we used these two different matrices to
study cell adhesion and cell spreading. First, as shown in Fig.
11, Cys at concentrations that blocked the formation of nonreducible HMW FN complexes was unable to prevent the TNF-
-induced reorganization of the FN matrix as studied by immunofluorescent microscopy (Fig. 11, A and B).
We next studied cell adhesion and cell spreading on these preformed
matrices. To do this, the original cell layers were gently removed with 0.5% sodium deoxycholate, and normal CPAE endothelial cells were then
seeded onto these preformed matrices. This allowed us to determine
whether CPAE cells adhere or spread differently on the cross-linked FN
matrix containing the nonreducible HMW multimers compared with the
non-cross-linked FN matrix. As shown in Fig. 11,
C-F, endothelial cell adhesion and cell
spreading were essentially identical on the subendothelial matrices
preformed by TNF-
-treated CPAE cells in the presence and absence of
the transglutaminase inhibitor (Fig. 11, C and
D). Cell adhesion to the FN network is shown by overlays of
staining for
5
1-integrins (green) and the matrix FN (red; Fig. 11, E and F). Direct
microscopic counting of 12 areas/slide confirmed that cell adhesion and
cell spreading were basically similar on these two different matrices
(Fig. 12).
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DISCUSSION |
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The present study indicates that TNF- causes an increase in the
formation of nonreducible HMW FN complexes in endothelial cell layers,
potentially due to an enhanced transglutaminase activity on the surface
of endothelial cells. This conclusion is supported by at least three
observations. First, both the increased formation of nonreducible HMW
matrix FN complexes and the increase in cellular transglutaminase
activity in the endothelial cell layers was TNF-
dose dependent.
Indeed, fibroblast cell layers with low basal levels of
transglutaminase activity contained a matrix with little nonreducible
HMW FN, and we observed no increase in transglutaminase activity in
fibroblast cell layers after TNF-
exposure. Second, inhibition of
transglutaminase activity abolished the TNF-
-induced increase in
nonreducible HMW matrix FN while also reducing the constitutive amounts
of such HMW FN complexes detected in control endothelial cells not
exposed to TNF-
. Third, TNF-
-treated endothelial cells displayed
high levels of cell surface transglutaminase activity and were capable
of cross-linking substrate FN precoated on culture plates at a much
greater level compared with that of untreated control endothelial cell layers.
TNF- is released from activated mobile monocytes and
macrophages, from sessile macrophages such as hepatic Kupffer cells (12), and from cells within the gut during bacteremia
and/or endotoxemia (30). TNF-
is believed to make a
positive contribution to the inflammatory and generalized host response
to sepsis (12, 30) by increasing endothelial
permeability, thus allowing certain plasma proteins important for both
wound repair and immune function to enter the extravascular space.
However, high levels of TNF-
can also elicit pathophysiological
effects in that they can disrupt the integrity of the lung endothelial
barrier (16, 50, 51) and elicit
apoptosis of endothelial as well as of tumor cells (36).
Intracellular transglutaminase activity is elevated when cells undergo
apoptosis, and transglutaminase-mediated cross-linking of proteins has
been suggested to contribute to the regulation of programmed cell death
(21, 27).
The present study documents an increase in extracellular
transglutaminase activity in the bovine lung endothelial cell layer after exposure to TNF-. It is not likely that this increase in transglutaminase activity was due to the nonspecific release of the
enzyme from apoptotic endothelial cells because it was only found
associated with the harvested CPAE cells and not in their conditioned
medium. Indeed, our findings indicate that the increased transglutaminase activity that was likely mediating the enhanced FN
multimerization after TNF-
exposure was on the cell surface and not
in the culture medium. Extracellular tissue transglutaminase has been
clearly documented in many cell types including endothelial cells
(1, 2, 5, 21,
32, 43). A recent study by Gaudry et al.
(22) has shown for the first time the secretion and
expression of transglutaminase on the surface of human umbilical vein
endothelial cells. Our studies not only confirm these novel findings
but also establish their pathological relevance, demonstrating an
increased surface expression of transglutaminase activity on endothelial cells after exposure to the inflammatory cytokine TNF-
.
For most adherent cell types, FN in the ECM is organized into HMW
complexes primarily by interchain disulfide bonds. However, the
cross-linking of FN by extracellular transglutaminase appears to
provide an additional mechanism especially available to endothelial cells, although it can be seen to a limited degree in fibroblast cell
layers. Transglutaminase-mediated cross-linking of FN (26, 32, 48), fibrinogen (33), and
proteoglycans (28) into HMW complexes can take place
constitutively in endothelial cells. TNF- has also been shown to
upregulate transglutaminase expression in other cell types such as
brain astrocytes (34). Our present findings, which suggest
increased cell surface transglutaminase in TNF-
-treated lung
endothelial cell layers, provide a mechanism for the enhanced FN
multimerization observed in the monolayers after exposure to this cytokine.
Our results show that the majority of matrix FN (~80%) was in the form of HMW complexes in the endothelial cell layers, with ~30% of these HMW FN multimers being stable under reducing conditions. This is in contrast to cultured fibroblasts where, although a major portion of the FN in the ECM also exists in HMW form, most of the HMW FN complexes in the ECM of fibroblasts will readily become monomeric (220 kDa) under reducing conditions. Consistent with this observation is the fact that it appears that fibroblast cell layers have very low levels of transglutaminase activity to mediate such FN multimerization compared with CPAE cells. However, Barry and Mosher (3, 4) have shown that activated factor XIII, also known as plasma transglutaminase, can stimulate the cross-linking of FN in the ECM of fibroblasts. They speculated that both FN already assembled in the ECM and FN in the process of being incorporated can be acted on by activated factor XIII. Based on studies with fibroblast cell layers, it has been proposed that the cross-linking of FN by transglutaminase may work in conjunction with disulfide-bonded FN multimerization to stabilize the assembly of the FN matrix (3).
An unexpected observation was that the increased transglutaminase
activity induced by TNF- did not cause additional HMW FN complexes
to be seen under nonreducing conditions. This finding suggests that
transglutaminase-mediated cross-linking of FN in CPAE cell layers is
primarily influencing those FN molecules already incorporated within
the matrix due to disulfide linkage. This potential extracellular
function of transglutaminase, which has been released and localized to
the cell surface of endothelial cells after exposure to TNF-
, may
enable the endothelium to stabilize its subendothelial matrix
especially in response to local vascular inflammation, causing the
release of TNF-
, a conclusion consistent with the report by Gentile
et al. (23) that indicates that cell-matrix interactions
can be stabilized by transglutaminase activity. The speculation that
constitutively expressed extracellular transglutaminase can stabilize
cell-matrix interactions is further supported by the findings that
cells deficient in transglutaminase are less able to adhere to an ECM
compared with normal cells. However, it is also possible that the
enhanced transglutaminase cross-linking of matrix FN may have
pathological effects, including alteration of the signaling properties
of the ECM or perhaps a reduction in FN turnover in the ECM.
TNF- has been shown to alter the subendothelial FN matrix and reduce
the barrier function of the endothelial cell monolayers (16, 17, 50, 51).
After exposure of CPAE cells to TNF-
, the matrix FN is characterized
by the formation of nonreducible HMW FN multimers and the appearance of
thick FN bundles deep within the matrix (16,
17). There is also reduced colocalization of endothelial
cell surface FN
5
1-integrins with the
fine FN fibers in the matrix (16, 17). Our
current findings suggest an important role for enhanced endothelial
cell surface transglutaminase activity in the formation of nonreducible
HMW complexes of FN in the matrix after TNF-
exposure. However, our
findings also suggest that such cross-linking of matrix FN is not the
basis for either the increase in endothelial protein permeability or the reorganization of the fine fibrillar FN matrix because
transglutaminase inhibitors did not prevent either of these
TNF-
-induced changes. Moreover, endothelial cell adhesion and cell
spreading on the FN-rich matrix was also not altered by Cys treatment
to block transglutaminase.
Another possible factor contributing to the altered ECM is the
degradation of matrix FN by proteases. Partridge et al.
(35) showed by zymography that medium from TNF--treated
microvessel endothelial cells contains metalloproteinase that can
cleave FN and other matrix proteins. TNF-
has also been shown to
induce the release of metalloproteinases such as gelatinase and
collagenase (35), which have the potential to cause matrix
degradation and/or matrix FN reorganization. But TNF-
also causes
endothelial cells to release protease inhibitors such as
plasminogen activator inhibitor-1 and -2 and tissue inhibitor of
metalloproteinases (25, 45), which can also
block matrix proteolysis. In this regard, the recent findings by Curtis
et al. (17) clearly document that local proteolysis of
matrix FN is not the biochemical mechanism causing disruption and/or
reorganization of FN in the ECM of CPAE monolayers after their 18-h
exposure to TNF-
, a protocol also used in the present study.
In the present study, we did not detect by Western blot analysis or
tracer 125I-FN experiments any significant increase in the
amount of FN fragments in CPAE cell layers or their culture medium
after TNF- exposure. These findings confirm and extend the recent
study from our laboratory (17) documenting that
although conditioned medium from TNF-
-treated CPAE cells contains
proteolytic activity that can degrade both gelatin (denatured collagen)
as well as FN, no FN fragments were found in either the cell lysate or
the conditioned medium. Obviously, the proteolytic balance within the
medium and/or matrix will determine the presence or absence of FN
fragments after TNF-
exposure.
The possibility that the increase in the cross-linking of FN matrix
could be a protective response of endothelial cells to the presence of
TNF- warrants consideration. For example, cross-linking of matrix
proteins by extracellular transglutaminase can promote cell-matrix
interaction (17) and potentially stabilize matrix integrity. Accordingly, increased extracellular transglutaminase activity leading to the enhanced cross-linking of FN assembled in the
matrix via disulfide-bonded multimer formation may provide endothelial
cells an additional mechanism to resist the cytotoxic effects of
TNF-
, especially in regard to the integrity of the ECM, which is
vital to cell adhesion and endothelial barrier function. The rapid
normalization of barrier function that can be seen after the addition
of excess soluble FN to the culture medium followed by its ECM
incorporation (16, 17, 50,
51) may reflect the fact that a large portion of the
endogenous FN already assembled in the ECM became cross-linked after
TNF-
exposure.
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ACKNOWLEDGEMENTS |
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The secretarial assistance of Debbie Moran and Wendy Ward is extremely appreciated.
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FOOTNOTES |
---|
These studies were supported primarily by National Institute of General Medical Sciences Grant GM-21447 (to T. M. Saba) and in part by American Lung Association Research Grant RG-133N (to B. Gao).
R. Chen was a postdoctoral fellow supported by National Heart, Lung, and Blood Institute Grant HL-07529 and is currently a Research Instructor in the Department of Medicine, Medical University of South Carolina (Charleston, SC). C. Huang and R. F. Rotundo were postdoctoral fellows supported by National Heart, Lung, and Blood Institute Grant HL-07529. C. Huang is currently a Research Associate in the Department of Microbiology and Immunology, Medical University of South Carolina. R. F. Rotundo is currently a Research Associate in the Department of Physiology and Cell Biology, Albany Medical College (Albany, NY).
Address for reprint requests and other correspondence: T. M. Saba, Dept. of Physiology and Cell Biology (MC-134), Albany Medical College, 47 New Scotland Ave., Albany, NY 12208 (E-mail: sabat{at}mail.amc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 7 June 1999; accepted in final form 3 February 2000.
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