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Transient and Prolonged Increase in Endothelial Permeability Induced by Histamine and Thrombin

Role of Protein Kinases, Calcium, and RhoA

Geerten P. van Nieuw Amerongen, Richard Draijer, Mario A. Vermeer, , Victor W. M. van Hinsbergh

From the Gaubius Laboratory TNO-PG (G.P.v.N.A., R.D., M.A.V., V.W.M.v.H.), Leiden, and Institute for Cardiovascular Research, Vrije Universiteit (V.W.M.v.H.), 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

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Abstract—In the present study, we differentiated between short- and long-term effects of vasoactive compounds on human endothelial permeability in an in vitro model. Histamine induced a rapid and transient (<3 minutes) decrease in barrier function, as evidenced by a decreased transendothelial electrical resistance and an increased passage of ²²Na ions. This increase in permeability was inhibited completely by chelation of intracellular calcium ions by BAPTA-AM and inhibition of calmodulin activity and myosin light chain (MLC) phosphorylation. The presence of serum factors prolonged the barrier dysfunction induced by histamine. Thrombin by itself induced a prolonged barrier dysfunction (>30 minutes) as evidenced by an increased passage of peroxidase and 40 kDa dextran. It was dependent only partially on calcium ions and calmodulin. The protein tyrosine kinase inhibitors genistein and herbimycin A, but not the inactive analogue daidzein, inhibited to a large extent the increase in permeability induced by thrombin. Genistein and BAPTA-AM inhibited the thrombin-induced permeability in an additive way, causing together an almost complete prevention of the thrombin-induced increase in permeability. Inhibition of protein tyrosine kinase was accompanied by a decrease in MLC phosphorylation and a reduction in the extent of F-actin fiber and focal attachment formation. Inhibition of RhoA by C3 transferase toxin reduced both the thrombin-induced barrier dysfunction and MLC phosphorylation. Genistein and C3 transferase toxin did not elevate the cellular cAMP levels. No evidence was found for a significant role of protein kinase C in the thrombin-induced increase in permeability or in the accompanying MLC phosphorylation. These data indicate that in endothelial cell monolayers that respond to histamine in a physiological way, thrombin induces a prolonged increase in permeability by "calcium sensitization," which involves protein tyrosine phosphorylation and RhoA activation.

Key Words: human endothelial cell • RhoA • protein tyrosine kinase • protein kinase C

	Introduction

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The endothelium is the main physical barrier in the extravasation of blood components to the surrounding tissue. Impairment of this barrier leads to an increase in permeability and formation of edema. Inflammatory mediators such as histamine, bradykinin, and substance P cause a rapid transient increase in permeability in vivo, which results from a rapid formation of endothelial gaps especially in the postcapillary venules.^{1 2} These gaps are thought to be due to endothelial cell (EC) contraction, a process that involves actin nonmuscle myosin interaction, and requires Ca²⁺, calmodulin (CaM) and ATP, and the phosphorylation of the myosin light chain (MLC).^{3 4 5} This process is regulated by Ca²⁺/CaM–dependent protein kinase I, the classic MLC kinase.^{4 5} Baluk et al¹ recently have shown that the half-life of the gaps induced by substance P in healthy rat tracheal venules is <2 minutes and that the presence of these gaps runs parallel to the increase in endothelial permeability. In frog mesenterium microvessels, a similar temporal relationship was found between the transient increase in cytoplasmic Ca²⁺ ions and the increase in endothelial permeability after stimulation with histamine.⁶

Additional mechanisms must occur to explain the prolonged vascular leakage that contributes to clinical forms of serious edema. These mechanisms include multiple release of vasoactive agents, endothelial binding and activation of leukocytes,⁷ and, occasionally, the participation of segments of the vascular bed other than postcapillary venules.^{8 9} In these circumstances, the endothelial barrier function is reduced by an interplay between actin nonmuscle myosin interaction,^{4 5} disintegration of endothelial junctions,¹⁰ and the formation of inter- and occasionally intracellular pores.^{11 12 13} However, the molecular mechanisms regulating prolonged leakage are still poorly understood.

ECs in vitro are helpful in providing biochemical information regarding molecular mechanisms contributing to endothelial permeability.^{4 5} Most information has been obtained using thrombin, a stimulus that induces a prolonged increase in permeability in endothelial monolayers,¹⁴ similar to the effect it elicits in vivo.¹⁵ In contrast to the transient effect of histamine and other vasoactive agents, the reduction of endothelial barrier function induced by thrombin extends over 1 hour and is far beyond the transient rise in cytoplasmic Ca²⁺ concentration that closely accompanies the leakage induced by histamine.¹⁶ Several authors have already pointed out additional factors that influence endothelial permeability other than the Ca²⁺/CaM–dependent phosphorylation of the MLC. These factors include activation of protein kinase C (PKC)^{17 18} and inhibition of MLC phosphatase.^{19 20} In this study, we investigated which pathways are involved in the different permeability-increasing effects of histamine and thrombin and provide evidence for the involvement of protein tyrosine phosphorylation and RhoA in addition to the Ca²⁺/CaM–dependent phosphorylation of MLC in the thrombin-induced permeability of these human endothelial monolayers.

The in vitro models frequently have been considered inadequate because most studies are limited to prolonged increases in endothelial permeability. Therefore, we also present data on the effect of histamine on human endothelial monolayers. Under the proper conditions, the response to histamine is identical as observed in postcapillary venules in vivo; its size and duration can be influenced by exposure of the cells to endotoxin or serum factors.

	Materials and Methods

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Materials
Tissue culture plastics and Transwells (diameter, 0.65 cm; pore size, 3 µm) were obtained from Costar; cell culture reagents were obtained as previously described.²¹ Human serum was obtained from the local blood bank and was prepared from fresh blood taken from healthy donors; this was pooled, heat-inactivated (30 minutes; 56°C), and stored at 4°C. Heat-inactivated newborn calf serum (NBCS) was obtained from Gibco BRL. Bovine thrombin (5000 NIH units) was from Leo Pharmaceutical Products. Fluorescein isothiocyanate-labeled dextran (molecular mass, 40 kDa), histamine, horseradish peroxidase (HRP), LPS from E. coli serotype O128:B12, phorbol 12-myristate-13-acetate (PMA), trifluoperazine (TFP), and anti-vinculin Ig were obtained from Sigma Chemical Co. BAPTA-AM and rhodamine phalloidin were from Molecular Probes. ML-7 was from Calbiochem Novabiochem Corporation. Calphostin C, genistein, herbimycine A, 1-oleoyl-2-acetylglycerol, thymeleatoxin and tyrphostin A47 were from Alexis Inc. Ranitidine, dimaprit, R--methylhistamine, and d-neobenodine were a kind gift from Professor H. Timmerman (Free University Amsterdam, the Netherlands). Ro31-8220 was a kind gift from Dr P.A. Brown (Roche Products Ltd, Welwyn Garden City, UK). C3 transferase toxin was kindly provided by Dr A. Ridley (Ludwig Institute, London, UK). [³²P]-Orthophosphoric acid and Tran ³⁵S label were from ICN Pharmaceuticals Inc. Anti-platelet myosin Ig (nonmuscle) was from Sanbio. Rabbit anti-mouse IgG–fluorescein isothiocyanate was from Dakopatts. ²²Na as sodium chloride was from Amersham Lifescience.

Evaluation of the Barrier Function
Human umbilical vein ECs (HUVEC) were isolated and cultured as previously indicated.²¹ For the evaluation of the barrier function, confluent monolayers of HUVEC (first and second passage) were released with trypsin-EDTA and seeded in high density on fibronectin-coated polycarbonate filters of the Transwell system and cultured. Medium was renewed every other day. Monolayers were used between 4 and 6 days after seeding. Exchange of macromolecules through the endothelial monolayers was investigated by assay of the transfer of HRP or dextran–fluorescein isothiocyanate and was performed as described previously.²¹ All passage experiments were performed in triplicate.

Passage of ²²Na, diluted in Medium 199 to 1% HSA to give a specific activity of 1250 cpm/µg, through EC monolayers was examined in a similar way. Passage of ²²Na was represented as a difference of histamine-induced and basal passage of ²²Na and was expressed in cpm/well.

Transendothelial Electrical Resistance
Transendothelial electrical resistance (TEER) was measured as described previously.²² In short, an alternating current (50 µA) was passed across the monolayer (2 pulses every minute). The electrical resistance was calculated by Ohm's law and was expressed as a percentage of the basal level. Basal TEER of HUVEC monolayers was 21.3±0.3 · cm² (mean±SEM; 37 determinations in 12 cultures). Basal TEER did not change significantly by pretreatment with the used compounds. Histamine (3x10^-6 mol/L) was used, which is intermediate between the half-maximal concentration (1.5x10^-6 mol/L) and the maximal effective concentration (10^-5 mol/L). Histaminergic agonists and antagonists were used at concentrations that were at least 10 times higher compared with their pD₂ and pA₂ values.

Phosphorylation of MLC
For analysis of MLC phosphorylation, a procedure was adapted from the work of Goeckeler and Wysolmerski²³ and Chrzanowska-Wodnicka and Burridge.²⁴ Confluent cells of passage 1 or 2, seeded in 12-well plates, were labeled with 0.5 mL 150 µCi/mL Tran ³⁵S label in low methionine medium (Medium 199 containing 10^-5 mol/L methionine, 10% human serum, 10% NBCS, 150 µg/mL crude EC growth factor, 5 U/mL heparin, 100 U/mL penicillin, and 0.1 mg/mL streptomycin) for 48 hours. Cells were washed with phosphate-free buffer (in mmol/L: NaCl 119, KCl 5, glucose 5.6, MgCl₂ 0.4 , CaCl₂ 1, and Pipes 25; pH 7.2) and labeled with 0.5 mL 150 µCi/mL [³²P]orthophosphoric acid in phosphate-free buffer for 2 hours and then stimulated with histamine or thrombin. Buffer was removed, and cells were lysed by scraping in 300 µL of ice-cold lysis-buffer (25 mmol/L Tris-HCl, 250 mmol/L NaCl, 75 mmol/L NaF, 5 mmol/L EGTA, 5 mmol/L EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.2 mmol/L phenylmethylsulfonyl fluoride, 0.5 mmol/L dithiothreitol, 10 µg/mL aprotinin, and 100 mmol/L Na₄P₂O₇). The lysates were centrifuged for 20 minutes in an Eppendorf centrifuge. The supernatants were incubated with 6 µL of polyclonal rabbit anti-platelet myosin IgG for 1 hour. Subsequently, 20 µL^1:1 of prewashed protein A-Sepharose 4B was added for an additional hour. Immune complexes bound to protein A Sepharose 4B were collected by centrifugation. Pellets were washed 3 times with PBS and resolved in 50 µL SDS sample buffer. The samples were electrophoresed on 16% SDS polyacrylamide gels. The gels were dried and exposed to a phosphoimaging screen. Quantitation of ³²P incorporation into MLC was performed using a Fuji BAS 1000 PhosphorImager as follows. Double-labeled samples were exposed to phosphoimaging screens directly, and the total amount of radioactivity (³⁵S+³²P) was quantitated for MLCs. A second exposure was obtained in which a filter of 4 layers of aluminum foil was present between the gel and the phosphoimaging screen to block ³⁵S radiation. The amount of ³²P and ³⁵S incorporation in the MLC band of each sample was calculated, and MLC phosphorylation was expressed as a percentage of control.

Extraction and Assay of cAMP
Intracellular cAMP levels were determined by radio-immunoassay (Amersham, Amersham, UK) as described previously.²¹

Immunocytochemistry
The presence of vinculin and F-actin were visualized by indirect immunofluorescence with mouse anti-vinculin antibody (1:300) and by direct staining with rhodamine-phalloidin (1:100).

Statistical Analysis
Data are reported as mean±SEM. Comparisons between >2 groups were made using the Kruskal-Wallis test, and individual groups comparisons were done using a Mann-Whitney test for post hoc comparisons. Comparisons of time curves of 2 groups were made using repeated measures ANOVA, and individual groups comparisons were done using a Student t test for post hoc comparisons of the means. Differences were considered significant at the P<0.05 level.

	Results

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Histamine Induces a Short-Term Decrease in Endothelial Barrier Function
In HUVEC, the addition of histamine (3x10^-6 mol/L) induced a rapid decrease in the real-time TEER, which was maximal after 1 minute (72±7% compared with basal level; 5 different cultures in triplicate) and lasted for <5 minutes (Figure 1A). An increase in the passage of ²²Na paralleled the decrease in TEER (Figure 1C). The effect of histamine on TEER was mimicked by the histamine H₁ agonist thiazolylethylamine, but not by the histamine H₂ agonist dimaprit (Figure 1B). Preincubation for 10 minutes with the histamine H₁ antagonist d-neobenodine blocked the decrease in TEER induced by histamine, while the histamine H₂ antagonist ranitidine had no effect (Figure 1A).

View larger version (34K): [in this window] [in a new window]   Figure 1. Histamine transiently decreases the barrier function of human umbilical vein endothelial monolayers by a Ca2+/CaM– and MLC kinase–dependent mechanism. A and B, Effect of histaminergic compounds on the TEER. A, Cells were pretreated for 10 minutes with 3x10-8 mol/L d-neobenodine (H1 antagonist), 10-6 mol/L ranitidine (H2 antagonist), or were sham treated and subsequently stimulated with 3x10-6 mol/L histamine. Values are mean±SEM of 2 different cultures in triplicate. *P<0.05, d-neobenodine-pretreated cells versus control cells. Basal TEER of HUVEC monolayers was 21.3±0.3 · cm2 (mean±SEM; 37 determinations in 12 cultures). B, Cells were equilibrated for 10 minutes and stimulated with 3x10-6 mol/L histamine, 10-5 mol/L thiazolylethylamine (H1 agonist), or 10-5 mol/L dimaprit (H2 agonist). Values are mean±SEM of 2 different cultures in triplicate. *P<0.05, dimaprit- versus histamine-stimulated cells. C, Effect of histamine on the passage of 22Na. Monolayers were equilibrated for 15 minutes and at 0 minutes were stimulated with 10-5 mol/L histamine or were sham treated. Passage of 22Na was expressed as a difference between histamine-stimulated and basal passage of 22Na. Values are mean±SEM of 2 different cultures in 6-fold. *P<0.05, histamine-induced versus basal 22Na passage. D, E, and F, Effect of various inhibitors on the histamine-induced decrease in TEER. D, Cells were pretreated for 1 hour with 3 µmol/L BAPTA-AM (), 10 µmol/L TFP (), or were sham treated (). After 1 hour, cells were exposed to 3x10-6 mol/L histamine. Values are mean±SEM of 2 different cultures at least in triplicate. *P<0.05, BAPTA-AM– or TFP-pretreated versus control cells. E, Cells were pretreated for 2 hours with 10 µmol/L ML-7 () or were sham treated (). After 2 hours, cells were stimulated with 3x10-6 mol/L histamine. Values are mean±SEM of 2 different cultures in triplicate. *P<0.05, ML-7–pretreated versus control cells. F, Cells were pretreated with 30 µg/mL genistein for 1 hour () or were sham treated (). After 1 hour, cells were stimulated with 3x10-6 mol/L histamine. Values are mean±SEM of 2 different cultures at least in triplicate.

The histamine-induced increase in EC permeability was inhibited completely by preincubation with the intracellular calcium chelator BAPTA-AM or the CaM inhibitor TFP (Figure 1D) and to a large part by the MLC kinase inhibitor ML-7 (Figure 1E). Addition of histamine to HUVEC increased MLC phosphorylation transiently (135±13% after 2 minutes; Figure 2, ). This effect extended slightly beyond the decrease in TEER. However, part of the MLC phosphorylation was inhibitable by the protein tyrosine kinase (PTK) inhibitor genistein (30 µg/mL for 1 hour). The genistein-insensitive MLC phosphorylation exactly paralleled the histamine-induced increase in permeability (Figure 2, ), as the histamine induced decrease in TEER was completely insensitive to inhibition of PTKs with genistein (Figure 1F). Preincubation of the cells with C3 transferase did not affect the histamine-induced increase in permeability (see RhoA Involvement in Thrombin-Enhanced Permeability). Together, these data are consistent with a role of Ca²⁺/CaM–dependent and genistein-insensitive phosphorylation of MLC in the transient increase in permeability induced by histamine.

View larger version (28K): [in this window] [in a new window]   Figure 2. MLC phosphorylation is stimulated transiently by histamine. A, Autoradiograph of MLCs immunoprecipitated from cells under basal conditions (0 minutes) and 1, 2, 5, and 10 minutes after stimulation with 10-5 mol/L histamine. Cells were labeled with 32P and 35S as described in Materials and Methods. Top, An exposure in which a filter was present to block 35S signal. Bottom, An exposure without filter. B, Quantitation of MLC phosphorylation. Cells were preincubated for 1 hour in the absence () or the presence () of 30 µg/mL genistein and stimulated with 10-5 mol/L histamine, and MLC phosphorylation was measured after the time points indicated. The level of 32P incorporation into MLCs was calculated relative to the amount of 35S incorporation into the MLCs of the same sample, as described in Materials and Methods. Histamine significantly induced MLC phosphorylation at 1, 2, and 5 minutes. *P<0.05. Genistein significantly lowered MLC phosphorylation (P=0.000). Values are mean±SEM of 2 different cultures at least in duplicate.

The Response of ECs to Histamine Can Be Modified
Pretreatment of HUVEC with 10 µg/mL LPS for 24 hours enlarged the decrease in TEER induced by histamine (Figure 3A) from 79±9% to 58±5% (mean±SEM of 3 different cultures at least in triplicate; P<0.001). Addition of human serum to our system considerably prolonged the response induced by histamine (Figure 3B).

View larger version (17K): [in this window] [in a new window]   Figure 3. Effect of LPS and human serum on the histamine-induced decrease in TEER. A, Endothelial monolayers were grown to confluence and cultured for 24 hours in absence () or presence () of 10 µg/mL LPS. Cells were stimulated with 3x10-6 mol/L histamine. B, Cells were stimulated with 3x10-6 mol/L histamine in the absence () or presence () of 20% human serum, and TEER was recorded. Values are mean±SEM of at least 2 experiments in triplicate. *P<0.05, pretreated versus control cells.

Thrombin Induces a Prolonged Decrease in Endothelial Barrier Function That Involves PTK Activity
A prolonged response also is induced by thrombin in the absence of serum. The thrombin-induced reduction in TEER lasted for at least 30 minutes. The decrease in TEER was paralleled by a rapid increase in the passage of macromolecules, which was sustained usually for at least 1 hour, as is shown for HRP in Figure 4.

View larger version (26K): [in this window] [in a new window]   Figure 4. Effect of genistein on the passage of HRP through human umbilical vein endothelial monolayers under basal conditions (, ) and after exposure to 1 U/mL thrombin (, , ). Cells were preincubated for 1 hour in Medium 199 to 1% HSA with 30 µg/mL genistein (, ) or with 30 µg/mL daidzein (+) or without addition (, ). The inset shows the early time points of the same set of experiments in more detail. Values are mean±SEM of 2 different cultures in triplicate. The interaction between time and genistein treatment was significant (P=0.000) for thrombin-stimulated cells. Treatment with genistein resulted in lower HRP passage compared with no treatment. Differences were significant from 4 minutes onward. *P<0.05, genistein-pretreated versus nonpretreated cells that were stimulated with thrombin.

In agreement with previous reports,^{21 25} the prolonged increase in permeability for macromolecules (HRP) induced by thrombin was inhibited partially (maximally 60%) by BAPTA-AM or by the CaM inhibitor TFP (not shown). Thus, in contrast to the transient increase in permeability induced by histamine, the prolonged increase induced by thrombin required additional activation or sensitization steps other than Ca²⁺/CaM–dependent MLC phosphorylation. Therefore, we evaluated whether inhibition of PKC or PTK affected the thrombin-induced permeability.

At 1 and 10 nmol/L, the PKC activator PMA (15 minutes' preincubation) reduced endothelial permeability by 25% to 50%, whereas at higher concentrations (100 nmol/L), which give an extreme activation of PKC compared with thrombin and histamine, it increased the permeability. In the presence of thrombin, 10 nmol/L PMA reduced the permeability in HUVEC significantly (Table). Similarly, the PKC-activating diacylglycerol analogue 1-oleoyl-2-acetylglycerol (50 µmol/L) and thymeleatoxin (10 nmol/L), which activates predominantly calcium-dependent PKCs, reduced thrombin-enhanced permeability of dextran by 42±13% (n=7) and 44±6% (n=6), respectively.

View this table: [in this window] [in a new window]   Table 1. Passage of Dextran (40 kDa) Through HUVEC Monolayers and MLC Phosphorylation in the Presence or Absence of PMA, Ro31-8220, and/or Thrombin

Subsequently, the PKC inhibitors Ro31-8220 and Calphostin C were used. Although Ro31-8220 completely counteracted the effect of PMA, it did not alter the thrombin-induced increase in permeability (Table). Calphostin C (100 nmol/L) slightly increased the basal permeability (from 0.82±0.17 to 1.21±0.36 µg · h^-1 · cm^-2), but the thrombin-stimulated permeability was not affected by 15 minutes' preincubation with Calphostin C, being 7.8±1.7 and 7.2±1.4 µg · h^-1 · cm^-2, respectively (4 cultures). The phosphorylation of MLC was not affected by PMA or Ro31-8220 under thrombin-stimulated conditions (Table). These data do not support a significant role for PKC activation in thrombin-induced increase in permeability of HUVEC monolayers.

Subsequently, we studied whether inhibition of PTK prevented the thrombin-enhanced permeability. As shown in Figure 4, preincubation of HUVEC monolayers with genistein, but not its inactive analogue daidzein (both preincubated at 30 µg/mL for 1 hour) attenuated the thrombin-induced HRP passage, which became significant after 5 minutes. The reduction of thrombin-induced permeability by genistein was dose-dependent (3 to 100 µg/mL tested; data not shown). Preincubation with another PTK inhibitor herbimycine A (0.3 µg/mL for 2 hours) resulted in a similar inhibition, whereas tyrphostin A47 (up to 10 µg/mL), which has a different substrate spectrum, had no effect on the thrombin-induced HRP passage.

Genistein reduced the basal MLC phosphorylation (Figure 5), parallel to its reduction of basal permeability. Thrombin induced a prolonged increase in the phosphorylation of MLC (Figure 5 inset, ), which was maximal 1 minute after exposure to thrombin and remained elevated for at least 30 minutes. Although the degree of MLC phosphorylation in thrombin-induced HUVEC was lower in genistein-treated cells, this decrease reflected the lower basal MLC phosphorylation state rather than the increase induced by thrombin itself (Figure 5 inset, ). Simultaneously, the thrombin-induced decrease in TEER attenuated by 36±17% (assayed after 10 minutes; P<0.01; 2 experiments in 5-fold) after preincubation of 30 µg/mL genistein, whereas genistein did not affect the basal TEER.

View larger version (50K): [in this window] [in a new window]   Figure 5. Parallel inhibition of MLC phosphorylation and HRP passage by genistein. Cells were preincubated for 1 hour in the absence or presence of 30 µg/mL genistein and stimulated with 1 U/mL thrombin, and MLC phosphorylation was determined after 10 minutes (cross-hatched bars). HRP passage data are derived from Figure 4 and represent the amount of HRP passed after 10 minutes and are expressed as a percentage of basal level (hatched bars). *P<0.05, genistein-pretreated versus control cells. #P<0.05, thrombin-stimulated cells in the presence versus absence of genistein. Inset, Time curve of inhibition by genistein of thrombin-induced MLC phosphorylation. Cells were preincubated for 1 hour in the absence () or presence () of 30 µg/mL genistein and stimulated with 1 U/mL thrombin, and MLC phosphorylation was determined after the time points indicated. MLC phosphorylation was measured as described in Materials and Methods. Thrombin significantly induced MLC phosphorylation at 1, 2, 5, and 10 minutes. *P<0.05. Genistein significantly lowered MLC phosphorylation (P=0.001). Values are mean±SEM of 2 different cultures at least in duplicate.

When HUVEC were preincubated with genistein, the cellular cAMP concentration remained unaltered both under basal conditions (2.3±0.2 and 2.3±0.1 pmol/3.5x10⁵ cells in control and genistein-preincubated cells) and after 10 minutes' stimulation of the cells with 1 U/mL thrombin (3.1±0.5 and 2.9±0.3 pmol/3.5x10⁵ cells, respectively; 3 experiments). This excludes the possibility that genistein could act on endothelial permeability and MLC phosphorylation via elevation of the cAMP concentration.

The inhibition of thrombin-induced permeability by genistein was additive to the inhibition by BAPTA-AM (Figure 6). Together, these compounds caused a nearly complete prevention of the thrombin-induced permeability. They also affected the basal permeability, probably because a low degree of endothelial activation occurs even under basal conditions. These data show that Ca²⁺-dependent and PTK-sensitive pathways cooperate in the regulation of endothelial permeability.

View larger version (23K): [in this window] [in a new window]   Figure 6. Effect of BAPTA-AM and genistein on basal (cross-hatched bars) and thrombin-enhanced permeability (hatched bars). Endothelial monolayers were pretreated for 1 hour with 3 µmol/L BAPTA-AM, 30 µg/mL genistein, or a combination of both. The HRP passage was determined 1 hour after sham treatment or exposure to 1 U/mL thrombin. Values are mean±SEM of 3 different cultures in triplicate. There was a significant difference between HRP passage of the various thrombin-stimulated groups (P=0.0008). *P<0.05, cells pretreated with BAPTA-AM or genistein versus nonpretreated cells. #P<0.05, cells pretreated with a combination of BAPTA-AM and genistein versus pretreatment with each compound alone.

PTKs Are Involved in Thrombin-Induced Cytoskeletal Rearrangements
Under basal conditions, F-actin fibers in HUVEC were arranged into a fine cortical network with a dense peripheral band evident at the cell boundaries (Figure 7A); occasionally, stress fibers were observed. When the monolayers were preincubated with genistein (30 µg/mL; Figure 7B) the cells became wrinkled, the cortical actin band was less obvious, and an increase in diffuse cytoplasmic actin staining was observed. After stimulation of the cells with 1 U/mL thrombin, many stress fibers were formed (Figure 7C); simultaneously, small gaps became visible at the cell-cell contact areas, and an increase in vinculin staining was observed, indicating that focal adhesion sites were formed (Figure 7E and 7G). The thrombin-induced cytoskeletal rearrangements were inhibited largely by preincubation with genistein (Figure 7D and 7H). This was reflected by a marked, but not complete reduction of stress fibers, a reduction of the occurrence of small gaps and a reduction in the extent of focal adhesion formation.

View larger version (114K): [in this window] [in a new window]   Figure 7. Immunocytochemical staining of actin (A through D) and vinculin (E through H) in HUVEC. ECs were preincubated for 1 hour with 30 µg/mL genistein and stimulated with 1 U/mL thrombin. The cells were stained as described in Materials and Methods. A and E, Basal condition. B, D, F, and H, Cells were preincubated with genistein. C, D, G, and H, Cells were stimulated for 30 minutes with thrombin.

RhoA Involvement in Thrombin-Enhanced Permeability
Recent data pointed to a role of RhoA in the formation of stress fibers²⁶ and cell contraction^{24 27 28} in nonvascular cells. Because thrombin-induced tyrosine phosphorylation may be involved in the activation of RhoA and subsequently in the regulation of stress fiber formation via RhoA, we used an inhibitor of RhoA, the toxin C3 transferase of C. botulinum to evaluate whether RhoA contributed to the thrombin-induced increase in permeability. Titration of the C3 transferase toxin revealed that 24 hours' incubation with 3 to 5 µg/mL toxin caused sufficient uptake without changing the permeability of the monolayers by itself. Preincubation with 5 µg/mL C3 transferase toxin markedly attenuated the thrombin-enhanced permeability as well as basal permeability (Figure 8A). This attenuation was accompanied by a reduced MLC phosphorylation (Figure 8B). The reduction in endothelial permeability by C3 transferase was abolished by heat treatment of the C3 transferase preparation (5 minutes at 95°C). Preincubation of the cells with 5 µg/mL C3 transferase did not change the cAMP level of thrombin-stimulated cells (2.5±0.6 versus 2.6±0.3 pmol/3.5x10⁵ cells, 2 cultures in duplicate).

View larger version (31K): [in this window] [in a new window]   Figure 8. Effect of C3 transferase on basal (cross-hatched bars) and thrombin-enhanced (hatched bars) permeability (A) and MLC-phosphorylation (B). Endothelial monolayers were pretreated for 24 hours with 5 µg/mL C3 transferase. The HRP passage was determined 1 hour after sham treatment or exposure to 1 U/mL thrombin. MLC phosphorylation was measured as described in Materials and Methods 10 minutes after addition of thrombin. Values are mean±SEM of 2 different cultures at least in duplicate. *P<0.05, thrombin-stimulated cells that were pretreated with C3 transferase versus nonpretreated cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we have provided evidence for the involvement of PTK activity and RhoA in the prolonged thrombin-induced increase in permeability. The thrombin-enhanced permeability was accompanied by an increase in MLC phosphorylation and the generation of F-actin fibers, both of which were reduced by the inhibition of PTK or RhoA. The transient increase in permeability induced by histamine was not affected by PTK or RhoA inhibition.

In in vivo experiments, stimulation of healthy vessels with vasoactive agents, such as histamine, bradykinin, and substance P, induces a transient increase in permeability of postcapillary venules.^{1 2} This involves a Ca²⁺/CaM–dependent activation of the EC⁶ and a transient formation of minute gaps at the cell-cell contacts leaking monastral blue during a period of only a few minutes.¹ In our in vitro model, the time course of the permeability increase induced by histamine is identical to the increase observed in vivo in leakage and involves a similar activation mechanism.⁶ However, under many pathological conditions, massive leakage can occur over a long period of time. We have demonstrated here that the increase in endothelial permeability induced by histamine was prolonged significantly in time by the addition of serum factors. These data support the idea that additional mechanisms contribute to the prolonged vascular leakage. At this moment, the additional factor(s) in serum responsible for the prolongation of the histamine effect are not yet known. Recently, Bloemers et al²⁹ reported a sensitization of H₁ receptor in embryonic cells by the presence of serum and leukotrienes. Alternatively, sensitization of the Ca²⁺/CaM–dependent activation of MLC phosphorylation, known from studies in smooth muscle cells,²⁸ may also contribute to the prolonged response.

The thrombin-induced increase in endothelial permeability provides a condition in which the Ca²⁺/CaM–dependent MLC phosphorylation is sensitized. Chelation of cytoplasmic Ca²⁺ ions by BAPTA-AM only partially reduces the thrombin-induced increase in endothelial permeability.^{21 25} Our data show that inhibition of PTK reduces thrombin-induced permeability by an additional mechanism and almost completely prevents this increase when added simultaneously with BAPTA-AM. Inhibition of PTK may act on endothelial permeability by various mechanisms. It has been shown that PTK inhibitors attenuate the agonist-induced cellular Ca²⁺ influx.^{30 31} However, our finding that coincubation of BAPTA-AM with genistein has an additive effect suggests that activation of PTKs by thrombin involves an additional pathway. Other reports have pointed to the disruption of adherens junctions and reorganization of focal adhesion plaques by thrombin. Rabiet et al³² found that thrombin disrupted the VE-cadherin-catenin complex in adherens junctions, which could be prevented by the PTK inhibitor herbimycin A. Schaphorst et al³³ reported the tyrosine phosphorylation of focal adhesion kinase p125^FAK, which accompanied the reorganization of focal adhesion sites induced by thrombin. These changes in cell-cell and cell-matrix interaction may contribute to the enhanced permeability induced by thrombin. Here, we show that inhibition of PTKs affects the formation of F-actin fibers and decreases the MLC phosphorylation. By these actions, it may influence the interaction of the F-actin cytoskeleton with cell-cell and cell-matrix attachment sites and reduces actin-nonmuscle myosin interaction causing local contractile forces probably in the margins of the ECs.

Another candidate molecule to be regulated by PTKs and involved in permeability is RhoA. Hippenstiel et al³⁴ recently have shown that RhoA plays a role in basal endothelial permeability. A well-established effect of RhoA is stress fiber formation, a process that is known to be activated by PTKs in ECs.³⁵ Furthermore, it is known that activation of RhoA induces MLC phosphorylation, probably via Rho kinase, which may act via inactivating the regulatory subunit of a myosin phosphatase.³⁶ Here, we show that inhibition of RhoA by C3 transferase attenuates both the basal and thrombin-induced EC barrier dysfunction and the accompanied increase in MLC-phosphorylation. This fits with the finding that the thrombin-mediated stress fiber formation is dependent on the activation of PTKs. The function of these stress fibers is not completely understood at present. In ECs, they seem to have a protective function against fluid shear stress by developing cell tension.³⁷ Goeckeler and Wysolmerski²³ and Moy et al¹⁶ have demonstrated that tensile forces are developed in thrombin-stimulated ECs, whereas these forces are insignificant in HUVEC exposed solely to histamine. In this context, it is likely that the F-actin fiber structures contribute to a state of prolonged EC contraction after thrombin challenge. Furthermore, it is known that in smooth muscle cells, activation of RhoA results in "calcium sensitization," ie, independently of a change in Ca²⁺, MLC phosphate levels increase by inhibition of the smooth muscle myosin phosphatase (SMPP-1) via activation of a RhoA-dependent kinase, resulting in force generation.²⁸ In addition to an effect of Rho kinase on the activity of myosin phosphatase 1, Rho kinase may also act itself as an MLC kinase.³⁶ In smooth muscle cells, the calcium sensitization is accompanied by a translocation of RhoA from the cytosol to the cell membrane.²⁸ Preliminary data indicate that activation of ECs by thrombin results in a similar translocation of RhoA. Verin et al²⁰ have shown that in ECs, the myosin phosphatase is inhibited by thrombin. Based on these observations, we suggest a model (Figure 9) in which transient EC barrier failure is mediated by an activation of Ca²⁺/CaM–dependent protein kinase I, whereas prolongation of EC barrier dysfunction results from a sensitization of this process by a parallel activation of PTK and RhoA.

View larger version (22K): [in this window] [in a new window]   Figure 9. Proposed mechanisms affecting MLC phosphorylation that are involved in transient and prolonged EC barrier dysfunction. Elevation of intracellular Ca2+ by agonist exposure causes a transient increase of MLC phosphorylation by activation of a Ca2+/CaM–dependent MLC kinase (MLCK). MLC phosphorylation can be prolonged by activation of RhoA in a PTK-dependent way. Rho kinase probably reduces the activity of myosin phosphatase type 1.20 In addition to an effect of Rho kinase on the activity of myosin phosphatase 1, Rho kinase itself may act also as a MLC kinase.36 It should be noted that RhoA also is involved in the organization of the actin cytoskeleton and in cell-cell and cell-matrix interactions. PLC indicates phospholipase C; PP1 M, myosin phosphatase type 1; and MLC-P, phosphorylated MLC.

An interesting finding of this study is that although inhibition of PTKs by genistein lowers the basal MLC phosphorylation status, it does not influence the relative increase in MLC phosphorylation induced by histamine. First, this indicates that activation of the Ca²⁺/CaM–dependent MLC kinase by histamine is not PTK-dependent under our experimental conditions. This is confirmed by the fact that inhibition of PTKs does not affect the histamine-induced EC contraction, as measured by TEER. In addition, this finding suggests the existence of different pools of MLC. The relative increase in MLC phosphorylation that is caused by histamine is enough to induce contraction of genistein-treated ECs, although total MLC phosphorylation levels were lowered by inhibition of PTKs. Under these conditions, MLC-phosphate levels did not exceed basal MLC-phosphate levels. This is of importance, because until now it was unclear how a generalized contraction of the EC could cause focal openings between the cells. One explanation may be that pools of MLCs located near the border of the cell are involved in the formation of the small gaps. These findings need further investigation.

Under our experimental conditions, we could not find evidence of an important contribution of PKC to the thrombin-induced increase in endothelial permeability of human EC. Several lines of evidence make it unlikely that PKC plays a major role in the prolonged increase in human endothelial permeability induced by thrombin. First, incubation of human EC with PMA caused a decrease in permeability at moderate concentrations (1 and 10 nmol/L), whereas the permeability increased only at very high concentrations, which give an extreme activation of PKC compared with thrombin and histamine. Second, Ro31-8220 and Calphostin C, inhibitors of PKC, did not prevent the thrombin-induced increase in permeability. PMA and Ro31-8220 also had no effect on thrombin-stimulated MLC phosphorylation. Third, histamine and thrombin induce a very similar rise in intracellular Ca²⁺ and give a comparable activation of PKC in human EC.^{38 39} For animal ECs, the role of PKC in endothelial permeability has been established by several investigators.^{17 18} However, in HUVEC, the contribution of PKC could not be demonstrated unequivocally. Yamada et al⁴⁰ showed that activation of PKC by PMA caused a decrease in endothelial permeability, whereas Bussolino et al⁴¹ found an increase in endothelial permeability by PKC activation. Garcia et al²⁵ did not find any direct effect of PMA on human endothelial permeability and MLC phosphorylation, whereas they found an increase of both by PMA in bovine ECs. Therefore, it seems that species differences are responsible for the discrepancies observed between the different studies.

In conclusion, we have demonstrated that in endothelial monolayers in vitro, which give a similar transient permeability response after exposure to histamine as expected from in vivo experiments,¹ thrombin induces a prolonged increase in permeability that involves protein tyrosine phosphorylation and RhoA activation. This mechanism appears comparable to the calcium sensitization observed in smooth muscle cells.²⁸ Future studies have to verify its occurrence in ECs in vivo and elucidate whether a similar sensitization mechanism underlies the increased permeability that is enhanced by leukocytes and humoral factors circulating in patients with prolonged edema.

	Acknowledgments

 
This study was financially supported by the Netherlands Heart Foundation (grant 94.048). We would like to thank Dr A.H. Zwinderman (Leiden University Medical Center) for advice and help regarding statistical analysis.

Received May 15, 1998; accepted September 15, 1998.

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