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Am J Physiol Cell Physiol 283: C1196-C1205, 2002. First published June 26, 2002; doi:10.1152/ajpcell.00064.2002
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Vol. 283, Issue 4, C1196-C1205, October 2002

Role of alpha vbeta 3-integrin in TNF-alpha -induced endothelial cell migration

Baochong Gao1,2,3, Thomas M. Saba1,2, and Min-Fu Tsan3

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tumor necrosis factor-alpha (TNF-alpha ), 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-alpha caused an increase in the formation of membrane protrusions and cell migration. Fluorescence microscopy revealed an increase in alpha vbeta 3 focal contacts but a decrease in alpha 5beta 1 focal contacts in TNF-alpha -treated cells. In addition, both cell-surface and total cellular expression of alpha vbeta 3-integrins increased significantly, whereas the expression of alpha 5beta 1-integrins was unaltered. Only focal contacts containing alpha vbeta 3- but not alpha 5beta 1-integrins were present in membrane protrusions of cells at the migration front. In contrast, robust focal contacts containing alpha 5beta 1-integrins were present in cells behind the migration front. A blocking antibody to alpha vbeta 3, but not a blocking antibody to alpha 5-integrins, significantly inhibited TNF-alpha -induced cell migration. These results indicate that in response to TNF-alpha , endothelial cells may increase the activation and ligation of alpha vbeta 3 while decreasing the activation and ligation of alpha 5beta 1-integrins to facilitate cell migration, a process essential for vascular wound healing and angiogenesis.

integrins; focal contacts; tumor necrosis factor-alpha


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha - and a beta -subunit. More than 20 integrin complexes have been identified representing different combinations of at least 16 alpha - and 8 beta -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 alpha 5beta 1-integrin complex essentially interacts only with fibronectin in the matrix, whereas the alpha 6beta 1 complex interacts preferentially with laminin (19, 28, 32). Some integrin complexes have multiple preferred substrates in the matrix. One example is alpha vbeta 3-integrin, which interacts with vitronectin and fibronectin, as well as laminin. Integrins alpha 5beta 1 and alpha vbeta 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-alpha (TNF-alpha ). TNF-alpha 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-alpha itself. Activated macrophages and monocytes are major sources of TNF-alpha , 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-alpha -induced apoptosis, whereas the mechanism of TNF-alpha -induced endothelial cell migration is relatively unknown. Studies show that TNF-alpha 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-alpha 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-alpha concentrations between 100 and 250 units/ml induced the highest levels of tubule formation, whereas tubule formation was significantly reduced at TNF-alpha concentrations higher than 500 units/ml (43). TNF-alpha 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-alpha at a concentration known to induce cell migration to identify the role of cell-surface integrins in TNF-alpha -induced endothelial cell migration.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Bovine pulmonary artery endothelial (CPAE) cells were obtained from American Type Culture Collection (Manassas, VA). Recombinant human TNF-alpha (20 units/ng) was obtained from Cellular Products (Buffalo, NY). Monoclonal antibodies to alpha vbeta 3 (clone LM609), alpha 5beta 1 (clone HA5), and actin (MAB1501) and polyclonal antibodies to alpha 5- (AB1928) and beta 3- (AB1932) integrins were obtained from Chemicon International, (Temecula, CA). The blocking antibody to alpha 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-alpha 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-alpha 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-alpha , 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-alpha -treated sample were quantified in each experiment. To detect integrins in focal contacts, the cells were fixed, permeabilized, and incubated with antibodies to alpha vbeta 3 (LM609) or alpha 5beta 1 (HA5) and fluorescence-labeled secondary antibodies (Molecular Probes, Eugene, OR).

To determine the effects of blocking antibodies on cell migration, confluent endothelial cells on glass coverslips were treated with or without TNF-alpha and scraped with a razor blade. The debris was removed by washing the cells with serum-free MEM. The cells were then incubated in a 37°C incubator for 5 h in the presence or absence of blocking antibodies to either alpha 5 (BIIG2)- or alpha vbeta 3 (LM609)-integrin complexes. The number of cells migrated into the wound area was determined as described above.

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 epsilon <UP><SUB>280</SUB><SUP>1%</SUP></UP> = 12.8.

Glass coverslips in 12-well plates were incubated overnight with purified fibronectin at 2 µg/ml in coating buffer (50 mM NaHCO3, pH 9.6) at 4°C. Endothelial cells treated with or without TNF-alpha were lifted into suspension with trypsin-EDTA buffer (GIBCO Invitrogen) and seeded onto fibronectin-coated or noncoated coverslips at 105 cells/well. The cells were incubated in either serum-free medium on fibronectin-coated surfaces or MEM containing 20% FBS on noncoated surfaces at 37°C for 30 min. Nonadhered cells were removed by washing with PBS, and adhered cells were examined and photographed under an inverted microscope. Cells with membrane protrusions were quantified as described above.

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 alpha vbeta 3 (LM609)- or alpha 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-alpha 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 alpha vbeta 3 or alpha 5beta 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 alpha 5 (AB1928) or beta 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-alpha were lysed in the lysis buffer, and alpha vbeta 3- or alpha 5beta 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 alpha 5- or beta 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 alpha 5beta 1-integrin (clone HA5) or an antibody to human alpha vbeta 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of TNF-alpha on endothelial cell migration and the formation of membrane protrusions. We examined the migration of endothelial cells treated with TNF-alpha 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-alpha exposure at this dosage. These functional changes include dissociation of alpha 5beta 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).

The migration of endothelial cells was evaluated by using a well-established in vitro wound-healing assay (10, 15, 34). In these experiments, endothelial cell monolayers treated with or without TNF-alpha were wounded with a razor blade. After a 5-h incubation, the number of cells migrated into the wound area was determine under an inverted microscope. Results showed that TNF-alpha -treated cells displayed a significant increase in cell migration (Fig. 1, A-C). In addition, increased formation of membrane protrusions was observed in TNF-alpha -treated cells at the migration front (Fig. 1, A, B, and D), suggesting a possible role of the membrane protrusions in the increased cell migration.


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  		Fig. 1.   Tumor necrosis factor-alpha (TNF-alpha ) induced increases in cell migration and in the formation of membrane protrusions. Confluent endothelial cell monolayers were treated with (B) or without (A) TNF-alpha at 200 units/ml for 18 h, and cell monolayers were wounded with a razor blade. The cells were then incubated for 5 h at 37°C in a serum-free medium. The number of cells migrated into the open area (C) and the percentage of cells with membrane protrusion (D) were determined under an inverted microscope. Results represent means ± SD of 5 experiments. *P < 0.05. Bar, 100 µm.

We next asked the question whether the increased formation of membrane protrusion was a characteristic of all TNF-alpha -treated cells, not only cells at the migration front. One way to answer this question is to determine the formation of membrane protrusions in a cell adhesion assay under subconfluent conditions. To determine the effect of matrix proteins, we determined the formation of membrane protrusions on surfaces coated with fibronectin, a common substrate for both alpha vbeta 3- and alpha 5beta 1-integrins. In this experiment, cells in monolayers treated with or without TNF-alpha were lifted into suspension and seeded onto glass coverslips coated with or without fibronectin. Cells were then incubated briefly at 37°C either in a serum-free medium on fibronectin-coated surfaces or in the presence of 20% serum on noncoated surfaces. Nonadhered cells were removed by a washing with PBS. Adhered cells were photographed under an inverted microscope. Results in Fig. 2 show clearly that TNF-alpha -treated cells display more membrane protrusions than control cells in this assay on both fibronectin-coated and noncoated surfaces. These observations under subconfluent conditions suggest that increased formation of membrane protrusions may be a characteristic of all TNF-alpha -treated cells, not only cells at the migration front in the wound-healing assay.


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  		Fig. 2.   Increased formation of membrane protrusions was a characteristic of all TNF-alpha -treated endothelial cells. Confluent endothelial cell monolayers treated with (C and D) or without (A and B) TNF-alpha (200 units/ml, 18 h) were lifted into suspension and seeded onto glass coverslips. Cells were incubated at 37°C for 30 min either in a serum-free medium on fibronectin-coated surfaces (Fn-coated, A and C) or in the presence of 20% serum on noncoated surfaces (noncoated, B and D). The percentage of cells with membrane protrusions was quantified (E) under an inverted microscope. Results represent means ± SD of 5 experiments. *P < 0.05. Bar, 100 µm.

Effect of TNF-alpha on the localization of alpha vbeta 3- and alpha 5beta 1-integrins in focal contacts. alpha vbeta 3 and alpha 5beta 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-alpha 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.

To test this possibility, endothelial cells treated with or without TNF-alpha were assayed for cell migration as in Fig. 1, and cells were fixed and stained with antibodies recognizing either alpha vbeta 3- or alpha 5beta 1-integrin complexes. As shown in Fig. 3, only alpha vbeta 3-containing focal contacts were detected in membrane protrusions of cells at the migration front (Fig. 3, B and D). In contrast, alpha 5beta 1-integrins in cells at the migration front were observed only in structures resembling endocytic vesicles (Fig. 3, A and C), not in focal contacts. However, focal contacts containing alpha 5beta 1-integrins were readily identified in cells immediately behind the migration front (Fig. 3, A and C). In comparison, control cells not treated with TNF-alpha had much fewer alpha vbeta 3 focal contacts and significantly lower levels of membrane protrusion formation (Fig. 3, compare B and D). These data support the notion that TNF-alpha -induced formation of membrane protrusion and cell migration may rely on the increase in the ligation of alpha vbeta 3-integrins.


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  		Fig. 3.   Focal contacts containing alpha vbeta 3- but not alpha 5beta 1-integrins were observed in cells at the migration front. Confluent endothelial cells on coverslips treated with (C and D) or without (A and B) TNF-alpha were wounded as described in Fig. 1. The cells were incubated for 5 h at 37°C in a serum-free medium before being fixed and incubated with antibodies to either alpha 5beta 1 (A and C)- or alpha vbeta 3 (B and D)-integrins. Arrows point to some of the focal contacts containing either alpha vbeta 3 (B and D)- or alpha 5beta 1 (A and C)-integrins. Arrowheads in A and C indicate antibody-labeled alpha 5beta 1-integrins in structures resembling endocytic vesicles. Bar, 50 µm.

To obtain a closer look at the formation of alpha vbeta 3 focal contacts on migrating cell, we carried out a time course of cell migration into the wound area and compared the rate of migration of control and TNF-alpha -treated endothelial cells. Results (Fig. 4) showed that cell migration could be detected in TNF-alpha -treated cell monolayers 1 h after wounding (Fig. 4E), whereas similar levels of cell migration were not observed until 4 h after wounding in control monolayers (Fig. 4C). In addition, focal contacts containing alpha vbeta 3-integrins formed in all migrating cells, especially on membrane protrusions (Fig. 4, C-H).


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  		Fig. 4.   Focal contacts containing alpha vbeta 3 were expressed on all migrating cells. Confluent endothelial cell monolayers on coverslips treated with (E-H) or without (A-D) TNF-alpha were wounded as in Fig. 1. The cells were incubated at 37°C for 1 (A and E), 2 (B and F), 4 (C and G), or 8 h (D and H) in a serum-free medium before being fixed and labeled with an antibody to alpha vbeta 3-integrins. Arrows in C-H point to some of the alpha vbeta 3-containing focal contacts. Bar, 100 µm.

An important question was whether the increased alpha vbeta 3 and decreased alpha 5beta 1 focal contacts occurred not only in cells at the migration front but also in cells in confluent monolayers. To answer this question, endothelial monolayers treated with or without TNF-alpha were fixed and stained with antibodies recognizing both activated and nonactivated form of either alpha vbeta 3- or alpha 5beta 1-integrins. Results show (Fig. 5) that the expression of alpha vbeta 3-integrins in control cells in monolayers is only detectable at cell-cell junctions (Fig. 5B), whereas alpha vbeta 3-containing focal contacts can be readily identified around the cell periphery in TNF-alpha -treated cells (Fig. 5D). In addition, TNF-alpha -treated cells also display increased gap formation, suggesting a loss of cell-cell interactions after cell monolayers were exposed to TNF-alpha . This is consistent with earlier observations under similar conditions (6, 14, 30). Increased alpha vbeta 3 focal contacts around the periphery of the TNF-alpha -treated cells may be a cellular response to increase cell adhesion in compensating the loss of cell-cell interactions. In contrast to alpha vbeta 3 focal contacts, robust alpha 5beta 1-containing focal contacts were detected in control cells (Fig. 5A), and an apparent decrease in alpha 5beta 1 focal contacts was observed in TNF-alpha -treated cells (Fig. 5C). These results indicate that TNF-alpha caused an increase in the activation/ligation of alpha vbeta 3 and a decrease in the activation/ligation of alpha 5beta 1-integrins in all TNF-alpha -treated endothelial cells, not only in cells at the migration front.


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  		Fig. 5.   TNF-alpha induced a decrease in alpha 5beta 1- but an increase in alpha vbeta 3- containing focal contacts on cells in monolayers. Confluent endothelial cells cultured on coverslips treated with (C and D) or without (A and B) TNF-alpha were fixed and stained with antibodies to either alpha 5beta 1 (A and C) or alpha vbeta 3 (B and D). Arrows point to some of the focal contacts containing either alpha 5beta 1 (A and C)- or alpha vbeta 3 (D)-integrins. Arrows in B indicate some of the alpha vbeta 3-integrins expressed at cell-cell junctions of control cells. Bar, 50 µm.

Effect of TNF-alpha on the expression of alpha vbeta 3- and alpha 5beta 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-alpha -induced changes in the expression of alpha vbeta 3- and alpha 5beta 1-integrins in endothelial cells using biochemical approaches.

Integrins can display different activation states, and the state of integrin activation is influenced by their interactions with ligands, antibodies, and cations such as Mn2+ (1, 24, 28, 41). Binding of a ligand or Mn2+ can switch an integrin complex from a "low-affinity state" (nonactivated form) to a "high-affinity state" (activated form). The transition of the affinity states involves conformational changes of the integrins, which can be detected by specific antibodies recognizing motifs exposed only when integrins are activated. To quantify the expression of all forms of integrins, we used antibodies to recognize both activated and nonactivated forms of integrins for immunoprecipitation.

To determine the surface expression of the integrins, the cell surface was first biotinylated and then alpha vbeta 3- or alpha 5beta 1-integrins were immunoprecipitated from the cell lysate. The immunoprecipitated integrins were then quantified by Western blotting using streptavidin conjugated to horseradish peroxidase (Fig. 6). To determine the total cellular expression of the integrins, alpha vbeta 3 or alpha 5beta 1 was immunoprecipitated from the whole cell lysate and the integrins were quantified by Western blotting using antibodies to either alpha 5- or beta 3-integrin subunit (Fig. 7). These antibodies were used to quantify alpha 5beta 1- and alpha vbeta 3-integrin complexes, because alpha 5-subunit has been found only in alpha 5beta 1 complexes and beta 3- subunit forms complexes only with alpha v in endothelial cells (9, 28, 31).


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  		Fig. 6.   TNF-alpha induced an increase in cell-surface expression of alpha vbeta 3- but not alpha 5beta 1-integrins. The surface of confluent endothelial cells treated with or without TNF-alpha was labeled with biotin, and alpha 5beta 1- or alpha vbeta 3-integrin complexes were immunoprecipitated from the cell lysate. Cell-surface integrins were quantified by Western blotting using horseradish peroxidase conjugated streptavidin (A). Total cellular integrins were determined on separate Western blots using antibodies to either alpha 5- or beta 3-integrins (B). Shown in A and B are representative Western blots. Results from densitometric scanning expressed as optical density (OD) ratios of cell-surface integrins to total integrins are plotted in C. Values represent means ± SD of 3 experiments. *P < 0.05.



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  		Fig. 7.   TNF-alpha induced an increase in total cellular expression of alpha vbeta 3- but not alpha 5beta 1-integrins. Endothelial cells treated with or without TNF-alpha were lysed, and alpha 5beta 1- or alpha vbeta 3-integrin complexes were immunoprecipitated from the cell lysate. Precipitated integrins were quantified by Western blotting using antibodies to alpha 5- or beta 3-integrins (A). The cell lysate used for immunoprecipitation was analyzed on a separate gel to probe for actin on Western blots (B). Shown in A and B are representative Western blots. Results from densitometric scanning expressed as OD ratios of integrins to total actin are plotted in C. Values represent means ± SD of 3 experiments. *P < 0.05.

Results (Figs. 6 and 7) indicated that TNF-alpha caused a significant increase in both the cell-surface and total cellular expression of alpha vbeta 3-integrins. In contrast, the expression of alpha 5beta 1-integrins did not change significantly despite the clear decrease in alpha 5beta 1-containing focal contacts observed (Fig. 5). Thus the increase in alpha vbeta 3-containing focal contacts observed in TNF-alpha -treated endothelial cells (Figs. 3-5) was at least partially due to the increased surface expression of the integrins. On the other hand, the data were consistent with the concept that an inactivation, rather than a decrease in surface expression of alpha 5beta 1-integrins, was the basis for the reduction of focal contacts containing alpha 5beta 1-integrins observed in TNF-alpha -treated endothelial cells (Fig. 5).

Effect of blocking antibodies to alpha vbeta 3- and alpha 5beta 1-integrins on TNF-alpha -induced cell migration. The above observations suggest that increased alpha vbeta 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 alpha vbeta 3-ligand interactions are blocked.

To test this hypothesis, the cell migration assay was performed in the presence of blocking antibodies to either alpha vbeta 3- or alpha 5beta 1-integrins. A blocking antibody to alpha 5-subunit was used to block the function of alpha 5beta 1-integrin complexes, because alpha 5-subunit has only been found in alpha 5beta 1-integrin complexes (28, 31). We first examined the effect of the antibodies on cell adhesion to determine the concentration at which the antibodies can act effectively. Because fibronectin is a substrate for both alpha vbeta 3- and alpha 5beta 1-integrins in the matrix, we determined whether the antibodies could block cell adhesion on fibronectin-coated surfaces. We observed that both blocking antibodies inhibited the adhesion of endothelial cells with significant blocking effects observed at 5 µg/ml for anti-alpha vbeta 3 and a fivefold dilution for anti-alpha 5 antibodies (Fig. 8).


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  		Fig. 8.   Blocking antibodies to alpha vbeta 3- or alpha 5-integrins inhibited the adhesion of endothelial cells in a concentration-dependent manner. Confluent endothelial cells were lifted into suspension and preincubated with or without blocking antibodies to either alpha vbeta 3 or to alpha 5 at indicated concentrations for 30 min on ice. Cells were diluted into serum-free MEM and seeded at the same cell density on glass coverslips precoated with 2 µg/ml fibronectin. Coverslips were coated with BSA as background controls. Nonadhering cells were removed by a washing with PBS after a 30-min incubation at 37°C. Adhered cells were counted under an inverted microscope. Results represent means ± SD of 3 experiments. *P < 0.05.

We next determined the effect of the blocking antibodies on cell migration. Results showed that cell migration was inhibited in TNF-alpha -treated cells by the blocking antibody to alpha vbeta 3-integrins in a concentration-dependent manner (Fig. 9). In contrast, the blocking antibody to alpha 5-integrins had little effect on TNF-alpha -induced cell migration (Fig. 10), even at concentrations that significantly inhibited cell adhesion (Fig. 8). These observations suggest that alpha vbeta 3-integrins play an important role in TNF-alpha -induced cell migration.


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  		Fig. 9.   The blocking antibody (Ab) to alpha vbeta 3-integrins significantly inhibited TNF-alpha -induced cell migration in a concentration-dependent manner. Confluent endothelial cells on coverslips were treated with (B, D, and F) or without (A, C, and E) TNF-alpha , and cell monolayers were wounded as in Fig. 1. Cells were then incubated at 37°C for 5 h in the presence of LM609, a blocking Ab to alpha vbeta 3-integrins, at indicated concentrations. The number of cells migrated into the wound area was determined under an inverted microscope (G). Results represent means ± SD of 3 experiments. *P < 0.05. Bar, 100 µm.



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  		Fig. 10.   The blocking Ab to alpha 5-integrins had no significant effect on TNF-alpha -induced cell migration. Confluent endothelial cells on coverslips were treated with (B, D, and F) or without (A, C, and E) TNF-alpha , and cell monolayers were wounded as in Fig. 1. Cells were then incubated at 37°C for 5 h in the presence of BIIG2, a blocking Ab to alpha 5-integrins, at indicated dilutions. The number of cells migrated into the wound area was determined under an inverted microscope (G). Results represent means ± SD of 3 experiments. Bar, 100 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The results presented in the current study demonstrated that TNF-alpha 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 alpha vbeta 3-integrins. In contrast, the expression of alpha 5beta 1-integrins remained unchanged. The increased formation of membrane protrusions and cell migration in TNF-alpha -treated cells was facilitated by the increased expression of alpha vbeta 3 on the cell surface and increased recruitment of alpha vbeta 3-integrin into focal contacts. Several lines of evidence presented in this study support these conclusions. First, a significant increase in alpha vbeta 3-integrin expression was detected on the surface of TNF-alpha -treated endothelial cells. Second, a marked increase in alpha vbeta 3-containing focal contacts was observed after cells were exposed to TNF-alpha . Third, only alpha vbeta 3-containing focal contacts, but not alpha 5beta 1-containing focal contacts, were detected in membrane protrusions of cells at the migration front. Fourth, a blocking antibody to alpha vbeta 3-integrins, but not a blocking antibody to alpha 5-integrin subunit, significantly inhibited TNF-alpha -induced cell migration.

The development of inflammation is mediated by cytokines released upon bacterial infection. Proinflammatory cytokines such as TNF-alpha 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-alpha 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-alpha 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-alpha -induced cell migration via a coordinated regulation between alpha vbeta 3- and alpha 5beta 1-integrins. On the other hand, it is well known that the angiogenic effect of TNF-alpha varies with cell lines and experimental conditions (12, 22, 26). Therefore it remains to be determined whether the TNF-alpha -induced coordinated regulation of alpha vbeta 3- and alpha 5beta 1-integrins observed in CPAE cells also occurs in other endothelial cell lines or under in vivo conditions.

The integrin complex alpha vbeta 3 interacts with a wide range of matrix proteins. It is, however, not expressed at high levels compared with alpha 5beta 1 on resting endothelial cells (37). A likely reason for its increased expression on TNF-alpha -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 alpha 5beta 1-integrins (8, 9). TNF-alpha 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 alpha 5beta 1-integrins and the increased localization of alpha vbeta 3-integrins in focal contacts.

The current study demonstrated changes in alpha vbeta 3 surface expression and focal contacts in endothelial cells after TNF-alpha 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 alpha vbeta 3 was detected only at cell-cell junctions in untreated cells, whereas focal contacts containing alpha vbeta 3-integrins were readily identified in cells after TNF-alpha exposure. In contrast, alpha 5beta 1-integrins were present in robust focal contacts in untreated cells, and the number of alpha 5beta 1 contacts was dramatically reduced after TNF-alpha exposure. No focal contacts containing alpha 5beta 1-integrins were observed in membrane protrusions of cells at the migration front. These coordinated changes in alpha vbeta 3- and alpha 5beta 1-integrins induced by TNF-alpha may mediate the observed membrane protrusion formation and cell migration.

Considerable evidence suggests that signaling among integrins is modulated by "cross talk" mediators. Integrin alpha vbeta 5-mediated vitronectin internalization appeared to require the ligation of alpha 5beta 1-integrins (27). Ligation of alpha vbeta 3-integrins was found to suppress alpha 5beta 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 alpha 5beta 1-integrin with fibronectin (3). Kim et al. (20) demonstrated that the ligation of alpha 5beta 1-integrins could potentiate alpha vbeta 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 alpha 5beta 1- and alpha vbeta 3-integrins induced by TNF-alpha 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-alpha induces the increase in alpha vbeta 3-integrin expression and endothelial cell migration, processes that may be essential for vascular wound healing and angiogenesis.


    ACKNOWLEDGEMENTS

We thank Kara L. Powell and Alice Damrau-Abney for technical assistance and Debbie Moran for administrative assistance.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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