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1Institute of Anatomy and Cell Biology, University of Würzburg, D-97070 Würzburg; 2Department of Pharmacology and Toxicology, Albert-Ludwigs-University, D-79104 Freiburg, Germany; and 3Department of Human Physiology, University of California, Davis, California 95616
Submitted 13 March 2003 ; accepted in final form 18 September 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-catenin as well as the formation of intercellular gaps. To understand the mechanism by which inhibition of Rac caused disassembly of adherens junctions, we used laser tweezers to quantify VE-cadherin-mediated adhesion. LT and cytochalasin D, an actin depolymerizing agent, both reduced adhesion of VE-cadherin-coated microbeads to the endothelial cell surface, whereas the inhibitor of Rho kinase Y-27632 did not. Stabilization of actin filaments by jasplakinolide completely blocked the effect of cytochalasin D but not of LT on bead adhesion. We conclude that Rac regulates endothelial barrier properties in vivo and in vitro by 1) modulation of actin filament polymerization and 2) acting directly on the tether between VE-cadherin and the cytoskeleton.
vascular endothelial cadherin; adherens junction; permeability; Rho proteins; actin
-, -, and 
-catenin. Local breakdown of the endothelial barrier is a hallmark of the vascular response to a variety of permeability-increasing inflammatory agents (21). Accumulating evidence indicates that members of the Ras homology (Rho) family of small GTPases, Rho, Rac, and Cdc42, are important regulators of endothelial barrier properties by influencing both the endothelial actin-based cytoskeleton and the integrity of interendothelial junctions (23). Activated Rho acts primarily via Rho kinase (ROCK) to stimulate myosin-based contractility by upregulating phosphorylation of myosin light chain (2). The roles of Rac and Cdc42 in endothelial barrier regulation are less well understood.
Previous studies have demonstrated that inhibitory glucosylation of Rho family proteins by Clostridium difficile toxin B (assumed to inhibit Rho, Rac, and Cdc42) caused breakdown of the endothelial barrier in vitro (17) and in vivo (1). In contrast, selective inhibition of Rho activity by Clostridium botulinum C3 transferase-mediated ADP ribosylation or inactivation of ROCK by compound Y-27632 improved endothelial barrier properties both in vitro and in vivo (1, 2, 28). This indicates that toxin B-mediated breakdown of the endothelial barrier is due to inactivation of Rac or Cdc42 rather than to inactivation of Rho. Overexpression of inactive as well as constitutively active Rac by transfection of cultured endothelial cells with adenovirus expressing these proteins caused, within 14–16 h after transfection, a significant increase of transfer of FITC-dextran through monolayers grown on filters, further supporting the notion that normal Rac activity is important for endothelial barrier function (29). In contrast, transfection studies with constitutively active and inactive Cdc42 displayed no significant effect on monolayer permeability and integrity (26, 28, 29).
On the basis of these observations and our own experience with the influence of toxin B and Y-27632 on capillary permeability, the present study was undertaken to selectively inhibit Rac in capillary endothelial cells and to analyze the functional consequences of Rac inactivation on endothelial barrier properties in vitro and in vivo. In vivo experiments require rapid inactivation of Rac within a time window of 2 h. For this purpose, we used lethal toxin (LT) from Clostridium sordellii (8) at concentrations shown by parallel biochemical experiments to selectively inactivate Rac but not Rho and Cdc42 within 60–120 min after application. Under these conditions, LT increased capillary hydraulic conductivity (Lp) in vivo and caused dissociation of endothelial junctions in vitro. Barrier breakdown was accompanied by simultaneous loss of F-actin.
To discriminate between the general (unspecific) consequences of F-actin breakdown on barrier integrity versus a direct modulatory role of Rac inhibition on VE-cadherin-mediated adhesion, we quantified VE-cadherin adhesion by a laser tweezer approach. This assay is based on microbeads coated with dimeric VE-cadherin ectodomains. The beads are allowed to settle on the surface of cultured microvascular endothelial cells, where the beads recruit endothelial VE-cadherin, -catenin, and actin and induce assembly of typical adherens-type contacts at the free cellular surface (5). Adhesion of beads was probed by forces exerted by trapping the beads into the focus of a laser beam placed by microscopic control immediately above individual microbeads.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Test reagents. C. sordellii LT was prepared as described previously (18, 25), and used at 200 ng/ml for different time intervals under cell culture conditions as described in Cell culture. Activity and specificity of toxin preparations were analyzed as described in Glucosylation assay (1, 19). The ROCK inhibitor Y-27632 (Calbiochem; La Jolla, CA) was used at 30 µM, jasplakinolide (Calbiochem) at 10 µM, and cytochalasin D (Sigma; St. Louis, MO) at 10 µg/ml.
Glucosylation assay. MyEnd cells were incubated with various concentrations of LT in culture medium for various time intervals. Afterward, cells were harvested and lysed (18). Lysates were incubated with LT in the presence of 10 µM [14C]UDP-glucose for 60 min. After precipitation with ice-cold trichloroacetic acid [20% (wt/vol)], proteins were subjected to SDS-PAGE (12.5%), and the incorporated radioactivity of the 20- to 30-kDa band was analyzed by phosphoimaging. To demonstrate Rac-specific glucosylation by the LT preparations used in this study, recombinant RhoA, Rac-1, and Cdc42 were subjected to in vitro glucosylation under the same conditions applied to endothelial proteins.
Cytochemistry. MyEnd cells were grown on coverslips coated with gelatin cross-linked with glutaraldehyde (1, 22). LT was added to the culture medium at 200 ng/ml for 60 min, Y-27632 at 30 µM for 90 min, and cytochalasin D at 10 µM for 50 min. After incubation with toxins, the culture medium was removed, and monolayers were fixed for 10 min at room temperature (RT) with 2% formaldehyde (freshly prepared from paraformaldehyde) in PBS (consisting of 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, and 1.5 mM KH2PO4; pH 7.4). Afterward, monolayers were treated with 0.1% Triton X-100 in PBS for 5 min. After being rinsed with PBS at RT, MyEnd cells were preincubated for 30 min with 10% normal goat serum and 1% BSA at RT and incubated for 16 h at 4°C with rat monoclonal antibody 11D4.1 (undiluted hybridoma supernatant) directed to the ectodomain of mouse VE-cadherin (16) or with polyclonal antibody against -catenin (Sigma; dilution 1:200 in PBS). After several rinses with PBS (3 x 5 min), monolayers were incubated for 60 min at RT with Cy3-labeled goat anti-rat IgG or Cy3-labeled goat anti-mouse IgG (Dianova; Hamburg, Germany, diluted 1:600 in PBS).
For visualization of F-actin, Alexa-phalloidin (Mobitec; Göttingen, Germany, diluted 1:60 in PBS) was used (incubation for 1 h at RT). Cells incubated with antibodies or Alexa-phalloidin were rinsed with PBS (3 x 5 min). Coverslips were mounted on glass slides with 60% glycerol in PBS containing 1.5% n-propyl gallate (Serva; Heidelberg, Germany) as an antifading compound.
Scanning electron microscopy. MyEnd monolayers with adhering VE-cadherin-coated beads were fixed overnight with 6.5% glutaraldehyde in HBSS (GIBCO-BRL). After dehydration with a graded acetone series, critical point drying, and sputtercoating with palladium-gold (CPD 030, Bal-Tec; Schalksmühle, Germany), cells were examined with a JSM-840 scanning electron microscope (Jeol; Tokyo, Japan).
Quantification of F-actin content in MyEnd cells. MyEnd monolayers (untreated or treated with various substances) were fixed at RT with 3% formaldehyde in PBS, rinsed in PBS briefly, and permeabilized with 0.1% (vol/vol) Triton X-100 in PBS for 5 min. Each coverslip was then incubated for 1 h at 37°C with 500 µl (1 µg/ml) phalloidin covalently labeled with tetramethyl-rhodamine isothiocyanate (TRITC). These conditions have been shown to allow complete saturation of binding to cellular F-actin (11, 12). Cells were washed 3 x 1 min in PBS to remove unbound TRITC-phalloidin. TRITC-phalloidin was extracted from cells by two incubation steps for 1 h each with 1 ml of methanol at 37°C. For control of complete extraction of TRITC-phalloidin, coverslips were routinely screened by fluorescence microscopy. Methanol supernatants were pooled, centrifuged at 100,000 g for 20 min, and quantified in a fluorescence spectrometer at an excitation wavelength of 540 nm and an emission wavelength of 563 nm.
Recombinant VE-cadherin-Fc. We used the VE-cadherin-Fc fusion protein consisting of the complete extracellular domain of mouse VE-cadherin (EC1-EC5) fused to the Fc portion of human IgG1, including the hinge region and Ig domains CH2 and CH3. The protein was expressed by stably transfected Chinese hamster ovary cells and purified from culture supernatants by affinity chromatography using protein A agarose (Oncogene; Cambridge, MA). Further details have been published (4, 16).
Coating of polystyrene beads. After being vortexed, a 10-µl solution of protein A-coated superparamagnetic polystyrene microbeads (Dynabeads, diameter 2.8 µm, Dynal; Oslo. Norway) containing 2 x 109 beads/ml was washed three times using 100 µl of buffer A (100 mM sodium phosphate buffer; pH 8.1). Washing was performed by immobilization of beads for 1 min in a magnetic tube holder (MPC-E-1, Dynal) and reuptake in the corresponding buffer. Washed beads were suspended in 100 µl of 100 mM of buffer A in HBSS containing 10 µg of either VE-cadherin-Fc or of the Fc part of human IgG (for control experiments) and allowed to react for 16 h at 4°C under permanent slow overhead rotation to avoid aggregation. After being washed 3 x 5 min in 100 µl of buffer A and 3 x 5 min in buffer B (100 mM sodium borate; pH 9.0), beads were incubated for 45 min at RT in 100 µl of buffer B containing 0.54 mg dimethyl pimelimidate dihydrochloride (DMP; Pierce) to covalently cross-link protein A and bound Fc parts. After being washed 2 x 5 min in buffer C (100 µl of 0.2 M ethanolamine; pH 8.0), beads were incubated in buffer C for 2 h at RT. Finally, beads were washed 3 x 5 min in HBSS and stored in HBSS at 4°C for up to 8 days under permanent slow overhead rotation to avoid aggregation of beads. The concentration of beads in these stocks was 
1.6 x 108 beads/ml.
Laser tweezer. The home-built laser tweezer setup consisted of a Nd:Yag laser (1,064 nm), the beam of which was expanded to fill the back aperture a high-numerical aperture objective (100 x 1.3 oil, Zeiss; Oberkochen, Germany), coupled through the epi-illumination port of an Axiovert 135 microscope (Zeiss) and reflected to the objective by a dicroic mirror (FT 510, Zeiss). Through all experiments, the laser intensity was 40 mW in the focal plane. Coated beads (10 µl of stock solution) were suspended in 500 µl of culture medium and allowed to interact with MyEnd monolayers for 30 min at 37°C before the initiation of experiments (addition of various compounds). Beads were considered tightly bound when resisting laser displacement at the 40-mW setting. For every condition, 100 beads were counted. The percentage of beads resisting laser displacement under various experimental conditions was normalized to control values.
Animal preparation. Rats were kept under conditions that conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and experiments were approved by the Institutional Animal Care and Use Committee of the University of California-Davis. Rats (male, Sprague-Dawley, 350–450 g, Hilltop Laboratory Animals) were anesthetized with pentobarbital sodium (65 mg/kg body wt) given subcutaneously. Anesthesia was maintained by giving additional pentobarbital (3 mg/dose) as needed. At the end of the procedure, rats were killed by pentobarbital overdose. We used male rats to avoid potential variability due to hormonal fluctuations in females.
Preparation of rats for Lp measurement. Anesthetized rats were placed on a heating pad to maintain normal body temperature. A midline surgical incision of 1 cm was made in the abdominal wall, and the mesentery was gently taken out and spread over a pillar. The upper surface of the mesentery was continuously superfused with mammalian Ringer solution (temperature 37°C). All the experiments were carried out in straight nonbranched segments of venular microvessels, which were typically 25–35 µm in diameter. All vessels selected had brisk blood flow and were free of white blood cells.
Measurement of Lp of the microvessel wall. Measurements were based on the modified Landis technique, which measures the volume flux of water across the wall of a microvessel perfused via a glass micropipette after occlusion of the vessel. The assumptions and limitations of the measurement have been evaluated in detail (21).The volume flux per unit surface area of the vessel wall (Jv/S) was estimated during single occlusions, lasting 10 s each, at constant hydraulic pressure (usually 50 cmH2O) with the assumption that the net effective pressure determining fluid flow (Peff) was equal to the applied hydraulic pressure minus 3 cm H2O, the approximate oncotic pressure contributed by BSA in all perfusates (10 mg/ml). Lp was estimated for each occlusion as (Jv/S)/Peff. All perfusates were mammalian Ringer solution additionally containing serum albumin at 10 mg/ml (Sigma A 4378). LT was added to the perfusate and delivered via the micropipette continuously. Measurements of initial (Jv/S) were made at 10-min intervals for up to 80 min in the presence and absence of LT. In preliminary experiments, LT at 100 ng/ml was perfused through two venules for 150 min with no change from baseline Lp. Therefore, we used 200 ng/ml LT in subsequent venule perfusion experiments.
Statistics. Values are expressed as means ± SE. Statistical significance was assumed for P < 0.05. Because of the small sample size, we used nonparametric ANOVA (Kruskall-Wallis test) to examine the effects of LT on Lp over time.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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40 min. This time corresponds to the time in cultured MyEnd cells where first changes in junctional integrity in VE-cadherin-mediated adhesion became detectable during LT treatment (see Fig. 3). These experiments indicate that Rac activity is important for maintenance of capillary barrier properties in vivo.
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Glucosylation of Rho proteins by LT. To exclude the possibility that the LT preparation used in the present study might not only inactivate Rac but also Rho and Cdc42, we incubated recombinant RhoA, Rac-1, and Cdc42 with LT (200 ng/ml) for 120 min, as described in detail previously (19). Under these conditions, LT selectively glucosylated Rac but only weakly glycosylated Cdc42 and not RhoA (Fig. 2A). The degree of glucosylation was determined from the incorporation of [14C]UDP-glucose. After 120 min, Rac-1 was glucosylated 6- to 10-fold more than other small GTPases, RhoA or Cdc42 (Fig. 2A, inset). In contrast, toxin B (100 ng/ml, 150 min) induced strong glucosylation of all GTPases, RhoA, Rac-1, and Cdc42. We also measured glucosylation of Rac-1 in cultured MyEnd cells after varying times of exposure to 200 ng/ml LT. After 90 min, 60% of cellular Rac-1 was glucosylated and unavailable for [14C]UDP-glucose incorporation in cellular lysates (Fig. 2B). Prominent cellular changes were observed within 60–90 min of incubation with LT (see Fig. 3).
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Effect of toxins on morphology and F-actin content of cultured endothelial cells. In untreated MyEnd monolayers, VE-cadherin and -catenin displayed continuous immunolabeling of intercellular adherens junctions (Fig. 3, a and e). This continuous adherens belt displayed multiple interruptions in response to treatment with LT (200 ng/ml) for 60 min (Fig. 3, c and g). The first changes became visible at 40 min of treatment. Junctional sites lacking VE-cadherin and -catenin immunoreactivity coincided with the appearance of intercellular gaps. Inhibition of ROCK with Y-27632 (30 µM, 90 min) neither caused interruptions of adherens belt nor the formation of intercellular gaps (Fig. 3, b and f). Cytochalasin D (10 µM, 60 min), an inhibitor of actin polymerization, caused interruptions of junctional VE-cadherin and -catenin and created gaps that were less pronounced than those seen in response to LT (Fig. 3, d and h).
LT, Y-27632, and cytochalasin D caused significant changes in cellular F-actin content and distribution. Control MyEnd cells showed abundant stress fibers throughout the cytoplasm (Fig. 3i), which were largely reduced after treatment with both Y-27632 (Fig. 3j) and cytochalasin D (Fig. 3l). In contrast to cytochalasin D, which also induced fragmentation of the cortical actin system (as indicated by the arrows in Fig. 3), Y-27632 completely preserved this subset of actin filaments. In fact, the cortical actin staining appeared to be more prominent compared with controls. In LT-treated cells, stress fibers were less numerous but appeared to be thicker and more straight compared with controls (Fig. 3k). At sites of gap formation, the staining of cortical actin filaments was also reduced (see arrows in Fig. 3). This underlines the specificity of LT for Rac-1 as inhibition of Rho would be expected to cause disappearance of stress fibers (as seen in Fig. 3j). These changes in F-actin content were quantified in the present study by phalloidin binding of formaldehyde-fixed monolayers permeabilized with Triton X-100. Compared with control levels, treatment with LT and cytochalasin D resulted in a 50% and 56% reduction of F-actin, respectively. Y-27632 reduced F-actin by only 20% (Table 1).
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Effect of LT, toxin B, cytochalasin D, and Y-27632 on VE-cadherin-mediated adhesion probed by laser tweezer. Microbeads coated with VE-cadherin-Fc were allowed to settle for 30 min on MyEnd monolayers before the addition of various compounds. During this period, one of several small filopodial processes was seen to connect individual beads to the endothelial cell surface (Fig. 4a). In control experiments (beads coated with albumin), no attachment structures were seen to develop between the beads and cell surface. In LT-treated cultures, only a few beads could be encountered attached to the endothelium by processes (Fig. 4b). In addition, LT caused the formation of numerous gaps between endothelial cells. As shown recently by immunostaining, typical adherens-type contacts containing VE-cadherin, -catenin, and F-actin were formed between the beads and surface of endothelial cells (5). In the present study, under control conditions, close to 70% (mean 66%) of beads resisted displacement by laser tweezer, whereas the remaining 30% of beads could be removed by laser trapping and thus were considered not specifically bound. In monolayers treated with either LT or cytochalasin D, microbeads resisting displacement by laser tweezer dropped to 55% and 46% of control values (Table 2), respectively. Interestingly, this was about the same value as measured after treatment with toxin B (50%). Inhibition of ROCK by Y-27632 did not significantly increase or decrease the VE-cadherin-mediated bead adhesion (113%) (Table 2). This indicates that the reduction of VE-cadherin binding was mainly caused by inhibition of Rac, because Rac-1 is the only GTPase that is inhibited by both LT and toxin B.
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To see whether the weakening of VE-cadherin-mediated adhesion by LT and cytochalasin D was caused by partial disassembly of the actin filament system or by direct effects of these compounds on the tether between VE-cadherin and F-actin, cells with attached VE-cadherin-coated beads were pretreated with jasplakinolide (10 µM) for 60 min to cause stabilization of F-actin. Jasplakinolide competes with phalloidin for the same binding site on F-actin. Thus actin filaments stabilized by jasplakinolide resist labeling with phalloidin (7). With the use of the phalloidin binding assay, only 7% of control level F-actin was detectable after jasplakinolide treatment (Table 1). Thus the remaining 93% of actin filament binding sites can be assumed to be occupied and stabilized by jasplakinolide (14). Jasplakinolide caused a significant 25% increase of firmly attached VE-cadherin beads (83% of all beads; Table 3). Subsequent treatment of monolayers with LT in the presence of jasplakinolide caused 25% of the bound beads to detach (75% of the attached beads remained bound; Table 3) during the after 60 min of treatment. In contrast, jasplakinolide treatment completely abolished bead release by cytochalasin D (101% remained bound; Table 3). As expected, Y-27632 neither caused weakening nor strengthening of bead adhesion in both controls and jasplakinolide-treated cultures. These experiments show that weakening of VE-cadherin-mediated adhesion by cytochalasin D is mainly caused by disassembly of F-actin, whereas only 50% of the antiadhesive activity of LT is caused by decay of F-actin. The remaining inhibitory action of LT on VE-cadherin-mediated adhesion is independent of F-actin and appears to act directly on the VE-cadherin anchoring complex.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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LT action on endothelial adherens junctions. LT has been previously shown to cause disorganization of endothelial junctions as well as final detachment of cells after prolonged treatment for 12–16 h (29). Because LT has been reported to also glucosylate Ras and Ral (8), it is possible that these long-term effects of LT may at least in part be caused by secondary actions on other signaling pathways involved in proliferation and cell survival.
To minimize the possibility of secondary phenomena, we confined our experiments to a short time course of 30–90 min, during which Rac became inactivated by 50–70%. Because the onset of both the increase of capillary permeability and formation of intercellular gaps in MyEnd monolayers became obvious after a lag phase of 30–60 min, we conclude that inactivation of 30–50% of cellular Rac by glucosylation is sufficient for both destabilization of adherens junctions and a significant weakening of adhesion of VE-cadherin-coated beads. Furthermore, LT and toxin B, when used under conditions of comparable Rho protein glucosylation, both reduced binding of VE-cadherin-coated beads by 50%. The fact that Rac-1 is the only GTPase inhibited by both toxins indicates that mainly inhibition of Rac-1 seems to be causing the described effects. Whether partial inhibition of Cdc42 and of still-unidentified other 20- to 30-kDa GTPases might also contribute to barrier breakdown cannot be excluded. Further insight into the role of Rac activity for the maintenance of endothelial barrier properties may come from experiments in which human umbilical vein endothelial cell (HUVEC) monolayers were transfected with recombinant adenoviruses expressing inactive as well as constitutively active Rac mutants (29). Furthermore, inhibition of Rac-regulated intercellular adhesion seems to be involved in barrier breakdown by thrombin in cultured endothelial cells (27).
There are also studies reporting that microinjected active and inactive Rac did not induce gap formation in HUVEC monolayers and that microinjected inactive Rac blocked TNF--induced gap formation (6, 26, 28). Although interpretation of these data is difficult because they depend on expression levels and assumed dominant negative effects of the transfected and microinjected proteins, the main conclusion to be drawn is that Rac activity needs to be precisely controlled to maintain endothelial barrier properties. A functional link between VE-cadherin and Rac can be also concluded from studies with VE-cadherin-deficient endothelial cells. Transfection of these cells with VE-cadherin resulted in both the upregulation and membrane recruitment of Rac and the Rac-specific guanosine exchange factor Tiam-1 (20).
Mechanism of LT-induced gap formation and role of the actin cytoskeleton in VE-cadherin-mediated adhesion. The experiments in the present study show that inhibition of Rac by LT has an immediate strong (30–60 min) effect on endothelial junctional integrity as shown by the severalfold increase of capillary permeability and opening of interendothelial junctions in cultured MyEnd cells.
This breakdown of junctional integrity was accompanied at the same time scale by an 50% reduction of adhesiveness of VE-cadherin-coated beads attached to the free endothelial cell surface. These bead-to-cell contacts can be considered ectopically located adherens-type junctions associated with F-actin, -catenin, and VE-cadherin (5). Unlike intercellular adhesive junctions, adherens junctions between beads and the free cell surface are not challenged by contractile forces so that contraction-dependent opening of intercellular junctions can be separated from junctional dissociation caused by weakening of cadherin binding.
In MyEnd cells, the main sites of the actin cytoskeleton are intercellular junctions (specialized section of the cortical cytoskeleton) and stress fibers. Treatment with both LT and cytochalasin D caused loss of stress fibers and fragmentations of the cortical actin cytoskeleton. However, exposure to Y-27632 resulted in loss of stress fibers only. The junction-associated actin system became even more prominent in response to Y-27632. It is reasonable to assume that the integrity of the junction-associated actin filament system is important for intercellular adhesion. This would explain why LT and cytochalasin D caused weakening of adhesion, whereas adhesion remained unchanged in response to treatment with Y-27632. Because the adhesive interaction (transinteraction) of VE-cadherin has been shown to be an extremely low-affinity reaction (millimolar range) with a very short half-life of cadherin-cadherin bonds (bonds dissociate almost twice a second), cadherins disconnected from the actin filament cytoskeleton will disappear from junctional sites by rapid lateral diffusion (3, 4). Lateral diffusion of VE-cadherin has been previously shown to increase 10-fold in response to treatment with cytochalasin D (from 0.017 to 0.17 µm2/s), and this increase of mobility was accompanied by a dispersal of cadherin clusters (5).
LT-induced weakening of bead adhesion was only partly caused by an LT-induced loss of F-actin, as demonstrated by experiments in which cellular F-actin was stabilized by pretreatment with jasplakinolide. Whereas cytochalasin D-induced weakening of VE-cadherin-mediated adhesion was completely blocked by jasplakinolide pretreatment, 50% of anti-adhesive activity of LT was independent of F-actin. This indicates that LT-induced inhibition of Rac exerts an additional direct effect on cadherin-mediated adhesion that is independent of the integrity of the actin filament cytoskeleton. Disconnection of cadherins from the actin filament system and depolymerization of F-actin are probably the two main mechanisms of how LT-induced inhibition of Rac leads to untethering of VE-cadherin, followed by disassembly of endothelial adherens junctions and subsequent endothelial barrier breakdown.
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ACKNOWLEDGMENTS |
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GRANTS
This study was supported in part by National Heart, Lung, and Blood Institute Grants HL-44485 and HL-28607 and by Deutsche Forschungsgemeinschaft Grant SFB 487 (TP B5).
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FOOTNOTES |
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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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REFERENCES |
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