1 Department of Physiology and 2 Center for Cell Biology and Cancer Research, Albany Medical College, Albany, New York 12208, and 3 Laboratory of Cell Physiology, Veterans Affairs Medical Center, Washington, District of Columbia 20422
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ABSTRACT |
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Tumor necrosis factor- (TNF-
), one
of the major inflammatory cytokines, is known to influence endothelial
cell migration. In this study, we demonstrate that exposure of calf
pulmonary artery endothelial cells to TNF-
caused an increase in the
formation of membrane protrusions and cell migration. Fluorescence
microscopy revealed an increase in
v
3
focal contacts but a decrease in
5
1 focal
contacts in TNF-
-treated cells. In addition, both cell-surface and
total cellular expression of
v
3-integrins
increased significantly, whereas the expression of
5
1-integrins was unaltered. Only focal
contacts containing
v
3- but not
5
1-integrins were present in membrane
protrusions of cells at the migration front. In contrast, robust focal
contacts containing
5
1-integrins were present in cells behind the migration front. A blocking antibody to
v
3, but not a blocking antibody to
5-integrins, significantly inhibited TNF-
-induced
cell migration. These results indicate that in response to TNF-
,
endothelial cells may increase the activation and ligation of
v
3 while decreasing the activation and
ligation of
5
1-integrins to facilitate
cell migration, a process essential for vascular wound healing and angiogenesis.
integrins; focal contacts; tumor necrosis factor-
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INTRODUCTION |
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ADHESION AND MIGRATION are distinct functions of endothelial cells essential for maintaining the integrity of the endothelium and repairing or forming blood vessels during wound healing or angiogenesis. The balance between adhesion and migration is precisely regulated in response to changing environments in the blood stream. Strong adhesion to the extracellular matrix is required for resting endothelial cells to maintain the integrity of the endothelium (8, 21, 31), whereas modulated adhesion to the matrix is necessary to facilitate cell migration (17, 18, 44). One way cells can modulate the strength of adhesion and facilitate migration is to change the expression and distribution of integrins on the cell surface.
Functional cell-surface integrins are complexes of an - and a
-subunit. More than 20 integrin complexes have been identified representing different combinations of at least 16
- and 8
-subunits (19, 31). The difference in the subunit
composition determines the specificity of the integrin complex for its
substrate in the matrix. For example, the
5
1-integrin complex essentially interacts only with fibronectin in the matrix, whereas the
6
1 complex interacts preferentially with
laminin (19, 28, 32). Some integrin complexes have
multiple preferred substrates in the matrix. One example is
v
3-integrin, which interacts with
vitronectin and fibronectin, as well as laminin. Integrins
5
1 and
v
3
are predominant integrin complexes expressed in endothelial cells (8, 37). Both integrin complexes have been implicated in endothelial cell adhesion and migration (17, 18, 33, 42).
The regulation of cell adhesion and migration involves coordinated
events including cell signaling, cytoskeleton rearrangement, and
surface integrin redistribution. These cellular events are known to be
influenced by inflammatory cytokines such as tumor necrosis factor-
(TNF-
). TNF-
is a 17-kDa polypeptide that forms homotrimers on
the cell surface. It is synthesized and secreted by many cell types
upon stimulation with a variety of toxins and cytokines including
TNF-
itself. Activated macrophages and monocytes are major sources
of TNF-
, and a primary target of this specific cytokine is the
endothelial cell (23, 25, 36, 38).
Over the past decade, considerable effort has been focused on
TNF--induced apoptosis, whereas the mechanism of
TNF-
-induced endothelial cell migration is relatively unknown.
Studies show that TNF-
can display either proangiogenic or
antiangiogenic effect depending on experimental conditions (12,
22, 26). One of these conditions appears to be the dosage or
concentration of TNF-
used in vivo or in vitro. It promotes the
formation of tubular structure at relatively low dosages but becomes
inhibitory to angiogenesis and induces apoptosis at relatively
high dosages (12, 22, 29). In vitro, TNF-
concentrations between 100 and 250 units/ml induced the highest levels
of tubule formation, whereas tubule formation was significantly reduced
at TNF-
concentrations higher than 500 units/ml (43).
TNF-
concentrations around 250 units/ml were also observed in the
blood of patients with serious inflammation and sepsis or in healthy
human subjects challenged with endotoxin (36, 39).
Accordingly, we used TNF-
at a concentration known to induce cell
migration to identify the role of cell-surface integrins in
TNF-
-induced endothelial cell migration.
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MATERIALS AND METHODS |
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Materials.
Bovine pulmonary artery endothelial (CPAE) cells were obtained from
American Type Culture Collection (Manassas, VA). Recombinant human
TNF- (20 units/ng) was obtained from Cellular Products (Buffalo,
NY). Monoclonal antibodies to
v
3 (clone
LM609),
5
1 (clone HA5), and actin
(MAB1501) and polyclonal antibodies to
5- (AB1928) and
3- (AB1932) integrins were obtained from Chemicon International, (Temecula, CA). The blocking antibody to
5 (clone BIIG2) was developed by C. H. Damsky and
obtained from the Developmental Studies Hybridoma Bank established
under the auspices of the National Institute of Child Health and Human
Development (NICHD) and maintained by the Department of Biological
Sciences, The University of Iowa, Iowa City, IA. All integrin
antibodies used in this study recognize both activated and nonactivated
form of integrins. Protease inhibitors phenylmethylsulfonyl fluoride
(PMSF) and N-p-tosyl-L-lysine
chloromethyl ketone (TLCK) were purchased from Sigma (St. Louis, MO).
Endothelial cell culture.
CPAE cells at passage 16 were cultured as described
previously (14). The cells were cultured in minimum
essential medium (MEM; GIBCO Invitrogen, Carlsbad, CA) containing 20%
fetal bovine serum (FBS; GIBCO Invitrogen). TNF- exposure was
carried out in MEM containing 5% FBS. All cells used in this study
were cultured to confluence and treated with or without TNF-
at 200 units/ml for 18 h before analysis (migration assay, adhesion
assay, immunofluorescence microscopy, or immunoprecipitation).
Determination of membrane protrusion formation and cell migration
with an in vitro wound-healing assay.
Confluent endothelial cells on glass coverslips were treated with or
without TNF-, and wounds were created on cell monolayers by using
the "scratch wound" protocol (10, 15, 34) with a razor
blade. The debris was removed by washing the cells with serum-free MEM,
and the cells were incubated in a 37°C incubator for 5 h in
serum-free MEM. The cells were photographed, and the number of
migrating cells and the percentage of cells with membrane protrusions
were determined under an inverted microscope. A total of nine areas
were selected randomly on each coverslip under a 40× objective. Cells
on three to six coverslips of either control or TNF-
-treated sample
were quantified in each experiment. To detect integrins in focal
contacts, the cells were fixed, permeabilized, and incubated with
antibodies to
v
3 (LM609) or
5
1 (HA5) and fluorescence-labeled
secondary antibodies (Molecular Probes, Eugene, OR).
Determination of membrane protrusion formation with cell adhesion
assay.
Human fibronectin was purified from cryoprecipitate (American Red
Cross) by using geletin-sepharose affinity chromatography according to
the procedure of Engvall and Ruoslahti (11). Human cryoprecipitate (15 ml) was diluted 1:1 with the column equilibration buffer and loaded onto a 10-ml gelatin-sepharose column (Pharmacia Biotech, Piscataway, NJ) at a flow rate of 0.5 ml/min. The column was
washed with 1 M NaCl in phosphate-buffered saline (PBS) and eluted with
4 M urea in the washing buffer. The eluted fraction was dialyzed
overnight in 0.2 M phosphate buffer, pH 7.4, and the fibronectin
concentration was determined by using the extinction coefficient
Determination of the effect of blocking antibodies on endothelial
cell adhesion on fibronectin-coated surfaces.
Endothelial cells in suspension were preincubated with blocking
antibodies to either v
3 (LM609)- or
5 (BIIG2)-integrins on ice for 30 min before being
seeded onto glass coverslips coated with 2 µg/ml fibronectin.
Coverslips coated with 10 µg/ml bovine serum albumin (BSA) were used
as controls for nonspecific adhesion. Cells were incubated in
serum-free medium at 37°C for 30 min. Nonadhered cells were removed
by washing with PBS. The number of adhered cells was determined by
counting under an inverted microscope as described above.
Determination of cell-surface integrin expression by surface
biotinylation, immunoprecipitation, and Western blotting.
Confluent CPAE cell monolayers treated with or without TNF- were
labeled with Biotin (Pierce, Rockford, IL) at 0.5 mg/ml in PBS for 60 min at 4°C. Cells were then lysed in the lysis buffer (150 mM NaCl, 5 mM EDTA, 1% sodium deoxycholate, 1% Triton X-100, and 20 mM Tris at
pH 7.4) containing protease inhibitors (0.3 mM PMSF and 0.1 mM TLCK).
The cell lysate was clarified by centrifugation in a Microfuge and
precleared by incubation with protein G agarose (GIBCO Invitrogen).
Integrins
v
3 or
5
1 were immunoprecipitated with
antibodies LM609 and HA5, respectively, followed by incubation with
protein G agarose. The agarose-bound integrins were solubilized in
boiled SDS-gel sample buffer under nonreducing conditions and clarified
by spinning in a Microfuge. Precipitated integrins were separated on
two identical 7.5% SDS gels and transferred onto two nitrocellulose
membranes. One membrane was used to determine cell-surface integrins
with streptavidin conjugated to horseradish peroxidase and enhanced
chemiluminescence (ECL) Western blotting detection solutions (both from
Amersham, Piscataway, NJ). The other membrane was used to
determine total cellular integrins in biotinylated cells with
antibodies to either
5 (AB1928) or
3
(AB1932) and ECL Western blotting detection solutions. The bands on
films were quantified by densitometric scanning using a BioRad imaging
densitometer (Bio-Rad, Hercules, CA).
Determination of total cellular integrin expression by
immunoprecipitation and Western blotting.
Confluent CPAE cells treated with or without TNF- were lysed in the
lysis buffer, and
v
3- or
5
1-integrins were immunoprecipitated from
the cell lysate by using monoclonal antibodies to the integrins as
described above. Precipitated integrins were separated on SDS gels and
transferred onto nitrocellulose membranes. The nitrocellulose membranes
were probed for either
5- or
3-integrins
by using polyclonal antibodies (AB1928 and AB1932). The integrins were quantified by densitometric scanning of Western blot films. The amount
of protein in cell lysate used in immunoprecipitation was determined on
a separate gel and Western blot probed for actin by using antiactin
antibody MAB1501.
Immunofluorescence microscopy.
CPAE cells cultured on coverslips were fixed with 3% formaldehyde,
permeabilized in 0.5% Triton, and stained with either an antibody
against human 5
1-integrin (clone HA5) or
an antibody to human
v
3-integrin (clone
LM609) at 2 µg/ml. This was followed by incubations with secondary
antibodies conjugated to Alexa-488 (Molecular Probes, Eugene, OR). The
coverslips were mounted with ProLong Anti-Fade (Molecular Probes) and
examined under a BX60 fluorescence microscope (Olympus, Melville, NY)
and photographed using a SPOT digital camera (Diagnostic Instruments,
Sterling Heights, MI).
Statistical analysis. All measurements were performed at least three times with duplicate samples. Results are presented as means ± SD. Levels of significance are determined by a two-tailed Student's t-test (13), and a confidence level of >95% (P < 0.05) was used to established statistical significance.
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RESULTS |
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Effect of TNF- on endothelial cell migration and the formation
of membrane protrusions.
We examined the migration of endothelial cells treated with TNF-
at
200 units/ml for 18 h, because previous studies indicate that
functional changes in endothelial monolayers occur between 12 and
24 h of TNF-
exposure at this dosage. These functional changes
include dissociation of
5
1-integrins from
focal contacts (14, 30), increased recycling of integrins
(14), reduced cell adhesion to fibronectin
(30), cell-cell gap formation (7, 14, 30),
and increase in protein permeability (6, 7, 40).
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Effect of TNF- on the localization of
v
3- and
5
1-integrins in focal contacts.
v
3 and
5
1
are predominant integrin complexes expressed in endothelial cells.
These integrin complexes have been shown to play important roles in
cell migration (31, 32, 37). It is possible that the
increased formation of membrane protrusions and cell migration after
TNF-
exposure were mediated by an increase in the ligation of these
integrins. If this were the case, one would expect to see
integrin-containing focal contacts in membrane protrusions, especially
in cells at the migration front.
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Effect of TNF- on the expression of
v
3- and
5
1-integrins.
Changes in focal contacts observed in Figs. 3 and 4 could have been
caused by changes in cell-surface expression and/or total cellular
expression of the integrins. However, individual integrins cannot be
detected by microscopy unless they have been recruited into focal
contacts. We therefore investigated TNF-
-induced changes in the
expression of
v
3- and
5
1-integrins in endothelial cells using
biochemical approaches.
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Effect of blocking antibodies to v
3-
and
5
1-integrins on TNF-
-induced cell
migration.
The above observations suggest that increased
v
3-containing focal contacts may have
served as anchors for membrane protrusions, without which membrane
protrusions may retract and cell migration may be abolished. If this
were true, one would expect to see an attenuation of cell migration
when the
v
3-ligand interactions are blocked.
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DISCUSSION |
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The results presented in the current study demonstrated that
TNF- at 200 units/ml, a concentration commonly found in severely septic patients, could cause endothelial cells to increase the formation of membrane protrusions and cell migration. These changes were accompanied by an increase in both cell-surface and total cellular
expression of
v
3-integrins. In contrast,
the expression of
5
1-integrins remained
unchanged. The increased formation of membrane protrusions and cell
migration in TNF-
-treated cells was facilitated by the increased
expression of
v
3 on the cell surface and
increased recruitment of
v
3-integrin into
focal contacts. Several lines of evidence presented in this study
support these conclusions. First, a significant increase in
v
3-integrin expression was detected on
the surface of TNF-
-treated endothelial cells. Second, a marked
increase in
v
3-containing focal contacts was observed after cells were exposed to TNF-
. Third, only
v
3-containing focal contacts, but not
5
1-containing focal contacts, were
detected in membrane protrusions of cells at the migration front.
Fourth, a blocking antibody to
v
3-integrins, but not a blocking antibody to
5-integrin subunit, significantly inhibited
TNF-
-induced cell migration.
The development of inflammation is mediated by cytokines released upon
bacterial infection. Proinflammatory cytokines such as TNF- mediate
vascular inflammation by inducing cell-cell and cell-matrix
dissociation of endothelial cells (7, 14, 23, 30). In
vitro, the dissociation of either cell-cell or cell-matrix interactions
can cause increased protein permeability across the endothelial
monolayer (4, 7, 30, 40). This may be the basis for the
increased endothelial protein permeability across the endothelium
observed in vivo with inflammation and sepsis. A similar process occurs
in the formation of new blood vessels. Angiogenic factors such as VEGF
cause cell-cell and cell-matrix dissociation followed by migration and
proliferation of endothelial cells (5). On the other hand,
many angiogenic factors have also been shown to cause increased
permeability across the endothelial monolayer and inflammatory response
(5, 9, 43). It is therefore likely that both processes
share a part of the same cell-signaling pathway.
TNF- has been shown to induce the release of metalloproteinases
(35), vascular endothelial growth factor A (VEGF-A), and interleukin-8 (43), all of which are potent angiogenic
factors. TNF-
has also been shown to modulate the expression of VEGF
receptors (16, 26). The current study has demonstrated a
possible involvement of integrin signaling in TNF-
-induced cell
migration via a coordinated regulation between
v
3- and
5
1-integrins. On the other hand, it is
well known that the angiogenic effect of TNF-
varies with cell lines
and experimental conditions (12, 22, 26). Therefore it
remains to be determined whether the TNF-
-induced coordinated regulation of
v
3- and
5
1-integrins observed in CPAE cells also
occurs in other endothelial cell lines or under in vivo conditions.
The integrin complex v
3 interacts with a
wide range of matrix proteins. It is, however, not expressed at high
levels compared with
5
1 on resting
endothelial cells (37). A likely reason for its increased
expression on TNF-
-treated cells is to allow cells to survive on a
changing matrix. Resting endothelial cells produce a fibronectin-rich
matrix both in vivo and in vitro, and their interactions with the
matrix are mediated predominately by
5
1-integrins (8, 9). TNF-
has been shown to cause the release of proteinases that can modify the
matrix of endothelial cells (35). This matrix modification
may be one reason for the observed decreased localization of
5
1-integrins and the increased localization of
v
3-integrins in focal contacts.
The current study demonstrated changes in
v
3 surface expression and focal contacts
in endothelial cells after TNF-
exposure. It also suggested a
possible coordinated regulation on the expression and ligation of two
different integrins. This is evident not only in protein expression but
also in the localization of these integrins in focal contacts. Integrin
v
3 was detected only at cell-cell junctions in untreated cells, whereas focal contacts containing
v
3-integrins were readily identified in
cells after TNF-
exposure. In contrast,
5
1-integrins were present in robust focal
contacts in untreated cells, and the number of
5
1 contacts was dramatically reduced
after TNF-
exposure. No focal contacts containing
5
1-integrins were observed in membrane
protrusions of cells at the migration front. These coordinated changes
in
v
3- and
5
1-integrins induced by TNF-
may
mediate the observed membrane protrusion formation and cell migration.
Considerable evidence suggests that signaling among integrins is
modulated by "cross talk" mediators. Integrin
v
5-mediated vitronectin internalization
appeared to require the ligation of
5
1-integrins (27). Ligation
of
v
3-integrins was found to suppress
5
1-mediated activation of
calcium/calmodulin-dependent protein kinase II (CamKII)
(2), which appeared to be required for integrin-mediated
phagocytosis and cell migration. CamKII at high levels, however, may
inhibit the interaction of
5
1-integrin with fibronectin (3). Kim et al. (20)
demonstrated that the ligation of
5
1-integrins could potentiate
v
3-mediated endothelial cell migration on
vitronectin by suppressing the activity of protein kinase A. It is
possible that the differential regulation on the expression of
5
1- and
v
3-integrins induced by TNF-
is
mediated by a cross-talk mediator. Future studies to identify such a
mediator may provide a better understanding of the mechanism by which
TNF-
induces the increase in
v
3-integrin expression and endothelial cell migration, processes that may be essential for vascular wound healing and angiogenesis.
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ACKNOWLEDGEMENTS |
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We thank Kara L. Powell and Alice Damrau-Abney for technical assistance and Debbie Moran for administrative assistance.
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FOOTNOTES |
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This study was supported by research grant RG-133N (B. Gao) from the American Lung Association of New York, National Institute of General Medical Sciences Grant GM-21447 (T. M. Saba), and a Veterans Affairs Merit Review Award (M.-F. Tsan).
Address for reprint requests and other correspondence: B. Gao, VA Medical Center (10R), 50 Irving St., N.W., Washington, DC 20422 (E-mail: baochong.gao{at}med.va.gov).
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. Section 1734 solely to indicate this fact.
June 26, 2002;10.1152/ajpcell.00064.2002
Received 12 February 2002; accepted in final form 19 June 2002.
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