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1Institute of Anatomy and Cell Biology, University of Würzburg, D-97070 Würzburg, Germany; 2Department of Physiology and Membrane Biology, School of Medicine, University of California, Davis, California 95616
Submitted 13 November 2003 ; accepted in final form 24 March 2004
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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permeability; Rho proteins; actin; stress fibers; vascular endothelial cadherin
In previous investigations, to understand the role of Rac-1 in permeability regulation, we carried out experiments to inhibit Rac-1 in cultured mouse endothelial cells parallel to those in intact microvessels. From studies using cultured epithelial cells, a role of Rac-1 in the stabilization of the adherens junction is known, and it has been demonstrated that Rac-1 is activated on the establishment of new adherens junctions via recruitment of a GTPase exchange factor which in turn activates Rac-1 (12). In cultured mouse endothelial cells we found similar strong evidence for a role for Rac-1 to regulate cell-cell adhesion (23). Inhibition of Rac-1 with LT reduced binding of vascular endothelial (VE)-cadherin-coated microbeads to the surface of endothelial cells as revealed by laser tweezer assays, suggesting that reduced VE-cadherin-mediated cell-cell adhesion takes part in the mechanisms to increase permeability. If similar mechanisms acted in intact microvessels, these experiments suggested that Rac-1-dependent mechanisms that control cell-cell adhesion may be necessary to maintain normal permeability. The limitation on this conclusion was that in cultured endothelial cells, we also found that both actin reorganization and contractile mechanisms contributed to the changes in the endothelial barrier after Rac-1 inhibition. The present experiments were designed to follow up these observations by investigating: 1) the effect of actin depolymerization in intact microvessels under conditions similar to those found with Rac-1 inhibition; 2) the effect of inhibition of contractile mechanisms on the LT-induced increase in microvesssel permeability, and 3) the idea that the formation of large gaps in cultured monolayers may not be the most reliable test for the contribution of myosin-based contractile mechanisms to increase permeability. Specifically, we have argued that large gaps may not be representative of physiological mechanisms regulating permeability and that an evaluation of the contribution of contractile mechanism based on the presence or absence of large gaps in the barrier may overestimate the contribution of contractile mechanisms to the action of small GTPases.
To test the action of Rac-1 inhibition on actin depolymerization, we build on previous investigations in the Würzburg laboratory (5–7), which demonstrated that the anchorage of VE-cadherin to the actin cytoskeleton (see also Fig. 6) is crucial for VE-cadherin-mediated adhesion because of the extremely low affinity (millimolar range) and half-life of single VE-cadherin bonds (about half a second). This was demonstrated, in part, by measuring adhesion of VE-cadherin-ectodomain-coated beads to the endothelial cell surface after actin breakdown by cytochalasin D. The effects of the actin-destabilizing agent cytochalasin D on VE-cadherin-mediated adhesion were completely inhibited by prior stabilization of actin filaments with jasplakinolide. From this result, we predicted that, if inhibition of Rac-1 decreased VE-cadherin-dependent adhesion by depolymerizing actin, the action of LT should be completely reversed by the actin-stabilizing agent jasplakinolide. One specific aim of the present investigations was to test this prediction in intact microvessels. We have also followed up the observation that, in cultured mouse endothelial cells, the effect of inhibiting Rac-1 with LT on VE-cadherin-coated bead adhesion could not be inhibited completely by jasplakinolide, suggesting additional mechanisms independent of actin depolymerization in vitro (23).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-, 
-, and 
-catenin, ZO-1, and claudin 5 have been verified by immunoblotting. Cultures were used for experiments when grown to confluent monolayers (day 3 up to day 7). Test reagents. C. sordellii LT was supplied by Dr. Holger Barth and Dr. Klaus Aktories (University of Freiburg, Germany) and used at 200 ng/ml for time intervals under cell culture conditions as described in Cell culture where significant glucosylation of Rac-1 by LT could be detected in our previous study (23). After 120 min of incubation with LT, 80% of Rac-1 in MyEnd cells was glucosylated and thereby inactivated. Specificity of toxin preparations was analyzed as described previously (23). Jasplakinolide (Calbiochem) was used at 0.1 µM and cytochalasin D (Sigma; St. Louis, MO) at 10 µg/ml. The Rho kinase inhibitor Y-27632 (Calbiochem; La Jolla, CA) was used at 30 µM, the MLCK inhibitor ML-7 at 10 µM, and the myosin ATPase inhibitor butanedione monoxime (BDM) at 5 mM (both from Sigma).
Cytochemistry. MyEnd cells were grown on coverslips coated with gelatin cross-linked with glutaraldehyde (1, 20). 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 phosphate-buffered saline (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 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 (15). After several rinses with PBS (3 x 5 min), monolayers were incubated for 60 min at RT with Cy3-labeled goat anti-rat IgG (diluted 1:600 in PBS, Dianova; Hamburg, Germany,).
For visualization of filamentous actin (F-actin), Alexa-phalloidin (diluted 1:60 in PBS, Mobitec; Göttingen, Germany) 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.
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 (6, 15).
Coating of polystyrene beads. After vortexing was completed, a 10-µl solution of protein A-coated superparamagnetic polystyrene microbeads (diameter 2.8 µm, Dynabeads; 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 sodium phosphate buffer, pH 8.1, in Hanks’ Balanced Salt Solution (HBSS; GIBCO, Karlsrühe) 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 buffer B containing 0.54 mg dimethyl pimelimidate dihydrochloride (DMP; Pierce, Rockford) to covalently cross-link protein A and bound Fc parts. After being washed 2 x 5 min in buffer C (100 µl 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 about 1.6 x 108 beads/ml.
Laser tweezer. As described previously (23), 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 of a high numerical aperature objective (x100, 1.3 oil, Zeiss; Oberkochen, Germany), coupled through the epiillumination 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 42 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 initiation of experiments (addition of various compounds). Beads were considered tightly bound when resisting laser displacement at 42 mW setting. For every condition, 100 beads were counted. Percentage of beads resisting laser displacement under various experimental conditions was normalized to control values.
Macromolecular flux across monolayers of cultured endothelial cells. MyEnd cells were seeded on polycarbonate transwell filters (3 µm pore size, 12 mm diameter, Costar; Corning, NY) in 12-well plates (Costar) and grown to confluency under culture conditions described in Cell culture. After being rinsed with fresh DMEM medium, cells were incubated with LT (200 ng/ml) in the presence or absence of BDM (5 mM) for 120 min. Lactate dehydrogenase (300 U/ml LDH, Sigma) was then added to the upper compartment. Aliquots of 10 µl were taken from the lower compartment at 0 and 5 min after addition of LDH. LDH activity was determined by using an in vitro LDH activity kit (Sigma) according to the manufacturer’s recommendations. Enzyme activity, measured spectrophotometrically by observing the rate of change of absorbance of NADH at 340 nm, was assumed to be proportional to LDH concentration. To rule out contribution of intracellular LDH by possible LT-induced cell damage, the activity of samples taken from the lower compartment before the addition of LDH to the upper chamber was measured and found to be similar for control and LT-treated monolayers.
Animal preparation. Rats were kept under conditions that conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals, approved by the Institutional Animal Care and Use Committee of the University of California, Davis, CA. Rats (male, 350–450 g, Sprague-Dawley, Hilltop Laboratory Animals) were anesthetized with pentobarbital sodium (65 mg/kg body wt) given subcutaneously. Anesthesia was maintained by giving additional pentobarbital (3 mg per dose) as needed. At the end of the procedure, rats were killed by pentobarbital overdose. Male rats were used to avoid 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 about 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 (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 cells. Mammalian Ringer solution was composed of (in mM) 132 NaCl, 4.6 KCl, 2 CaCl2, 1.2 MgSO4, 5.5 glucose, 5.0 NaHCO3, and 20 HEPES and Na-HEPES. The ratio of acid-HEPES to Na-HEPES was adjusted to achieve pH 7.40–7.45 for Ringer solutions. All perfusates were mammalian Ringer solution additionally containing BSA (10 mg/ml).
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 following occlusion of the vessel. The assumptions and limitations of the measurement have been evaluated in detail (17). The initial transcapillary water flow per unit area of the capillary wall [(Jv/S)0] was measured at predetermined capillary pressures of 30–60 cmH2O. Microvessel Lp was calculated as the slope of the relation between (Jv/S)0 and pressure. The volume flux per unit surface area of vessel wall (Jv/S) was estimated during single occlusions, lasting about 10 s each, at one 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 cmH2O, the approximate oncotic pressure contributed by the 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 (Jv/S)0 were made at 
10-min intervals for up to 90 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 for subsequent venule perfusion experiments as described previously (23). Jasplakinolide (0.1 µM), Y-27632 (30 µM), and BDM (5 mM) were applied simultaneously or before LT without significant differences. BDM and jasplakinolide were used alone for control experiments as indicated. Cytochalasin D was used at 10 µM because two preliminary experiments indicated no difference between 1 and 10 µM, and we previously used the latter concentration for our in vitro experiments (5, 23).
Statistics. Values throughout are expressed as means ± SE. Baseline Lp distributions are non-Gaussian in both frog mesentery capillaries and rat mesentery venules (16, 18). Because Lp distribution under conditions of the present experiments has not been investigated, we used the nonparametric Mann-Whitney statistic to test for differences in Lp between groups. Possible differences between groups in bead binding and macromolecular flux across cultured endothelial cell monolayers were assessed using unpaired Student's t-test. Statistical significance is assumed for P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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BDM reduces LT-induced loss of barrier function of cultured endothelial cells in vitro. To test the idea that actin-myosin contraction was an important mechanism to increase permeability in cultured endothelial cells after LT inhibition and to better compare the relative contribution of myosin-dependent contractile mechanisms to the LT-induced permeability changes in vivo and in vitro, transwell filter experiments using cultured MyEnd monolayers were performed. The activity of LDH in the lower chamber of control monolayers at 5 min was 0.003 ± 0.013 (n = 6) units. For monolayers exposed to LT for 120 min before the flux assay, the activity was 1.01 ± 0.156 (n = 6) units, about 340 times the control value. This high increase reflected the large gaps visible by immunostaining of MyEnd monolayers in parallel experiments (Fig. 4b). When MyEnd cells were incubated with LT in the presence of BDM (120 min), LDH activity was 0.105 ± 0.076 (n = 6), 35 times above control but nearly 10-fold less than with LT alone. No large gaps were found in the monolayers treated with BDM (Fig. 4e). This accumulation of LDH was significantly different from control values and demonstrates that contraction-independent mechanisms to increase monolayer clearance are present and may not be identified if only large gap formation is used as a criterion for changes in endothelial barrier integrity.
The effect of LT on VE-cadherin-mediated adhesion is not modulated by ML-7, BDM, or Y-27632. We tested whether inhibitors of myosin-dependent contraction modified the adhesion of VE-coated beads to endothelial cells in culture. LT (200 ng/ml, 120 min) reduced binding of VE-cadherin-coated microbeads to MyEnd cells to 35 ± 9% (n = 5) of control (Fig. 5). When Y-27632 (30 µM) or BDM (5 mM) were used for sequential or simultaneous treatment in addition to LT, bead binding was not significantly different from the results after LT treatment alone. The amount of bound beads changed to 37 ± 9% (n = 5) and 42 ± 5% (n = 5) following treatment in combination with Y-27632 and BDM, respectively. In the same way, ML-7 did not block the effect of LT on adhesion of VE-cadherin because only 50 ± 4% (n = 5) of the beads remained bound to the cellular surface. This demonstrates that Y-27632, ML-7, and BDM did not change the effect of Rac-1 inhibition on VE-cadherin-mediated adhesion. Also these mediators had no effect on bead adhesion in untreated cells. This demonstrated that VE-cadherin-mediated adhesion itself is not affected by myosin inhibitors as measured by cell-to-bead adhesion, even though in cultured endothelial cells, myosin-based contraction leads to formation of large intercellular gaps and significantly contributes to the LT-induced permeability increase (see above).
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DISCUSSION |
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Rac-1 stabilizes the endothelial barrier by regulating the anchorage between VE-cadherin and the actin cytoskeleton. Figure 6 summarizes current ideas how both types of intercellular junction, the adherens and the occludens junction, are linked to the actin cytoskeleton, and the integrity of the actin cytoskeleton is critical for maintenance of endothelial barrier properties. Rac-1 may act principally by depolymerizing actin, or it may act by regulating a cascade of pathways, some of which involve mechanisms such as the anchorage of VE-cadherin to the actin cytoskeleton. To investigate these possibilities, we tested the action of the actin-depolymerizing agent cytochalasin D at a concentration where it produced an increase in microvessel permeability similar to that caused by LT, and they both decreased cellular actin content by about 50% (23). As expected, stabilization of actin filaments with jasplakinolide blunted the effect of cytochalasin D on microvessel permeability. This conforms to our previous studies where jasplakinolide completely inhibited the weakening of VE-cadherin-coated bead adhesion in vitro (7, 23). The fact that jasplakinolide did not completely inhibit the effect of cytochalasin D might be attributed to the lower dose we applied for the in vivo experiments. Microvessel endothelium was found to be extremely sensitive to jasplakinolide leading to destabilizing effects of jasplakinolide on barrier functions when applied at the higher concentrations used in our previous study. In contrast, actin stabilization did not inhibit the effect of LT on microvessel Lp and only partially blocked the effect of LT on VE-cadherin-mediated adhesion. This indicates that the signaling pathway regulated by Rac-1 is, at least in part, separate from an action on the actin cytoskeleton, and it is likely that this involves the anchorage of VE-cadherin to the cytoskeleton via the adaptor molecules of the catenin family (Fig. 6). One possible mechanism is that Rac-1 regulates anchorage via the protein IQGAP-1. On the basis of experiments in cultured epithelial cells, Fukata et al. (12) proposed that IQGAP-1 may be sequestered by Rac-1, thereby preventing IQGAP-1 from intercalating between the cadherin-catenin complex and the actin cytoskeleton. Further experiments are needed to test whether this mechanism contributes to the action of Rac-1 in the endothelium in vitro and in vivo. We note however that inhibition of Rac-1 reduced cellular F-actin by 50% (23) in MyEnd cells, and this may also contribute to the effect of LT in vivo (Fig. 6).
Permeability increase in vivo does not require myosin-dependent contraction. Our study indicates that myosin-dependent contraction is not needed to increase permeability in microvessels in response to LT. Neither blocking Rho A via its effector kinase (ROCK) with Y-27632 nor directly inhibiting the myosin ATPase with BDM modulated the Lp increase induced by the inhibition of Rac-1 in the perfused microvessels. On the other hand, when vessels were perfused with BDM alone, resting levels of Lp decreased to about 50% of control values. This indicates that a contractile tonus is acting on the resting capillary endothelium with a tendency to destabilize baseline endothelial barrier properties. However, this contractile tonus does obviously not significantly contribute to the mechanisms that increase permeability in response to LT. This result is consistent with other recent investigations from this laboratory which indicated that Y-27632 and ML-7 reduced baseline permeability in rat microvessels but did not block the permeability increase induced by PAF or bradykinin (1, 2). However, inhibition of MLCK increased the rate of recovery toward base line in vessels treated with PAF, again suggesting that contractile forces can destabilize endothelial junctions.
In contrast to the results in microvessels, contractile mechanisms take part in the destabilization of endothelial barrier functions in response to inhibition of Rac-1 in cultured endothelial cells. When the myosin ATPase was inhibited by BDM, the LT-induced flux across MyEnd monolayers was reduced by 90% compared with experiments using LT alone. Accordingly, ML-7 and BDM completely blocked the formation of large intercellular gaps that were observed when MyEnd cells were treated with LT alone. On the other hand, permeability of MyEnd cells treated with LT in the presence of BDM was still 35 times higher than that in control monolayers, indicating that additional mechanisms different from contractile forces also contribute to maintenance of barrier integrity. In this context it is interesting that neither inhibitors of myosin activation had any effect on VE-cadherin-mediated adhesion because they did not alter binding of VE-cadherin-coated microbeads to the surface of resting MyEnd cells nor did they alter binding of VE-cadherin-coated beads to monolayers treated with LT. This indicates that myosin-dependent contraction is required for large gap formation in cultured endothelial cells and contributes to barrier dysfunction induced by inhibition of Rac-1. This result is also consistent with studies showing that contractile mechanisms play a key role in the increase of monolayer permeability in response to inflammatory mediators such as histamine and thrombin in cultured endothelial cells (3, 24) but not in microperfused capillaries (2). Specifically, in cultured endothelial monolayers the increase in permeability after treatment with histamine and thrombin could be prevented by inhibition of either ROCK (11, 24) or MLCK (22). This is generally understood in terms of actin-myosin contraction being driven by MLCK or ROCK. Both mechanisms increase the level of phosphorylated MLC. This phosphorylation at serine-19 is critical for interaction with actin filaments and activation of the myosin ATPase (8).
It should be noted that other mechanisms may also regulate actin-myosin contraction, and these may be involved in the action of Rac-1. In cultured MyEnd cells, thick stress fiber bundles appeared in response to inhibition of Rac-1. This remodeling of the stress fiber system was inhibited by treatment with ML-7 along with the inhibition of gap formation. However, stress fiber remodeling and gap formation was not inhibited by Y-27632. This suggests that stress fiber formation is not caused by activation of Rho but by direct signaling events through Rac-1 that may lead to activation of MLCK. It has been suggested that p21-activated kinase, an effector kinase of Rac-1, takes part in this process (13, 25).
Taken together, the results obtained from experiments with inhibitors of contractile mechanisms are consistent with the conclusion that the role of myosin-dependent contraction in the regulation of endothelial barrier properties seems to be different in vivo and in vitro and once more underlines the importance of parallel in vivo/in vitro studies in this field. Thus interpretation of experiments to investigate the mechanisms regulating endothelial barrier permeability must be done with caution using cultured endothelial cells because there may be upregulation of contractile mechanisms under some culture conditions. Another conclusion is that microscopic studies using immunofluorescence only reveal limited information about the functional integrity of intercellular junctions and that large intercellular gaps as seen in many cell culture studies contribute to permeability changes of monolayers but seem not to be the morphological equivalent of states of increased permeability in the in vivo situation. Functional assays such as the binding of microbeads coated with adhesion molecules can contribute to understand molecular mechanisms involved in the regulation of endothelial barrier functions. Our study shows that the physiological mechanism regulating permeability of microvessels is understood better from the quantification of VE-cadherin-mediated adhesion of microbeads than from the morphology of large gap formation in cultured endothelial cell monolayers.
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ACKNOWLEDGMENTS |
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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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