VCAM-1-mediated Rac signaling controls endothelial cell-cell contacts and leukocyte transmigration

Sandra van Wetering, Nadia van den Berk, Jaap D. van Buul, Frederik P. J. Mul, Ingrid Lommerse, Rogier Mous, Jean-Paul ten Klooster, Jaap-Jan Zwaginga, and Peter L. Hordijk

Department of Experimental Immunohematology, Sanquin Research at CLB and Laboratory for Clinical and Experimental Immunology, Academic Medical Center, 1066 CX Amsterdam, The Netherlands

Submitted 4 February 2003 ; accepted in final form 13 April 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Leukocyte adhesion is mediated totally and transendothelial migration partially by heterotypic interactions between the {beta}1- and {beta}2-integrins on the leukocytes and their ligands, Ig-like cell adhesion molecules (Ig-CAM), VCAM-1, and ICAM-1, on the endothelium. Both integrins and Ig-CAMs are known to have signaling capacities. In this study we analyzed the role of VCAM-1-mediated signaling in the control of endothelial cell-cell adhesion and leukocyte transendothelial migration. Antibody-mediated cross-linking of VCAM-1 on IL-1{beta}-activated primary human umbilical vein endothelial cells (pHUVEC) induced actin stress fiber formation, contractility, and intercellular gaps. The effects induced by VCAM-1 cross-linking were inhibited by C3 toxin, indicating that the small GTPase p21Rho is involved. In addition, the effects of VCAM-1 were accompanied by activation of Rac, which we recently showed induce intercellular gaps in pHUVEC in a Rho-dependent fashion. With the use of a cell-permeable peptide inhibitor, it was shown that Rac signaling is required for VCAM-1-mediated loss of cell-cell adhesion. Furthermore, VCAM-1-mediated signaling toward cell-cell junctions was accompanied by, and dependent on, Rac-mediated production of reactive oxygen species and activation of p38 MAPK. In addition, it was found that inhibition of Rac-mediated signaling blocks transendothelial migration of monocytic U937 cells. Together, these data indicate that VCAM-1-induced, Rac-dependent signaling plays a key role in the modulation of vascular-endothelial cadherin-mediated endothelial cell-cell adhesion and leukocyte extravasation.

human umbilical vein endothelial cells; vascular-endothelial cadherin; F-actin; reactive oxygen species; p38 mitogen-activated protein kinase; vascular cell adhesion molecule


THE ENDOTHELIAL LINING of the vasculature represents an important and dynamic barrier that controls diffusion and transport of plasma proteins and solutes and migration of leukocytes (4, 17). Under a variety of (patho)-physiological conditions, such as immune surveillance or inflammation, activated leukocytes are recruited from the circulation into the tissues. This extravasation process is composed of a tightly controlled sequence of events, driven by chemokines that are presented on the endothelial cell surface, and by adhesion molecules. Extravasation is initiated by selectin-mediated rolling and integrin-dependent adhesion of the leukocytes to the endothelium. This adhesion is followed by spreading, crawling, and the actual transendothelial migration of the leukocyte (10, 53, 63).

It is well established that leukocyte integrins are essential for stable adhesion to the endothelium and can also mediate rolling interactions (2, 8, 18, 27, 51). Various studies have demonstrated that leukocyte-endothelium interactions are accompanied by changes in intracellular Ca2+ levels (30, 38), the organization of the endothelial actin cytoskeleton (38, 46), and phosphorylation of myosin light chain kinase (23, 26, 46). Evidence that this signaling is initiated by Ig-like cell adhesion molecules (Ig-CAM) has come from studies showing endothelial cytoskeletal rearrangements, protein phosphorylation, and activation of transcription factors upon intercellular adhesion molecule (ICAM)-1 or vascular cell adhesion molecule (VCAM)-1 cross-linking (19, 21, 36, 40, 44). Furthermore, ICAM-1-mediated lymphocyte migration across endothelium appears to require the activation of the small GTP-binding protein Rho (1), which is a member of the family of Rho-like GTPases that are known to control cytoskeletal rearrangements and cadherin function (6, 29, 54, 57).

Previous studies from various laboratories, including our own, have shown that endothelial cell-cell junctions play an important role in the control of leukocyte transmigration (17, 24, 28, 50, 56). Endothelial cell-cell adhesion is largely dependent on the homotypic cell adhesion molecule vascular-endothelial cadherin (VE-cadherin, CD144) (15). VE-cadherin is a transmembrane glycoprotein, complexed via its cytoplasmic tail to armadillo family members such as {beta}- or {gamma}-catenin, which, in turn, are associated with {alpha}-catenin and the actin cytoskeleton (14, 16). Inhibition of VE-cadherin function increases neutrophil transmigration in vitro (28) and promotes neutrophil extravasation in a murine model for acute peritonitis (24). Furthermore, leukocyte adherence to endothelial cells results in the formation of intercellular gaps and a transient and focal loss of VE-cadherin localization between adjacent endothelial cells (50, 56). This gap formation is important for efficient transendothelial migration (56) and suggests that leukocyte adhesion induces endothelial signaling toward the cell-cell junctions.

Recently, we demonstrated that in human bone marrow endothelial cells, VCAM-1 cross-linking results in the formation of gaps and increased endothelial permeability (56). Furthermore, we showed that in primary endothelial cells, VE-cadherin-mediated cell-cell adhesion is controlled, at least in part, by the Rho-like GTPase Rac1 and involves Rac-mediated formation of reactive oxygen species (ROS) (57). In the present study, we further investigated the intracellular signaling events that occur upon VCAM-1 cross-linking and that might control VE-cadherin function. We show that VCAM-1 cross-linking results in the formation of gaps and a concomitant focal loss of VE-cadherin staining. This effect was dependent on the activation of the small GTPase Rac1 and the formation of ROS. Moreover, we identified p38 mitogen-activated protein kinase (p38 MAPK) as a downstream component in this VCAM-1-induced signaling pathway toward VE-cadherin. These data reveal a VCAM-1-mediated signaling cascade that plays an important role in the modulation of endothelial junctions during leukocyte extravasation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies and reagents. Mouse monoclonal antibodies (MAb) against VE-cadherin (cl75), {beta}-catenin, phosphotyrosine (PY-20), or Rac were from Transduction Laboratories (Becton Dickinson, Amsterdam, The Netherlands). VCAM-1 antibody (1G11 [PDB] ) was purchased from Immunotech SA (Marseille, France), and the phospho-p38 MAPK and p38 MAPK rabbit polyclonal antibodies were purchased from Cell Signaling Technology (Beverly, MA). Texas red-phalloidin, FITC-Dextran 3000, dihydrorhodamine-123 (DHR), and Alexa 488 goat-anti-mouse Ig secondary antibody were all from Molecular Probes (Leiden, The Netherlands). Cross-linking studies were performed with goat-anti-mouse-Ig F(ab)2 fragments from Jackson ImmunoResearch (Baltimore, MD). Goat-anti-mouse-Ig conjugated to horseradish peroxidase (HRP) was purchased from CLB (Amsterdam, The Netherlands). N-acetyl-L-cysteine (NAC) and catalase were from Sigma (Sigma Chemical, St. Louis, MO). The inhibitor of p38 MAPK (SB-203580) was purchased from Calbiochem (La Jolla, CA). Clostridium botulinum exoenzyme C3 was obtained from Kordia Laboratory Supplies (Leiden, The Netherlands). The Rac17–32 peptide that was recently shown to inhibit Rac function (58) was designed in combination with the protein transduction domain of the human immunodeficiency virus TAT-protein (41). The isolated transduction domain (YGRKKRRQRRRG) as well as the resulting fusion peptide (YGRKKRRQRRRGTCLLISYTTNAFPGEY) was synthesized at the Netherlands Cancer Institute (Amsterdam, The Netherlands). Peptides were dissolved in dimethyl sulfoxide (100 mg/ml) and used in a final concentration of 0.2 mg/ml.

Cell culture. Primary human umbilical vein endothelial cells (pHUVEC) were harvested as described (7) and maintained in RPMI 1640 (GIBCO, Grand Island, NY) supplemented with 10% (vol/vol) heat-inactivated human serum (HAS; CLB), 2 mM glutamine (GIBCO), 100 U/ml penicillin, and 100 µg/ml streptomycin (GIBCO) in fibronectin-coated culture flasks. The cells were used from passages 2–4.

Primary human monocytes were isolated from fresh buffy coats, using centrifugal elutriation as described previously (25). U-937 cells (a monocytic cell line) derived from a human histiocytic lymphoma were purchased from American Type Culture Collection (Manassas, VA). The cells were grown in culture flasks in a humidified, 5% CO2-95% air atmosphere in RPMI 1640 (GIBCO) containing 10% heat-inactivated fetal calf serum (GIBCO), 2 mM L-glutamine, 50 IU/ml penicillin, and 50 µg/ml streptomycin (complete medium).

Rac activation. The Rac activity assay was performed as previously described (48) with minor modifications. Briefly, pHUVEC cultured in fibronectin-coated 80-cm2 cell culture dishes until confluency were stimulated overnight with IL-1{beta} (10 ng/ml) and incubated for 45 min with anti-VCAM-1 antibodies. After VCAM-1 was cross-linked with the secondary antibody for the indicated periods, cells were washed with ice-cold PBS containing 2 mM Ca2+-0.5 mM Mg2+ and subsequently lysed for 10 min on ice in lysis buffer [50 mM Tris · HCl, pH 7.2, 5 mM MgCl2, 1% NP-40, 10% glycerin, 150 mM NaCl, and a protease inhibitor cocktail (Roche)]. Cleared lysates were incubated for 30 min at 4°C with glutathione S-transferase-p21-activated kinase (GST-PAK) protein, after which glutathione-Sepharose beads were added to precipitate GTP-bound Rac. Total lysates and precipitates were analyzed on Western blot with the MAb against Rac1.

Immunocytochemistry. Cells, cultured on fibronectin-coated glass coverslips, were treated with IL-1{beta} overnight, and VCAM-1 was cross-linked as described in Rac activation for various periods. Next, cells were fixed and permeabilized with 2% paraformaldehyde and 0.5% (vol/vol) Triton X-100 in washing buffer [PBS containing 0.5% (vol/vol) HAS and 1 mM Ca2+] for 20 min at room temperature. Cells were stained with the indicated mouse monoclonal or polyclonal antibodies, washed, and incubated with Alexa 488-conjugated goat-anti-mouse-Ig antibodies. F-actin was visualized by Texas red-phalloidin (1 U/ml). Images were recorded with a Zeiss LSM 510 confocal laser scanning microscope. For time-lapse confocal microscopy, cells were kept in culture medium in a temperature-controlled incubation chamber at 37°C. To assess the role of Rho, we pretreated monolayers for 18 h with the C3 toxin (5 µg/ml).

Detection of ROS production. To measure generation of ROS in endothelial cells, we cultured pHUVEC on fibronectin-coated glass coverslips and stimulated them with IL-1{beta} (10 ng/ml) for 6 h. Next, the VCAM-1 antibody was added for 30 min, and cells were simultaneously loaded with DHR (30 µM) for 30 min. After the cells were washed, VCAM-1 was cross-linked, and the fluorescence of DHR was quantitated by time-lapse confocal microscopy. For ROS scavenging or inactivation, cells were incubated overnight with 5 mM NAC or 3 mg/ml catalase as described (12). We found no effects of these pretreatments on the expression of VCAM-1 by the endothelial cells.

Detection of p38 MAPK by Western blotting. Endothelial cells were grown to confluency in 60-mm dishes, stimulated overnight with IL-1{beta} (10 ng/ml), and treated with anti-VCAM-1 antibody for 30 min. Next, cells were washed and incubated with the cross-linking secondary antibody [F(ab)2 fragments]. At the indicated times, cells were washed with ice-cold PBS containing 1 mM CaCl2 and 0.5 mM MgCl2 and lysed in 100 µl of SDS-sample buffer. Samples were run on 12.5% SDS-PAGE and transferred onto 0.2-µm nitrocellulose filters (Schleicher and Schuell, Dassel, Germany). The blots were blocked with 5% dried milk protein in TBST buffer [10 mM Tris · HCl, pH 7.4, 150 mM NaCl, and 0.5% (vol/vol) Tween 20], washed with TBST, and incubated with anti-phospho-p38 MAPK antibody (1:500 in TBST) or anti-total p38 MAPK antibody (1:500 in TBST) to assess equal loading. This was followed by incubation with goat-anti-rabbit-IgG-HRP (1:4,000; Amersham) at room temperature. p38 MAPK proteins were visualized using the ECL kit (Amersham).

Transmigration assay. Migration assays were performed in Transwell plates (Costar, Cambridge, MA) of 6.5 mm in diameter, with 5-µm pore filters. Before the assays were performed, U-937 cells were pretreated for 24 h with 1 mM dibutyryl cAMP to induce expression of the C5a receptor. Confluent endothelial monolayers on the filters were treated with IL-1{beta} (10 ng/ml) and, where indicated, pretreated in parallel with C3 toxin (5 µg/ml, 18 h), the Tat-Rac17–32 peptide (30 min), or carrier. Monolayers were then washed with transmigration medium (RPMI with 0.25% human serum albumin). At the start of the assay, 105 U-937 cells were placed in the upper compartment of the Transwells and allowed to migrate toward 10 nM C5a in the lower compartment for 120 min in a tissue culture incubator. Next, cells in the lower compartment were harvested and quantitated by fluorescence-activated cell sorter (FACS) analysis in the presence of a known amount of fluorescent beads (Molecular Probes). The percentage of migrated cells was calculated as a fraction of the input. Statistical analysis was performed using a Student's t-test.

Electric cell-substrate impedance sensing. Endothelial cells were added at 100,000 cells per array to fibronectin-coated electrode arrays (surface area 0.8 cm2) and grown to confluency. Each array contains a 0.001-cm2-diameter gold electrode and a 1-cm2 gold counter electrode. The arrays are connected to a phase-sensitive lock-in amplifier that allows continuous recordings of the electrical resistance of the monolayers, which was on average between 10 and 15 x 103 {Omega}. After electrode check of the arrays and of the basal electrical resistance of the endothelial monolayers under resting conditions, VCAM-1 was cross-linked as described in Rac activation and the electrical resistance was continuously monitored at 37°C at 5% CO2 with the electric cell-substrate impedance sensing (ECIS) model 100 controller (BioPhysics, Troy, NY) as described previously (61).


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
VCAM-1 cross-linking induces intercellular gaps in pHUVEC. To document the morphological changes within endothelial monolayers in response to VCAM-1-mediated signaling, we incubated IL-1{beta}-treated pHUVEC with anti-VCAM-1 antibody that was subsequently cross-linked for 30 min. This cross-linking mimics the clustering of integrins on the leukocyte and leads to the clustering of their ligands after leukocyte cell adhesion (47). Because of the lack of an appropriate VE-cadherin polyclonal antibody, endothelial cell-cell junctions were stained with a polyclonal anti-{beta}-catenin antibody. As shown in Fig. 1A, VCAM-1 cross-linking resulted in stress fiber formation, intercellular gaps, and loss of cell-cell adhesion. At sites of gap formation, concomitant loss of {beta}-catenin staining was observed. This effect was transient, because these events were completely restored after ~6 h of cross-linking. Treatment of endothelial cells with only the anti-VCAM-1 or the cross-linking F(ab)2 antibody did not alter the morphology of the monolayer (Fig. 1A). Figure 1B shows the surface distribution of cross-linked VCAM-1, which can be seen in small clusters covering the endothelial cell surface. Complementary analysis of monolayer electrical resistance shows that VCAM-1 cross-linking induces a transient loss of resistance (Fig. 1C). The effect of VCAM-1 is restored after 2 h, in line with our earlier data (56), obtained with permeability assays. The difference with the experiments in Fig. 1A, which required more time for restoration of the VCAM-1 effects, are likely due to the difference in substrate; we found that cells grown on fibronectin-coated glass coverslips consistently require more time to reseal their junctions compared with cells cultured on fibronectin-coated ECIS electrodes or fibronectin-coated tissue culture plastic.



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Fig. 1. Effect of VCAM-1 cross-linking on the actin cytoskeleton and cell-cell contacts. A: primary human umbilical vein endothelial cells (pHUVEC) were incubated for 30 min with medium (control), the monoclonal anti-VCAM-1 antibody, the cross-linking F(ab)2 antibodies, or with both of these in sequence to cross-link endothelial VCAM-1 [VCAM-1/F(ab)2]. Only under conditions of cross-linking of VCAM-1 was prominent formation of stress fibers observed, accompanied by the appearance of intercellular gaps (asterisks). Endothelial cells were stained for F-actin (red) and {beta}-catenin (green). Images are representative of at least 3 independent experiments. Bar, 20 µm. B: monolayers of pHUVEC were incubated with antibodies to VCAM-1 followed by cross-linking or were incubated with control antibodies (control), after which the cells were fixed and stained with antibodies to VCAM and with Texas red-phalloidin to visualize F-actin. VCAM-1 cross-linking induced intercellular gaps (asterisks) and clustering of VCAM-1 on the endothelial cell surface, as concluded from the punctate staining of VCAM-1 compared with the control. Bar, 20 µm. X-L, cross-linking. C: monolayer electrical resistance was monitored, as described in MATERIALS AND METHODS, for a period of 2 h after the addition of a cross-linking antibody to endothelial cells that were preincubated with the anti-VCAM-1 ({blacksquare}) or a control antibody ({bullet}). The drop in resistance surpasses the effect of addition of reagents to the cells, as shown for the control. The effect of VCAM-1 cross-linking was transient, and endothelial resistance was back to normal after 2 h. D: pHUVEC were pretreated with the Rho-inactivating C3 toxin, after which VCAM-1 was cross-linked for 30 min. C3 pretreatment prevented both the VCAM-1-mediated induction of F-actin stress fibers and intercellular gaps (asterisks). Cells were stained for F-actin (red) and phosphotyrosine (green), to detect focal adhesions. Images are representative of at least 3 independent experiments. Bar, 20 µm.

 

Activation of ICAM-1 has been shown to induce activation of p21Rho (49). Because VCAM-1 activation leads to a Rho phenotype in the pHUVEC (i.e., induction of stress fiber formation), we tested for a role for Rho by treatment of the cells with the Rho-inactivating C3 toxin, before VCAM-1 cross-linking. Figure 1D shows that C3 prevented the VCAM-1-induced formation of actin stress fibers, focal adhesions, and intercellular gaps, indicating that Rho activity is required for this response.

VCAM-1 cross-linking induces Rac activation in endothelial cells. Recently, we showed that transduction of endothelial cells with a cell-penetrating constitutively active form of Rac (Tat-RacV12) induced (Rho-dependent) formation of stress fibers and concomitant loss of VE-cadherin-mediated cell-cell adhesion (57), although transduction with a cell-permeable Tat-RhoV14 protein only partially mimicked the effects of Tat-RacV12. Because these effects were very similar to those of VCAM-1 cross-linking, we analyzed whether VCAM-1-mediated loss of cell-cell adhesion involves activation of Rac using a pull-down assay. As shown in Fig. 2A, cross-linking of VCAM-1 resulted in Rac activation within 5 min that lasted up to 15 min. At later time points (>30 min), Rac activity decreased to control levels (not shown).



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Fig. 2. Role of Rac in the VCAM-1 signaling. A: after cross-linking of VCAM-1, pHUVEC were lysed and Rac GTP was isolated using the GST-PAK pull-down assay, as described in MATERIALS AND METHODS. VCAM-1 cross-linking induced an increase in the levels of activated Rac, as shown by Western blotting of the samples with an anti-Rac MAb (top). Cell lysates were blotted in parallel to control for differences in the total amount of Rac protein (bottom). B: VCAM-1 cross-linking was performed after pretreatment with a cell-permeable peptide inhibitor of Rac (Tat-Rac17–32). Cells were stained with Texas red-phalloidin to detect F-actin (red) and with polyclonal antibodies for {beta}-catenin to visualize cell-cell junctions (green). The Tat-Rac17–32 peptide prevented VCAM-1-induced intercellular gap formation (asterisks). Bars, 20 µm. Data are representative of 3–4 independent experiments.

 

To test whether Rac activity was required for the VCAM-1-mediated loss of cell-cell adhesion, we performed VCAM-1 cross-linking in the presence of a cell-permeable peptide inhibitor of Rac, Tat-Rac17–32. This peptide represents a part of the effector loop of Rac1 and will compete in the cells with Rac-effector interactions, thereby preventing downstream signaling (58). In parallel experiments, we confirmed that this peptide blocked membrane ruffling in epithelial cells, induced by hepatocyte growth factor (not shown). In the presence of this Tat-Rac17–32 peptide, VCAM-1-mediated loss of cell-cell adhesion and focal loss of {beta}-catenin staining was completely abolished (Fig. 2B). These data indicate that Rac activity is involved in VCAM-1-mediated signaling, resulting in loss of cell-cell adhesion.

VCAM-1-mediated loss of cell-cell adhesion requires formation of ROS. ROS, e.g., H2O2, have been shown to reduce cadherin-based cell-cell adhesion and to affect endothelial cell function (39). In addition, our group recently showed that ROS mediate the formation of gaps in pHUVEC transduced with the cell-permeable Tat-RacV12 protein (57). In line with these data, we observed a rapid increase (within 5–15 min) in ROS production upon VCAM-1 cross-linking in pHUVEC (Fig. 3A). This effect lasted for ~30–45 min, after which the response declined. Moreover, Rac activity was shown to be essential for the formation of ROS upon VCAM-1 cross-linking, because the effect was blocked completely in the presence of the inhibitory Tat-Rac17–32 peptide (Fig. 3B). This peptide does not inhibit Rac activation (data not shown). As expected, formation of ROS was also prevented in the presence of the oxygen scavengers NAC (not shown) and catalase (Fig. 3B). Moreover, we found that after preincubation with NAC or catalase, which by itself left the actin cytoskeleton and {beta}-catenin distribution unaltered, VCAM-1-mediated gap formation and concomitant loss of {beta}-catenin staining was prevented (Fig. 3C). This finding is in line with our recent findings in human bone-marrow endothelial cells (56). The induction of actin stress fibers was not reduced by NAC or catalase, indicating that ROS are not required for this effect and that the induction of stress fibers is not sufficient to induce intercellular gaps. To test whether these effects also accompanied direct leukocyte-endothelium interactions, we seeded primary human monocytes for 30 min on monolayers of IL-1{beta}-activated pHUVEC. These experiments showed that scavenging of ROS also prevented interendothelial gap formation, induced by adhesion of primary monocytes (Fig. 3D). ROS scavenging did not interfere with the adhesion of the monocytes to the endothelium. Together, these results point to a role for ROS as important signaling molecules involved in VCAM-1-mediated and monocyte adhesion-induced loss of cell-cell adhesion.



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Fig. 3. Role of ROS in the effects of VCAM-1 in pHUVEC. A: pHUVEC were labeled with dihydrorhodamine-123 (DHR) before VCAM-1 cross-linking, as described in MATERIALS AND METHODS. VCAM-1 induced a time-dependent increase in endothelial ROS ({blacktriangleup}), which was detectable for 30–45 min. Control cells do not show increased production of ROS ({blacksquare}). Data are representative of 3 independent experiments and are presented as the percent increase over basal values. B: the VCAM-1-induced increase in endothelial ROS was blocked by preincubation of pHUVEC with the Tat-Rac17–32 peptide. As a control, cells were preincubated overnight with catalase, which prevented the VCAM-1-induced increase in ROS production. Data are presented as percentages of maximum increase, measured 30 min after cross-linking of VCAM-1. Data are means of 2–3 independent experiments. C: preincubation of pHUVEC with the oxygen scavenger N-acetyl-L-cysteine (NAC) prevented VCAM-1-induced intercellular gap formation (asterisks), indicating that ROS are required for this effect. Preincubation of pHUVEC monolayers with catalase had a comparable effect. Endothelial cells were stained for F-actin (red) and {beta}-catenin (green). Stress fiber formation was still induced; this was not caused by the NAC or Tiron pretreatments. Bar, 20 µm. Cat, catalase. D: monocytes were seeded for 30 min on monolayers of activated pHUVEC, pretreated or not with NAC to scavenge ROS. Phase-contrast images show the induction of intercellular gaps (asterisks) in the endothelial monolayer; this is prevented by the NAC pretreatment. Images are representative of 3 independent experiments. Adherent monocytes appear as phase bright. Bar, 50 µm. EC, endothelial cells.

 

VCAM-1 cross-linking results in phosphorylation of p38 MAPK. MAPKs, such as p42/p44 MAPK, JNK, and p38 MAPK, are essential signaling molecules implicated in cell growth, differentiation, and cellular responses to environmental stress. Furthermore, p38 MAPK is required for oxidant-induced rearrangement of the endothelial cytoskeleton (31) and VEGF-mediated stress fiber formation (45). Because these events are also induced by VCAM-1-mediated signaling, we analyzed whether p38 MAPK is activated by VCAM-1. As shown in Fig. 4A, cross-linking of VCAM-1 induced a maximal phosphorylation of p38 MAPK within 2–5 min, followed by a decline after 10 min. The VCAM-1-mediated phosphorylation of p38 MAPK was prevented by the Tat-Rac17–32 peptide, indicating that activation of Rac is required (Fig. 4B). Moreover, pretreatment of endothelial cells with NAC also largely prevented VCAM-1-induced phosphorylation of p38 MAPK, providing evidence that p38 MAPK activation occurs downstream of Rac and ROS (Fig. 4C). To test whether p38 MAPK is involved in VCAM-1-mediated loss of cell-cell adhesion, we pretreated endothelial cells with SB-203580 (a pharmacological inhibitor of p38 MAPK) for 30 min, after which VCAM-1 was cross-linked, also for 30 min in the presence of the inhibitor. Figure 4D shows that in the presence of SB-203580, VCAM-1-induced gap-formation and loss of cell-cell adhesion was partially reduced, confirming the notion that p38 MAPK is involved in VCAM-1-mediated loss of cell-cell adhesion.



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Fig. 4. Phosphorylation of p38 MAPK after VCAM-1 activation. A: VCAM-1 was cross-linked for various periods as indicated, and cell lysates were blotted with either phospho-specific antibodies to p38 MAPK (pp38; top) or antibodies that detect all p38 in the lysate, as a loading control (bottom). VCAM-1 cross-linking induced a rapid and transient phosphorylation of p38 MAPK. This phosphorylation of p38 MAPK by VCAM-1 activation was prevented by the Tat-Rac17–32 peptide (B; VCAM-1 cross-linking for 2 min) as well as by preincubation of the cells with NAC (C), indicating that the phosphorylation of p38 MAPK is downstream of Rac as well as ROS. D: preincubation of the endothelial monolayers with the p38 inhibitor SB-203580 (SB) largely prevented the effects of VCAM-1 on induction of F-actin stress fibers and intercellular gap formation (asterisks). Endothelial cells were stained for F-actin (red) and {beta}-catenin (green). Blots and images are representative of 3–5 independent experiments. Bar, 20 µm.

 

Role for the VCAM-1-mediated Rac signaling in leukocyte transendothelial migration. To further establish the contribution of VCAM-1-mediated activation of Rac to leukocyte transendothelial migration, we analyzed C5a-induced migration of monocytic U-937 cells across pHUVEC in an in vitro Transwell-based assay. The migration of U-937 cells across monolayers of activated HUVEC is mediated by very late antigen (VLA)-4 and VCAM-1 when HUVEC are activated by IL-1 and C5a is the chemotactic factor (9). Before the induction of transendothelial migration, the HUVEC monolayers were pretreated either with carrier, the isolated protein transduction domain (Tat-peptide), or the Tat-Rac17–32 peptide. The pHUVEC were washed to remove excess peptide, and migration of U-937 cells toward a gradient of C5a was analyzed. These experiments showed that inhibition of endothelial Rac strongly reduced the efficiency of U-937 transendothelial migration (Fig. 5). This inhibition was more pronounced compared with the effect of C3, used to inactivate endothelial Rho, suggesting that the role of Rac is dominant over Rho in the control of leukocyte transmigration. This is in line with earlier data showing that activated Rac can induce loss of endothelial cell-cell adhesion, whereas active Rho is less effective in this respect (57).



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Fig. 5. Role of endothelial Rac and Rho proteins in transendothelial migration of monocytic U-937 cells. As indicated, monolayers of pHUVEC on Transwell filters were pretreated with carrier, the Tat control peptide, or the Tat-Rac17–32 peptide for 30 min or with the Rho-inactivating C3 toxin for 18 h. After the monolayers were washed, C5a-induced transendothelial migration of U-937 cells was assayed as described in MATERIALS AND METHODS. Data represent means ± SE of 3 independent experiments performed in duplicate. *P < 0.01; **P < 0.002 vs. Tat control.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The interplay between activated leukocytes and the vascular wall is an important aspect of various (patho)-physiological processes, including immune surveillance, (chronic) inflammatory disorders, and stem cell homing. Since the initial studies of Huang et al. (30), who showed that endothelial calcium signaling contributes importantly to neutrophil transendothelial migration, the insights in the active role of the endothelium in the extravasation process have been growing steadily. The identification of ICAM-1-mediated activation of the small GTPase Rho (1, 19, 20), together with the studies that showed neutrophil-induced activation of endothelial myosin light chain kinase and its role in transendothelial migration (23, 26, 46), further underscored the role for endothelial signaling in the transmigration process. In the present study we provide evidence for a signaling pathway that controls VE-cadherin-mediated endothelial cell-cell adhesion during the process of leukocyte transendothelial migration.

Given the close interaction and interdependence of the VE-cadherin-catenin complex with the actin cytoskeleton, it is apparent that increased stress fiber formation, mediated by Rho, will impose contractile strength on the cadherins, counteracting their adhesive function. In line with this, it has been shown that Rho activity is required for the modulation of cadherin function (60, 62). However, transduction of pHUVEC with a cell-permeable, activated form of RhoA is not sufficient to induce large intercellular gaps (57) and induces only a partial increase in endothelial permeability. Similarly, activating Rho by the cytotoxic necrotizing factor from Escherichia coli also did not impair endothelial integrity or the distribution of VE-cadherin, despite the induction of prominent stress fibers (60). Finally, the effect of Rho inactivation with the C3 toxin on leukocyte transendothelial migration are limited (Fig. 5 and Ref. 56). Together, these data indicate that additional signaling must be involved in the induction of reduced endothelial cell-cell adhesion after activation of endothelial adhesion molecules.

Our current data suggest that the production of ROS by the Rac1 GTPase may constitute this additional signaling toward endothelial cell-cell junctions. In an earlier study we showed that protein transduction of an activated form of Rac is sufficient to induce (Rho dependent) loss of endothelial cell-cell adhesion and that this effect was mediated by ROS (57). The addition of H2O2 to endothelial (as well as epithelial) cells leads to a rapid and dramatic loss of cadherin-based cell-cell adhesion (data not shown; Ref. 59). In the current study we have shown that inactivation of ROS with catalase or scavenging of ROS with NAC prevents VCAM-1 or monocyte adhesion-induced interendothelial gap formation in pHUVEC. Finally, scavenging of ROS (40, 56) as well as inhibition of Rac signaling (Fig. 5) reduces monocyte transendothelial migration, indicating that the Rac-ROS pathway is an essential component of endothelial signaling during transendothelial migration. The work of Matheny et al. (40) is of particular relevance because these authors showed that VCAM initiates production of ROS in the endothelium, which was found to mediate efficient lymphocyte transendothelial migration. It is important to emphasize that it is not clear to what extent this pathway is used by transmigrating leukocytes in general. This will likely depend on the adhesion molecules used by various types of leukocyte. In agreement with this, we found that neutrophil transendothelial migration, which is mainly dependent on {beta}2-integrin-ICAM-1 interactions (52), does not seem to require ROS-mediated signaling in endothelial cells.

The current data suggest the following model (Fig. 6). Upon integrin-mediated adhesion of leukocytes to endothelial cells, Ig-CAMs such as VCAM-1 are clustered and initiate intracellular signaling via an as yet unidentified mechanism. This signaling involves activation of a Rac-dependent pathway, which results in the production of ROS and activation of p38 MAPK. The link among cadherin function, Rac, and ROS has been established in several different cell systems (6, 28, 29, 54, 57, 59), but the molecular details of the connection between p38 MAPK and cadherin function have yet to be explored. However, there are various studies that provide evidence that p38 MAPK is involved in reorganization of the endothelial actin cytoskeleton (31, 33) and in TNF-induced vascular permeability (22, 34, 43). Our data indicate that inhibition of p38 MAPK also reduces the effects of VCAM-1 on actin reorganization (Fig. 4D), although this does not appear to occur after ROS scavenging, which reduces p38 MAPK activity (Fig. 3C). This discrepancy might be due to the different means of interfering with the pathway. For instance, NAC pretreatment blocks activation of MAPK but leaves basal activity intact. In addition, this signaling pathway may not be completely linear, and it may be that p38 MAPK is required for Rho to be fully active. Future experiments are required to clarify the role of p38 MAPK in this signaling pathway.



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Fig. 6. Schematic representation of VCAM-1-induced endothelial signaling pathways. VE-cad, vascular-endothelial cadherin; {alpha}, {beta}, and {gamma}: {alpha}-, {beta}-, and {gamma}-catenin. Dashed arrows from p38 MAPK indicated the lack of knowledge about the link between this kinase and endothelial contractility or tyrosine phosphatases that could link p38 MAPK to the control of VE-cadherin. For further details, see text.

 

VCAM-1 cross-linking also leads to Rho-dependent formation of actin stress fibers and contractility, which is required but is not sufficient (57) to disrupt cell-cell adhesion. The Rac-ROS pathway, in combination with Rho-mediated contractility, may regulate leukocyte adhesion (42) and transendothelial migration, in part because of its role in inducing a transient and focal loss of endothelial cell-cell adhesion. In this regard it is of interest that shear stress has recently been identified as a transmigration-promoting stimulus and that cell tension, which will be affected by fluid shear, can induce Rac activation (11, 32).

Having identified the Rac-mediated signaling in the control of VE-cadherin-mediated cell-cell adhesion, a number of important questions require further analysis. First, it is unclear how adhesion molecules such as VCAM-1 may transduce signals into the cell. The COOH-terminal domains of ICAM-1 and VCAM-1 are only 29 and 19 amino acids long, respectively, and no signaling motifs have been identified, other than a series of basic amino acids in the juxtamembrane region that have been implicated in binding ezrin/radixin/moesin (ERM) proteins (64). Interestingly, the association of VCAM-1 with ERM proteins was recently described by Barreiro et al. (3) to occur in an endothelial docking structure for adherent leukocytes that contains adhesion, signaling, and structural proteins, suggesting that formation of this structure may be required for the induction of adhesion-induced endothelial signaling. Ig-CAM deletion mutants and transfection studies with (domains of) ERM proteins are required to provide more insight in this topic.

Second, the link between Rac and endothelial ROS is unclear. Endothelial cells have been reported to express the Rac-controlled neutrophil NADPH oxidase in association with the actin cytoskeleton (37), but whether this complex is implicated in the control of VE-cadherin function and cell-cell adhesion is presently unknown. A group of related enzymes (NADPH oxidases, or NOXs) has recently been identified (35), and preliminary data, based on RT-PCR analysis, show that a subset of these proteins is expressed in HUVEC as well. However, the mode of regulation and possible function of these enzymes in endothelial cells remain to be established.

Finally, it is unknown how activation of Rac ultimately contributes to reduced cadherin function. The production of ROS may play an important role in that ROS can inactivate tyrosine phosphatases (5), leading to a net increase in tyrosine kinase activity. In this respect it is of interest that the SHP-2 tyrosine phosphatase was found to be associated with the VE-cadherin complex and becomes dissociated upon cell treatment with thrombin (55). Regulation of the composition of these protein complexes may be another mechanism to control the tyrosine phosphorylation status and function of the cadherins and associated molecules.

In conclusion, the present study provides new data on endothelial signaling, initiated by cross-linking of VCAM-1. This signaling modulates endothelial cell-cell adhesion, monolayer integrity, and, probably as a result, leukocyte transendothelial migration. Given the importance of VCAM-1 in the induction of atherosclerosis (13), it is very likely that the relevance of VCAM-1-induced Rac activation and production of ROS extends beyond the process of leukocyte transendothelial migration and that this pathway has an important, more general role in the control of endothelial cell function.


    DISCLOSURES
 
This work was supported by grants from the Landsteiner Foundation for Blood Transfusion Research (9910), the Netherlands Cancer Foundation (99-2000), and the Netherlands Organisation for Scientific Research (902-26-221).


    ACKNOWLEDGMENTS
 
P. L. Hordijk is a fellow of the Landsteiner Foundation for Blood Transfusion Research.

Present address of S. van Wetering: Dept. of Pulmonology, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands.


    FOOTNOTES
 

Address for reprint requests and other correspondence: P. L. Hordijk, Sanquin Research at CLB, Dept. of Experimental Immunohematology, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands (E-mail: p.hordijk{at}sanquin.nl).

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.


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