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(Circulation Research. 2000;87:335.)
© 2000 American Heart Association, Inc.

Molecular Medicine

Activation of RhoA by Thrombin in Endothelial Hyperpermeability

Role of Rho Kinase and Protein Tyrosine Kinases

Geerten P. van Nieuw Amerongen, Sanne van Delft, Mario A. Vermeer, John G. Collard, Victor W. M. van Hinsbergh

From the Gaubius Laboratory TNO Health and Prevention (G.P.v.N.A., M.A.V., V.W.M.v.H.), Leiden; Division of Cell Biology (S.v.D., J.G.C.), The Netherlands Cancer Institute, Amsterdam; and Institute for Cardiovascular Research (G.P.v.N.A., V.W.M.v.H.), Vrije Universiteit, Amsterdam, the Netherlands.

Correspondence to Prof Dr V.W.M. van Hinsbergh, Gaubius Laboratory TNO-PG, PO Box 2215, 2301 CE Leiden, the Netherlands. E-mail vwm.vanhinsbergh{at}pg.tno.nl

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract—Endothelial cells (ECs) actively regulate the extravasation of blood constituents. On stimulation by vasoactive agents and thrombin, ECs change their cytoskeletal architecture and small gaps are formed between neighboring cells. These changes partly depend on a rise in [Ca²⁺]_i and activation of the Ca²⁺/calmodulin-dependent myosin light chain kinase. In this study, mechanisms that contribute to the thrombin-enhanced endothelial permeability were further investigated. We provide direct evidence that thrombin induces a rapid and transient activation of RhoA in human umbilical vein ECs. Under the same conditions, the activity of the related protein Rac was not affected. This was accompanied by an increase in myosin light chain phosphorylation, the generation of F-actin stress fibers, and a prolonged increase in endothelial permeability. Inhibition of the RhoA target Rho kinase with the specific inhibitor Y-27632 reduced all of these effects markedly. In the presence of Y-27632, the thrombin-enhanced permeability was additionally reduced by chelation of [Ca²⁺]_i by BAPTA. These data indicate that RhoA/Rho kinase and Ca²⁺ represent 2 pathways that act on endothelial permeability. In addition, the protein tyrosine kinase inhibitor genistein reduced thrombin-induced endothelial permeability without affecting activation of RhoA by thrombin. Our data support a model of thrombin-induced endothelial permeability that is regulated by 3 cellular signal transduction pathways.

Key Words: human endothelial cells • RhoA • protein tyrosine kinases • calcium • phosphorylation

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The endothelium is the main barrier for blood constituents and actively regulates the extravasation of blood components to the surrounding tissues. Formation of minute gaps between endothelial cells (ECs), for instance during inflammation, leads to extravasation of fluid and macromolecules and may cause life-threatening edema. Analogous to smooth muscle cell contraction, phosphorylation of the myosin light chain (MLC) by the Ca²⁺/calmodulin-dependent kinase I, the classic MLC kinase, directs the actin-myosin–based contraction process in ECs and is dependent on calcium ions and calmodulin.¹ In addition to a Ca²⁺/calmodulin-dependent transient increase in permeability induced by histamine and substance P,² thrombin induces a prolonged disturbance of endothelial barrier function.³ This is associated with a reorientation of the F-actin cytoskeleton, a prolonged MLC phosphorylation, and the formation of intercellular gaps.⁴ In contrast to smooth muscle cells, MLC phosphorylation in ECs in vivo does not result in a general contraction of the cells, but a contraction process does occur at the margins of a cell.²

In the last few years it has become clear that MLC phosphorylation is a highly regulated process in which small G proteins of the Rho family play a crucial role. Most attention has been paid to the small GTPase RhoA. In smooth muscle cells, activation of RhoA results in "calcium sensitization"; ie, independently of changes in [Ca²⁺]_i, MLC phosphate levels increase by inhibition of the smooth muscle myosin phosphatase, resulting in force generation.⁵ Other proteins of the Rho family of small GTPases also could be involved in MLC phosphorylation. Activation of the p21-activated kinase (Pak), an enzyme that is activated by the small G proteins Cdc42 and Rac, was shown to affect MLC phosphorylation.^{6 7} In HeLa cells, overexpression of Pak reduces MLC kinase activity.⁷ In contrast, in ECs Pak appears to increase MLC phosphorylation by being an MLC kinase itself.^{6 8}

It is a matter of debate which Rho-like small GTPases are involved in endothelial permeability. In human umbilical vein ECs (HUVECs), the thrombin-induced endothelial hyperpermeability was reduced by C3 transferase from Clostridium botulinum, a specific inhibitor of Rho.^{4 9} C3 transferase also reduced the thrombin-induced MLC phosphorylation.^{4 9 10} Several investigators have doubted a role of RhoA in thrombin-enhanced permeability¹¹ or have suggested that Rac participates in cytoskeletal remodeling by thrombin in ECs.¹²

In this study, we investigated whether thrombin induces an activation of RhoA and Rac using 2 newly developed assays for activation of RhoA^{13 14} and Rac.¹⁵ Protein tyrosine kinases (PTKs) have been implicated in intracellular signaling in thrombin-enhanced barrier dysfunction by many investigators.^{4 10 16 17} Because a genistein-sensitive PTK has been reported to act upstream of RhoA in lysophosphatidic acid–stimulated ECs,¹⁸ we subsequently studied whether PTKs also are required for activation of RhoA by thrombin. Furthermore, we evaluated the involvement of Rho kinase, a downstream target of RhoA, in the thrombin-enhanced endothelial permeability and MLC phosphorylation using the Rho kinase inhibitor Y-27632.^{19 20}

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
Cell culture reagents were obtained as described previously.⁴ Bovine thrombin was from Leo Pharmaceutical Products. Horseradish peroxidase (HRP) was obtained from Sigma Chemical Company. BAPTA–acetoxymethyl ester and rhodamine-phalloidin were from Molecular Probes. Genistein was from Alexis Inc. Y-27632 was supplied by Yoshitomi Pharmaceutical Industries. [³²P]Orthophosphoric acid and Tran³⁵S (a mixture of ³⁵S-labeled amino acids) were from ICN Pharmaceuticals, Inc. Antibody against Rho kinase was provided by Dr L Lim (Institute for Molecular and Cell Biology, Singapore).²¹ Antibody against RhoA was from Santa Cruz Biotechnology, Inc. Antibody against Rac was from Transduction Laboratories. Anti–platelet myosin Ig (nonmuscle) was from Sanbio. Secondary antibodies were from Dakopatts.

Cell Culture and Evaluation of Barrier Function
HUVECs were isolated and cultured as previously indicated.²² Barrier function was evaluated by the transfer of HRP across HUVEC monolayers grown on fibronectin-coated polycarbonate filters of the Transwell system (Costar).⁴

MLC Phosphorylation
MLC phosphorylation was measured by a double labeling technique. To that end, HUVECs were incubated for 24 hours with 150 µCi/mL Tran³⁵S label and for 2 hours with 150 µCi/mL [³²P]orthophosphoric acid in phosphate-free buffer before stimulation of the cells. Details have been given previously.⁴

Rho Activity Assay
Rhotekin binding assays were essentially performed as described.^{13 14} Briefly, 20 cm² confluent HUVECs were preincubated for 1 hour in medium 199+1% human serum albumin (HSA). Cells were stimulated and lysed. Lysates were cleared by centrifugation and incubated with bacterially produced glutathione-S-transferase (GST)—Rho binding domain of Rhotekin (RBD) immobilized on glutathione-coupled Sepharose beads for 30 minutes at 40°C. Beads were washed, eluted in Laemmli sample buffer, and analyzed by Western blotting using a rabbit polyclonal anti-RhoA antibody.

Rac Activity Assay
Pak binding assays were performed as described.¹⁵ Confluent HUVECs (20 cm²) were preincubated for 1 hour in medium 199+1% HSA. Cells were stimulated and lysed. Lysates were cleared by centrifugation and incubated with GST-Pak–Cdc42/rac1 interactive binding domain (CRIB, ie, the GTPase binding domain) immobilized on glutathione-coupled Sepharose beads for 30 minutes at 40°C. Beads were washed, eluted in Laemmli sample buffer, and analyzed by Western blotting using a mouse monoclonal anti-Rac1 antibody.

Detection of Rho Kinase by Immunoblotting
Detection of Rho kinase by immunoblotting was performed as indicated by Lim et al.²¹

Immunocytochemistry
The presence of F-actin was visualized by direct staining with rhodamine-phalloidin and photographed using a digital Nikon Coolpix 900 camera. Total gap area was quantified from these pictures using QWin image analysis software (Leica Imaging Systems).

Statistical Analysis
Data are reported as mean±SEM. Comparisons among >2 groups were made by 1-way ANOVA, followed by the Bonferroni-adjusted ² test. Comparisons of time curves of 2 groups were made using repeated-measures ANOVA. Individual group comparisons were done using a Student t test for post hoc comparisons of the means. Differences were considered significant at P<0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Activation of RhoA but Not Rac by Thrombin
To evaluate whether thrombin directly activates RhoA and Rac in ECs, we used 2 newly developed assays for RhoA and Rac activity. The assay for RhoA activity is based on binding of activated RhoA (RhoA-GTP) to a GST-Rhotekin fusion protein,^{13 15} which is reflected in the pull-down fraction of Figure 1 (top). Under control conditions hardly any RhoA-GTP could be detected. The amount of RhoA-GTP in the pull-down fraction was greatly enhanced 30 seconds after stimulation with 1 U/mL thrombin and diminished after 5 minutes. The overall amount of RhoA did not change (Figure 1, bottom). In contrast, the activity of Rac, which was measured by use of a GST-Pak fusion protein,¹⁵ did not change or even slightly decreased after thrombin stimulation (Figure 1, middle). With the same reagents, activation of Rac by bradykinin in rat PC12 cells was demonstrated previously (see Reference ¹⁵ ). Thus, thrombin rapidly and transiently activates RhoA but not Rac in HUVECs.

View larger version (40K): [in this window] [in a new window]   Figure 1. Thrombin transiently activates RhoA and has no effect on Rac in human ECs. Top, Immunoblot showing activation of RhoA after exposure of various time periods (in minutes) of HUVECs to 1 U/mL thrombin. Cell lysates were incubated with GST-RBD beads as described in Materials and Methods, the beads were washed, and the bound protein was analyzed by Western blotting using an antibody specific for RhoA. Middle, Immunoblot showing that thrombin has no effect on Rac in the same samples. Cell lysates were incubated with GST-Pak beads. The bound protein was analyzed by Western blotting using an antibody specific for Rac. Bottom, Before performing the GST pull-down, 1/40 of each sample was analyzed on Western blot using an antibody specific for RhoA, showing that equal amounts of protein were present in all samples. Similar results were obtained in 3 independent EC cultures.

Rho Kinase Is Involved in the Thrombin-Enhanced Permeability
To investigate whether RhoA acts in endothelial permeability via Rho kinase, the cell-permeant Rho kinase inhibitor Y-27632 was used at a high concentration of 10 µmol/L that was shown to completely inhibit Rho kinase.¹⁹ We first checked by Western blotting whether Rho kinase was present in ECs and found 1 single band at the expected molecular mass of 160 kDa in HUVECs (Figure 2A, inset) and other types of human macro- and microvascular ECs (glomerular and foreskin microvascular ECs, iliac artery and vein ECs, and aorta ECs; data not shown). The Rho kinase inhibitor Y-27632 was used to study the role of Rho kinase in the thrombin-induced endothelial barrier dysfunction. Preincubation for 1 hour with 10 µmol/L Y-27632¹⁹ had no effect on basal permeability (Figure 2A) but significantly attenuated the thrombin-enhanced endothelial permeability, as evidenced by a decreased passage of the marker molecule HRP through the endothelial monolayer. This attenuation was partial, even when Y-27632 was used at a higher concentration (up to 100 µmol/L was tested, which had the same effect as 10 µmol/L) or when its preincubation time was prolonged to 3 hours (data not shown).

View larger version (22K): [in this window] [in a new window]   Figure 2. Involvement of Rho kinase in thrombin-enhanced endothelial permeability. A, Effect of the Rho kinase inhibitor Y-27632 on the passage of HRP through HUVEC monolayers under basal conditions (, ) and after exposure to 1 U/mL thrombin (•, ). Cells were preincubated for 1 hour in medium 199+1% HSA without (, •) or with 10 µmol/L Y-27632 (, ), and HRP passage was measured as described in Materials and Methods. Values are mean±SEM of 9 cultures in 3 independent experiments. The interaction between time and Y-27632 treatment was significant (P=0.000) for thrombin-stimulated cells. Treatment with Y-27632 resulted in lower HRP passage compared with no treatment. *P<0.05, Y-27632-pretreated vs nonpretreated cells that were stimulated with thrombin. Inset, Detection of Rho kinase by Western blot in HUVEC extracts. Estimated molecular mass of the protein was 160 kDa. B, Effect of BAPTA and Y-27632 on basal (open bars) and thrombin-induced HRP passage (solid bars) across HUVEC monolayers. Cells were pretreated for 1 hour with 3 µmol/L BAPTA, 10 µmol/L Y-27632, or both. HRP passage was measured 2 hours after sham treatment or exposure to 1 U/mL thrombin. Values are mean±SD of 6 cultures in 2 experiments. *P<0.05, cells pretreated with BAPTA or Y-27632 vs nonpretreated cells. #P<0.05, cells pretreated with a combination of BAPTA and Y-27632 vs pretreatment with each compound alone.

Inhibition of Rho kinase by Y-27632, which was maximal at 10 µmol/L, together with chelation of intracellular Ca²⁺ ions by BAPTA (Figure 2B), nearly completely blocked the thrombin-enhanced HRP passage, suggesting that RhoA/Rho kinase- and Ca²⁺- dependent processes act on EC permeability by separate pathways.

Inhibition of Rho Kinase Reduces MLC Phosphorylation and Cytoskeletal Reorganization
Alterations in endothelial permeability are accompanied by actin nonmuscle myosin interaction, which is regulated by MLC phosphorylation. MLC phosphorylation, as determined by ³²P incorporation in MLC, was increased on activation of ECs by thrombin in agreement with previous observations (Figure 3).^{3 4 9 23} Preincubation with the Rho kinase inhibitor Y-27632 significantly reduced MLC phosphorylation under both basal and thrombin-stimulated conditions (Figure 3). Formation of both thrombin-induced F-actin stress fibers and small gaps between neighboring ECs was significantly prevented by Y-27632 (Figure 4). Thus, activation of Rho kinase by thrombin is involved in the thrombin-induced MLC phosphorylation and is required for the proper formation of stress fibers and small gaps.

View larger version (25K): [in this window] [in a new window]   Figure 3. MLC phosphorylation is inhibited by Y-27632. A, Autoradiograph of MLCs immunoprecipitated from cells under basal conditions (-) and 10 minutes after stimulation with 1 U/mL thrombin (+). Cells were labeled with 32P and 35S as described previously4 and preincubated with 10 µmol/L Y-27632 for 1 hour. MLC phosphorylation was measured as described in Materials and Methods. Top, Exposure in which a filter was present to block 35S signal. Bottom, Exposure without filter. B, Quantification of MLC phosphorylation. Shown is effect of 10 µmol/L Y-27632 on basal (open bars) and thrombin-enhanced (solid bars) MLC phosphorylation. The level of 32P incorporation into MLCs was calculated relative to the amount of 35S incorporation into MLCs of the same sample, as was described previously.4 Values are mean±SEM of 3 different cultures in duplicate. *P<0.05 Y-27632–pretreated vs nonpretreated cells.

View larger version (88K): [in this window] [in a new window]   Figure 4. Y-27632 reduces thrombin-induced cytoskeletal reorganization and gap formation. A, Immunocytochemical staining of F-actin in HUVECs grown on glass cover slips. ECs were preincubated for 1 hour in medium 199+1% HSA in the absence (a and b) or the presence (c and d) of 10 µmol/L Y-27632 and stimulated for 30 minutes with 1 U/mL thrombin (b and d). Gaps between cells are indicated with black arrows in panels b and d. B, Quantification of endothelial gap formation. Gap area is expressed as a percentage of total area covered by ECs grown on glass cover slips. Y-27632 significantly reduced thrombin-induced gap formation. *P<0.05. Values are mean±SEM of 3 independent experiments with HUVEC cultures from different donors.

Inhibition of PTKs Reduces Thrombin-Induced Permeability but Not RhoA Activation
To investigate whether the PTKs involved in endothelial permeability act upstream of Rho activation, ECs were preincubated for 1 hour with 30 µg/mL of the PTK inhibitor genistein. This dose of genistein is a maximally effective dose for inhibiting endothelial barrier dysfunction.⁴ Genistein did not affect the thrombin-induced activation of RhoA (Figure 5, inset). In parallel cultures genistein, but not its inactive analogue daidzein (data not shown; see also Reference ⁴ ), inhibited the thrombin-enhanced passage of HRP through the endothelial monolayer (Figure 5). This indicates that the genistein-sensitive PTKs, which are involved in endothelial barrier dysfunction, do not act upstream of RhoA activation.

View larger version (24K): [in this window] [in a new window]   Figure 5. Effect of genistein and Y-27632 on basal (open bars) and thrombin-enhanced (solid bars) permeability. Endothelial monolayers were pretreated for 1 hour with 30 µg/mL genistein, 10 µmol/L Y-27632, or both. HRP passage was determined 1 hour after sham treatment or exposure to 1 U/mL thrombin. Values are mean±SD of 6 cultures in 2 experiments. *P<0.05, cells pretreated with genistein or Y-27632 vs nonpretreated cells. #P<0.05, cells pretreated with a combination of genistein and Y-27632 vs pretreatment with each compound alone. Inset, Genistein has no effect on thrombin-induced activation of RhoA. HUVECs were preincubated with 30 µg/mL genistein for 1 hour and stimulated with 1 U/mL thrombin for 1 minute. Western blot of the pull-down of activated RhoA and 1/40 of total RhoA is shown.

Subsequently, endothelial monolayers were coincubated with genistein and Y-27632 to test whether PTKs act downstream of Rho kinase in inducing endothelial barrier dysfunction. The inhibition of the thrombin-induced HRP passage by genistein was in addition to the inhibition by Y-27632 (Figure 5). This indicates that a PTK-dependent process also acts on a RhoA/Rho kinase-independent pathway that affects EC permeability.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we provide direct evidence that thrombin potently activates RhoA in ECs but does not affect the activity of Rac. Activity of the PTKs involved in endothelial hyperpermeability was not required for the thrombin-induced activation of RhoA. Furthermore, it was shown that thrombin increases endothelial permeability via Rho kinase independently from a rise in [Ca²⁺]_i and that PTKs and Rho kinase have additive effects on endothelial hyperpermeability.

To our knowledge, we provide the first direct demonstration that RhoA is activated by thrombin in confluent endothelial monolayers. Its rapid onset was comparable with the rise in [Ca²⁺]_i. However, RhoA activation extended beyond the transient (5-minute) rise in [Ca²⁺]_i; some RhoA-GTP was still detectable after 30 minutes. Under basal conditions, hardly any active RhoA was detectable, suggesting that RhoA, unless activated, has little or no effect on basal barrier integrity. Previous studies indirectly suggested the involvement of RhoA in endothelial permeability largely on the basis of the use of C3 transferase. This toxin penetrates the cell with difficulty and requires long preincubation times. Thus, it may interfere with gene regulation. Other investigators have used toxin B, which inhibits RhoA, Rac, and Cdc42. Inactivation of RhoA, Rac, and Cdc42 disrupts the endothelial barrier,²⁴ whereas inactivation of RhoA alone enhances endothelial barrier function.¹¹ It is therefore likely that toxin B does not exert its disruptive effect via RhoA, but acts either via Rac or Cdc42.

In addition to RhoA, thrombin-enhanced endothelial permeability requires Ca²⁺-ions.^{4 9} Similarly, as previously found with the Rho inhibitor C3 transferase, inhibition of Rho kinase inhibited the increased permeability, but only partly at the maximally effective dose of Y-27632. In combination with the chelation of Ca²⁺ ions, Y-27632 almost completely prevented the thrombin-induced HRP passage. This indicates not only that both Ca²⁺ ions and the RhoA/Rho kinase pathway are necessary for the full thrombin response but also that RhoA/Rho kinase signaling and Ca²⁺-dependent processes additionally contribute barrier dysfunction.

MLC phosphorylation plays a pivotal role in initiating actin-myosin interaction and in the development of barrier dysfunction.⁴ Here, we extend our previous observation on RhoA⁴ and show that Rho kinase contributes to MLC phosphorylation in ECs. This is comparable with the situation in smooth muscle cells²⁵ and blood platelets,^{26 27 28} in which Rho kinase has recently been shown to be involved in MLC phosphorylation. Rho kinase can increase MLC phosphorylation by inhibiting the myosin phosphatase.²⁹ It is likely that the same mechanism of Rho kinase–induced MLC phosphorylation acts in ECs, as it was shown that in ECs the myosin phosphatase is inhibited by thrombin^{30 31} and that the inhibition of Rho by C3 transferase prevents the inhibition of the phosphatase induced by thrombin.⁹ In addition to an effect on MLC phosphorylation, Rho kinase also phosphorylates other proteins. Several of these proteins are involved in stress fiber formation and may act on EC barrier function, including LIM kinase,³² adducin, and members of the ERM (ezrin/radixin/moesin) family.³³ Their involvement in endothelial permeability remains to be demonstrated. The contribution of additional factors activated by Rho cannot be excluded. Phosphatidylinositol 4,5-biphosphate (PIP₂) may be involved, as intracellular levels of PIP₂ can be increased after activation of Rho³⁴ and PIP₂ is known to interfere with the actin cytoskeleton.³⁵

We have shown previously that, in addition to a rise in intracellular Ca²⁺ ions, PTKs are involved in the thrombin-enhanced endothelial barrier dysfunction.⁴ Other investigators demonstrated that a genistein-sensitive PTK acts upstream of lysophosphatidic acid–induced RhoA activation in ECs.¹⁸ The present study shows that inhibition of PTKs with genistein does not influence activation of RhoA by thrombin, indicating that PTKs do not act upstream of the activation of RhoA in thrombin-enhanced endothelial permeability. This suggests differences between activation of RhoA by either lysophosphatidic acid or thrombin, which may be the involvement of G₁₃. G₁₃ is involved in the activation of RhoA by lysophosphatidic acid but has not been demonstrated to be involved in thrombin-induced signal transduction.³⁶

Inhibition of PTKs with genistein reduced the thrombin-enhanced barrier dysfunction in addition to the effect of Y-27632. This indicates that PTKs and Rho kinase at least act by separate pathways. Interestingly, this study and our previous study show that chelation of Ca²⁺ by BAPTA acts in addition to the inhibition of both PTKs and Rho kinase. This suggests that the Ca²⁺-, RhoA-, and PTK-mediated pathways induced by thrombin reflect separate pathways and that all converge in increased permeability. For the RhoA- and Ca²⁺-dependent pathways, this convergence point is probably the MLC phosphorylation (compare Figure 6, left).

View larger version (24K): [in this window] [in a new window]   Figure 6. Proposed mechanisms involved in thrombin-induced endothelial barrier dysfunction. Thrombin induces Ca2+-dependent activation of MLC kinase as well as RhoA/Rho kinase– dependent inhibition of the myosin phosphatase. Both facilitate MLC phosphorylation, which causes an increased actin-myosin interaction, and a contraction process at the margins of the cells, which finally results in a disturbed endothelial barrier function. In addition to Ca2+- and RhoA-dependent processes, thrombin-induced PTK activities act on phosphorylation of junctional proteins as VE-cadherin and its associated catenins, which results in disruption of intercellular junctions.40 CaM indicates calmodulin; MLCK, MLC kinase; MLC-P, phosphorylated MLC; and PP1M, myosin phosphatase type 1.

Inhibition of PTKs may act on endothelial permeability by various mechanisms. PTK inhibitors attenuate agonist-induced increases in [Ca²⁺]_i.^{37 38} In accordance with this, Garcia et al¹⁰ recently reported that activity of an EC-specific MLC kinase is regulated by tyrosine phosphorylation in a RhoA-dependent manner. However, additional mechanisms must exist, as PTKs are involved in endothelial barrier dysfunction independent of changes in [Ca²⁺]_i⁴ and RhoA signaling (present study). A likely mechanism is the destabilization or disruption of junctions by tyrosine phosphorylation of junctional proteins. Tyrosine phosphorylation of occludin and ZO-1 can occur under conditions of hyperpermeability.³⁹ The agonist-induced disruption of adherens junctions is accompanied by tyrosine phosphorylation of vascular endothelial cadherin (VE-cadherin) and the associated catenins, resulting in the dissociation of VE-cadherin/catenins.^{40 41} Disintegration of junctional complexes and the actin-nonmuscle myosin interaction in the periphery of ECs may thus act in concert in prolonged thrombin-induced endothelial permeability (Figure 6).

Our data point to an important role for RhoA and Rho kinase in the regulation of endothelial permeability. Future studies have to demonstrate whether and when these factors are involved in altered endothelial barrier function in vivo. In large-vessel ECs, in particular in areas with altered shear forces, stress fibers are found and Rho-mediated processes are likely to be involved. The recent finding of Essler et al,⁴² that mildly oxidized LDL activates Rho/Rho kinase signaling in ECs, suggests the involvement of RhoA and Rho kinase in vascular leakage during the development of atherosclerosis. No information is presently available on microvascular ECs in vivo. However, it should be noticed that Rho kinase plays a role in cell migration and that prolonged permeability might be a reflection of the altered behavior of ECs during cell migration and angiogenesis, which occur in wound healing and pathological conditions.

	Acknowledgments

 
This study was financially supported by the Netherlands Heart Foundation (Grant 94.048).

Received February 21, 2000; revision received June 22, 2000; accepted June 22, 2000.

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