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British Heart Foundation Laboratories, Department of Medicine, University College London; and Institute of Child Health, London, United Kingdom
Submitted 24 September 2004 ; accepted in final form 2 December 2004
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ABSTRACT |
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reactive oxygen species; adherens junctions; actin cytoskeleton
Increase in endothelial permeability induced by hypoxia is accompanied by actin polymerization and formation of stress fibers, but the signaling pathways leading to these changes are still poorly understood (15, 20, 25, 31). An increase in endothelial permeability induced by vasoactive agents is regulated by Rho GTPases, well-known regulators of the actin cytoskeleton (45). Whether or not Rho GTPases play a role in hypoxia and reoxygenation-induced changes in permeability is not known. Moreover, Rho GTPases have also been implicated in oxygen sensing. Rac1 is a component of the NADPH oxidase complex (10, 28, 47) and has been shown to contribute to the reactive oxygen species (ROS) production required for integrin-mediated cell adhesion and spreading in HeLa cells (23). Cdc42 plays a role in hypoxia-induced activation of hypoxia-inducible factor (HIF)-1
in renal carcinoma Caki-1 cells (34). The individual contributions of Rac1, RhoA, and Cdc42 in the regulation of endothelial permeability still remain controversial. Rac1 is required for the maintenance of intercellular adherens and tight junctions (41, 32), but its activation has also been associated with a loss of junctional integrity (39, 40). RhoA promotes formation of stress fibers and contributes to breaking of intercellular adhesions as a result of an increased centripetal tension (38). It has been shown, however, that activation of RhoA with bacterial toxins (40) or with its constitutively active form, V14RhoA, is not sufficient for the breakdown of intercellular junctions (39). Some authors have shown that dispersion of endothelial intercellular junctions requires simultaneous activation of both Rac1 and RhoA (44), whereas others describe activation of one GTPase and inactivation of the other (40). The role of Cdc42 in the regulation of endothelial barrier function is also not fully understood. Transfection studies with its constitutively active and dominant negative forms showed no significant effect on endothelial permeability (40, 45), but other studies demonstrated that Cdc42 is required for the restoration of adherens junctions following exposure to thrombin (16).
Here we investigated the roles of Rho GTPases Rac1, RhoA, and Cdc42, as well as their upstream activators and downstream effectors, in endothelial responses to oxygenative stress. We show that Rho GTPases Rac1 and RhoA respond rapidly to changes in oxygen tension in a phosphatidylinositol 3-kinase (PI3-kinase)- and NADPH oxidase-dependent way and that their coordinated actions regulate endothelial barrier function in endothelial cells of conduit pulmonary arteries.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Porcine pulmonary artery endothelial cells (PPAECs) were isolated by collagenase digestion from conduit pulmonary arteries of adult Large White pigs. The cells were cultured in flasks covered with 10 µg/ml bovine fibronectin in RPMI (PAA Laboratories, Pasching, Austria) supplemented with 10% FCS and antibiotics and used between passages 2 and 5. They were grown until confluence on fibronectin-covered (bovine fibronectin, Sigma) 6-cm petri dishes, Thermanox plastic coverslips, and 96-well TC microwell plates (Nunc, Roskilde, Denmark) or polyester Transwell-Clear filters (3-µm pore size, 12-mm diameter, Costar Corning; Costar, High Wycombe, UK). Cell cultures were then either incubated in a normoxic incubator (20% O2, 5% CO2, 37°C, humidified atmosphere) or exposed to hypoxia (3% O2, 5% CO2, 92% N2) for 5 min–4 h. After 1 h, some hypoxic cultures were then returned to normoxic conditions for 1 h of reoxygenation. After exposure to normoxia, hypoxia, and hypoxia/reoxygenation, the cells were used for permeability assays, confocal microscopy, measurement of ROS, or GTPase activity assays.
Adenoviral gene transfer was used to express green fluorescent protein (GFP), dominant negative RhoA, Rac1, and Cdc42 (N19RhoA, N17Rac1, N17Cdc42), and constitutively active Rac1 (V12Rac1) proteins in PPAECs (45). Experiments were performed 16–18 h after adenoviral infection. The Rho kinase inhibitor Y-27632 (5 µM, Yoshitomi Pharmaceutical Industries), the PI3-kinase inhibitor LY-294002 (10 µM; Calbiochem, Nottingham, UK), lysophosphatidic acid (LPA, 1 µg/ml; Sigma), the flavoprotein inhibitor diphenylene iodonium (DPI, 10 µM; Calbiochem), the NADPH oxidase inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF, 2 mM; Sigma), and Rho GTPase inhibitor Clostridium difficile toxin B (50 ng/ml, Calbiochem) were added to cell cultures 1 h before the experiments were carried out. The RhoA inhibitor C3 transferase was expressed in Escherichia coli from the pGEX-2T vector as glutathione S-transferase (GST) fusion protein and purified as described previously (30). C3 transferase was added to the cells for overnight incubation at 10 µg/ml.
Transfection of 90–95% confluent PPAECs with GFP-tagged L63RhoA construct was carried out with the transfection reagent Metafectene (Biontex, Munich, Germany) according to the manufacturer's protocol.
GST-C3 transferase, pCDNA3-GFP, and pCDNA3-GFP-L63RhoA constructs and adenoviral constructs encoding N19RhoA, N17Cdc42, N17Rac1, V12Rac1, and GFP were a kind gift from Prof. Anne Ridley (Ludwig Institute for Cancer Research, London, UK).
Transendothelial Permeability Assays
Transendothelial permeability was measured as previously described (45). FITC-dextran (molecular wt 42,000, 1 mg/ml; Sigma) was added to the upper compartment of Transwell-Clear chambers (3-µm pore size, 12-mm diameter, Costar Corning; Costar), and cells were incubated in normoxic or hypoxic conditions. Samples were taken from the lower compartment after 15 min, 30 min, 1 h, 2 h, 3 h, and 4 h of incubation, and the amount of FITC-dextran in the lower compartment was determined with a TECAN GeNios microplate reader (TECAN, Reading, UK) using an excitation wavelength of 485 nm and emission at 510 nm. In experiments with inhibitors or with adenovirally infected cells, the flux of FITC dextran was compared with that of the control cells over the first hour of exposure to hypoxia. To determine endothelial permeability following reoxygenation, we added FITC-dextran to the upper chambers of Transwell filters after 1-h exposure to hypoxia, and then flux was measured over the next 2 h in normoxic conditions.
Immunofluorescence and Localization of F-Actin
To visualize the distributions of F-actin and myc-tagged proteins, PPAECs grown on Thermanox plastic coverslips were fixed with 4% formaldehyde dissolved in PBS for 15 min and permeabilized for 3 min in 0.1% Triton X-100 (Sigma). Cells were incubated with 1% BSA in PBS to block nonspecific binding and then incubated with rabbit polyclonal anti-c-myc antibody (1:200; Santa Cruz Biotechnology, Santa Cruz, CA) and mouse monoclonal anti-VE-cadherin antibody (1:200, Santa Cruz Biotechnology) for 1 h. Cells were then incubated with FITC-labeled donkey anti-mouse antibody (1:100; Jackson Immunoresearch Laboratories, West Grove, PA), Cy5-labeled donkey anti-rabbit antibody (1:100; Jackson Immunoresearch Laboratories), and 1 µg/ml TRITC-phalloidin (Sigma) for 1 h. The specimens were mounted in Mowiol (Calbiochem). Single plain images of the cells were obtained by confocal laser scanning microscopy (Bio-Rad Radiance 2100) with Bio-Rad software (LaserSharp 2000 version 5.1).
Rho, Rac, and Cdc42 Pull-down Assays
The cells were grown on 6-cm petri dishes until confluence and then incubated overnight in medium with 5% serum before exposure to hypoxia. RhoA activity was measured with recombinant GST-Rho binding domain bound to glutathione beads (Upstate Biotechnology), the activity of Rac1 was measured with GST-p21-activated kinase binding domain (PAK1-PBD, Upstate Biotechnology), and the activity of Cdc42 was measured with GST-Wiscott-Aldrich syndrome protein (WASP)-PBD (37). GST-WASP-PBD was a kind gift of Prof. Ridley. Affinity-precipitated RhoA, Rac1, and Cdc42 proteins were resolved by SDS-PAGE and detected by Western blotting. Horseradish peroxidase-conjugated mouse IgG used for Western blotting was from DAKO (Glostrup, Denmark). Western blots were analyzed by densitometry. The detailed protocol of Rac and Rho pull-downs was provided by Upstate Biotechnology (Rac and Rho activation assay kits).
Measurement of ROS
ROS generation in PPAECs was assessed using 2',7'-dichlorofluorescein diacetate (DCFH-DA, 5 µM; Calbiochem). In the presence of H2O2, this probe is oxidized to 2',7'-dichlorofluorescein, which was quantified using a TECAN GeNios microplate reader at excitation wavelength of 485 nm and emission at 530 nm. Cells were grown until confluence on 96-well plates, and the inhibitors were added to some of the wells before the experiment as described above. After preincubation, culture medium was replaced with fresh Krebs saline, pH 7.4, with or without inhibitors of Rho GTPases and containing the fluorescent probe DCFH-DA. Krebs saline was prepared as described in Ref. 3, with a modified glucose concentration at 5.6 mM. Cell cultures were placed in normoxic or hypoxic incubators, and ROS production was assessed after 1-h incubation.
Statistical Analysis
Experiments were performed in triplicate. Data are presented as means ± SD. Comparisons between more than two groups were performed using a one-way ANOVA test followed by Dunnett's posttest. Tukey posttest for multiple comparisons was used for the analysis of data obtained with the use of various inhibitors (permeability and ROS production). The choice between parametric and nonparametric tests was based on the Bartlett's test for homogeneity of variances. Statistical significance was accepted for P < 0.05, and all tests were performed with GraphPad Prism version 3.0.
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RESULTS |
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Confluent pulmonary artery endothelial cells cultured under normal oxygen tension had F-actin localized mainly at the cell periphery (Fig. 1A) where VE-cadherin, the major component of adherens junctions, was also localized (Fig. 1B). Hypoxia (3% oxygen) induced formation of thick bundles of actomyosin filaments (stress fibers) in the cell center (Fig. 1C) accompanied by dispersion of VE-cadherin from intercellular junctions (Fig. 1D). Formation of stress fibers was first noticed at 15 min and was maximal between 1–2 h of hypoxic exposure. After 3–4 h of hypoxia, junctional localization of VE-cadherin was partially restored, but the level of stress fibers remained higher than in normoxic controls (data not shown).
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Hypoxia and Reoxygenation Have Opposing Effects on Endothelial Permeability and the Activity of the Rho GTPases, RhoA, Rac1, and Cdc42
Hypoxia-induced formation of stress fibers and dispersion of VE-cadherin correlated with an increase in endothelial permeability measured as passage of fluorescently labeled dextran through PPAECs grown on Transwell filters. The passage rate of FITC-dextran through control PPAEC monolayers was 7.2 µg·h–1·cm–2 (SD 0.4). Hypoxia induced a transient breakdown of endothelial permeability, which reached a maximal value of 11 µg·h–1·cm–2 (SD 0.5) at 1 h of hypoxic exposure (Fig. 2A) (P < 0.05, comparison with normal controls). Reoxygenation of PPAECs for 1 h was sufficient for the restoration of endothelial barrier function.
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Rac1 activity was transiently reduced by hypoxia and reached its minimal value (twofold decrease) after 1 h of exposure and then gradually returned to the basal levels at 2–4 h of exposure (Fig. 2B). Reoxygenation of the cells for 1 h following 1-h exposure to hypoxia induced a significant increase in Rac1 activity to a level 2.5-fold higher than in controls (Fig. 2B). We chose 1 h as a time point at which to assess events in future studies, by which time endothelial barrier function was restored, although it took 2 h of reoxygenation for Rac1 activity to fall to the control level.
Unlike Rac1, hypoxia induced a transient activation of RhoA, first noticed at 15 min of exposure and reaching a maximal level at 1 h (2.5-fold increase). RhoA activity gradually decreased but was still significantly higher than in controls after 3 h of exposure (Fig. 2C). Reoxygenation of the cells for 1 h reduced RhoA activity to control levels (Fig. 2C). Cdc42 activity was increased by hypoxia and reduced to control levels by 1-h reoxygenation, a response comparable to that for RhoA (Fig. 2D). Expression levels of Rac1, RhoA, and Cdc42 remained unchanged during acute hypoxia and reoxygenation (Fig. 2, B–D).
RhoA and Rac1 Play Opposing Roles in the Regulation of Hypoxia/Reoxygenation-Induced Changes in Endothelial Barrier Function
RhoA/Rho kinase are required for the hypoxia-induced increase in endothelial permeability, stress-fiber formation, and dispersion of adherens junctions, whereas Rac1 is required for endothelial recovery during reoxygenation. To investigate the contributions of RhoA, Rac1, and Cdc42 to hypoxia-induced responses in PPAECs, we used the recombinant adenoviruses to express myc-tagged dominant negative mutants N19RhoA, N17Rac1, and N17Cdc42 and constitutively active Rac1, V12Rac1. The infection efficiency in PPAECs was evaluated with the use of adenovirus containing GFP and showed that 18 h after infection 76% (SD 8) of cells expressed GFP. Adenoviral infection did not have a significant effect on endothelial permeability in PPAECs (Fig. 3). The inhibitors of RhoA, N19RhoA, and C3-transferase, as well as the inhibitor of Rho kinase, Y-27632, prevented the hypoxia-induced increase in endothelial permeability but had no effect on endothelial permeability in normoxia or reoxygenation (Fig. 3). N17Rac1 had no significant effect on hypoxia-induced permeability but induced endothelial leakage in cells cultured in normoxic conditions and prevented the recovery of endothelial barrier function during reoxygenation (Fig. 3). In contrast, constitutively activated Rac1, V12Rac1, had a protective effect on endothelial barrier function in hypoxia (Fig. 3) and had no significant effect in normoxia and reoxygenation.
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The effects of the inhibitors of Rho GTPases on endothelial permeability correlated with their effects on the actin cytoskeleton and adherens junctions in PPAECs (Fig. 4). The inhibitors of RhoA/Rho kinase prevented hypoxia-induced formation of stress fibers and dispersion of VE-cadherin (Fig. 4, A–D) but had no effect on the changes in the actin cytoskeleton or adherens junctions in normoxia (data not shown) or during posthypoxic recovery (Fig. 4G). In contrast, N17Rac1 had no effect on hypoxia-induced effects (Fig. 4E) but induced dispersion of VE-cadherin in normoxia (data not shown) and completely prevented relocalization of F-actin to the cell cortex and the restoration of adherens junctions during reoxygenation (Fig. 4H). N17Cdc42 had no effect on the stability of adherens junctions or F-actin remodeling during normoxia (data not shown), hypoxia, or reoxygenation (Fig. 4, F and I).
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Constitutively activated Rac1, V12Rac1, inhibited hypoxia-induced formation of stress fibers and dispersion of intercellular junctions (Fig. 4P). In normoxia and reoxygenation V12Rac1-expressing cells showed strong junctional staining of VE-cadherin (Fig. 4O and data not shown). However, after prolonged expression (48 h), V12Rac1 had a damaging effect on endothelial integrity as well as inducing a loss of cortical F-actin and dispersion of VE-cadherin from intercellular junctions (Fig. 4R).
Rac1 acts upstream of RhoA. To address whether Rac1 and RhoA regulate their activities in a reciprocal manner, we studied the activity of those GTPases in PPAECs expressing N17Rac1, N19RhoA, or cells treated with the inhibitor of RhoA, C3 transferase. We observed that RhoA activity was significantly increased by N17Rac1, whereas the inhibitors of RhoA, N19RhoA, and C3 transferase, as well as the inhibitor of Rho kinase, Y-27632 (data not shown), did not have any effect on the activity of Rac1 (Fig. 5).
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NADPH Oxidase and PI3 Kinase Are Required for Hypoxia/Reoxygenation-Induced Changes in Rho GTPases Activity and Endothelial Remodeling
NADPH-oxidase-mediated changes in ROS production are upstream of Rho GTPases in the regulation of endothelial responses to hypoxia/reoxygenation. To examine whether changes in ROS production correlate with changes in the activity of Rac1 and RhoA, we measured ROS levels in PPAECs in normoxic and hypoxic conditions and on reoxygenation. The cells were untreated (controls) or treated with the inhibitors of Rho GTPases or the inhibitors of Rho kinase, NADPH oxidase, and PI3 kinase. ROS production decreased significantly in hypoxia (1.7-fold decrease P < 0.05, comparison with controls) and returned to control levels during reoxygenation (Fig. 6A). The inhibitor of NADPH oxidase, DPI, and the inhibitor of PI3 kinase, LY-294002, significantly reduced ROS production in PPAECs in normoxia, did not alter ROS levels in hypoxia, but completely prevented an increase in ROS levels following reoxygenation (Fig. 6A). To confirm the involvement of NADPH oxidase in ROS production, we used another inhibitor, AEBSF, structurally unrelated to DPI. DPI impairs NADPH oxidase by flavoprotein inhibition (8), whereas AEBSF inhibits NADPH oxidase activity by preventing its assembly at the cell membrane (6). We observed that AEBSF reduced ROS production to the same extent as DPI (Fig. 6A).
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Inhibition of NADPH oxidase and PI3 kinase mimics the effects of hypoxia and prevents the effects of reoxygenation on the actin cytoskeleton, adherens junctions, and endothelial permeability in PPAECs. The effects of the NADPH inhibitor DPI on F-actin remodeling, distribution of VE-cadherin, and endothelial permeability in control, normoxic PPAECs resembled the effects of hypoxia (Fig. 7, D and J). DPI reduced the amount of cortical F-actin and promoted formation of stress fibers and dispersion of intercellular junctions (Fig. 7D) and increased endothelial permeability (Fig. 7J). Although we did not observe any effects of DPI on endothelial remodeling and permeability in response to hypoxia (Fig. 7, E and J), the inhibitor completely prevented cortical redistribution of F-actin and the restoration of adherens junctions and endothelial barrier function during reoxygenation (Fig. 7, F and J). Another inhibitor of NADPH oxidase, AEBSF, had similar effects to DPI on F-actin and VE-cadherin distribution (data not shown) and endothelial permeability (Fig. 7J). Cells treated with the PI3 kinase inhibitor LY-294002 in normoxic conditions lost cortical distribution of F-actin and junctional localization of VE-cadherin (Fig. 7G) and showed an increase in endothelial permeability (Fig. 7J). Inhibition of PI3 kinase did not have any effect on hypoxia-induced changes (Fig. 7, H and J) but strongly inhibited endothelial recovery following reoxygenation (Fig. 7, I and J).
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DISCUSSION |
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We found that Rac1/RhoA activity changes and the restoration of adherens junctions during reoxygenation in PPAECs were dependent on the NADPH oxidase-regulated production of oxygen radicals. This result was confirmed using two unrelated inhibitors of NADPH oxidase, DPI and AEBSF. ROS acted upstream of Rac1 in PPAECs, as it has been shown to do in hypoxia-induced activation of HIF-1 (35). ROS production was also inhibited by the Rac1 inhibitor N17Rac1. A feed-forward mechanism of ROS-dependent upregulation of NADPH oxidase activity was reported in smooth muscle cells and fibroblasts (17). Delineation of the signals upstream and downstream of Rac1 induced by hypoxia and reoxygenation will require further study.
Several studies have shown that changes in the level of ROS alter endothelial permeability, but controversy still exists as to when and where ROS are produced, from an NAD(P)H oxidase system or the mitochondrial electron transport chain (26). Our results show that NADPH oxidase plays a major part in oxygen sensing in PPAECs and is the main source of ROS generation in these cells, consistent with other reports (36, 2). In these as in other cell types it remains to be established whether other sources of intracellular ROS such as mitochondria also play a role (43). Generation of ROS by PPAECs decreased during hypoxia, consistent with the NADPH oxidase theory of ROS generation, not the mitochondrial model (4). Both the contribution of NADPH oxidase to ROS production and the functional effects of ROS depend on the origin of the endothelial cells. NADPH activity of cultured human microvascular cells is substantially higher than that of cultured human umbilical vein endothelial cells (HUVECs) cultured under similar conditions (17), and hypoxia-induced production of ROS causes vasodilatation in renal arteries while reducing ROS production and causing vasoconstriction in pulmonary arteries (21). An optimal level of ROS production appears to be necessary for the maintenance of endothelial barrier function, as in the case of Rac1 activity levels. After reoxygenation of PPAECs, a modest 2- to 2.2-fold increase in ROS production from the relatively low end hypoxic level leads to endothelial recovery and restoration of intercellular junctional integrity, which seems to be a teleologically appropriate phenomenon. But following overexpression of V12Rac1 in HUVECs, a six- to sevenfold increase in ROS production induced breakdown of intercellular junctions (39). Growth factors and cytokines known to affect endothelial barrier function such as PDGF, EGF, and TNF- have also been shown to induce a high (up to 10-fold) increase in ROS production in NIH/3T3 cells (34).
The inhibitor of PI3 kinase, LY-294002, mimicked the effects of hypoxia in PPAECs, like DPI. This is likely to result from the effects of PI3 kinase on NADPH activity and ROS production. PI3 kinase has previously been shown to regulate the activity of NADPH oxidase (5, 7) and to act upstream of Rac1 in hypoxia-induced activation of HIF-1 in HEP3B and HEK-293 cell lines (12). It has also been shown to regulate formation of cadherin-mediated cell-cell contacts (13, 14, 22, 33) and to act upstream of Rac1 in cell-cell contact formation induced by the activation of N-cadherin (10).
Another issue that needs to be addressed is the mechanism by which Rho GTPase activation can occur in conditions such as hypoxia when ATP and GTP levels are low. We found that in oxygen-starved cells, Rac1 activity initially decreased but then increased again. A similar phenomenon was reported in porcine proximal tubule cells where Rac1 activity decreased following ATP and GTP depletion but then increased again even while ATP and GTP levels continued to fall (11). This recovery could result from selective impacts of ATP and GTP depletion on the activities of GAP and GEF proteins (11).
In this study we also observed a transient activation of Cdc42 during hypoxia and its deactivation during reoxygenation, generally resembling the activity time course of RhoA. However, the activation of Cdc42 did not have a significant effect either on the Rac1/RhoA-regulated actin remodeling or on the permeability changes seen in PPAECs. This suggests that Cdc42 did not act upstream of these GTPases in the present studies, as it appears to do in TNF- signaling in HUVECs and in growth factor stimulation of Swiss 3T3 fibroblasts (44, 24, 30). In addition, we observed that Rac1 and not Cdc42 was required for the reoxygenation-induced reconstitution of adherens junctions in PPAECs. By contrast, Kouklis et al. (16) recently reported that Cdc42 is more important than Rac1 in the reformation of adherens junctions after thrombin treatment in human microvascular and pulmonary artery endothelial cells. As Rac1 and Cdc42 act in concert in mediating cell spreading and migration and activate some of the same downstream effectors such as PAKs (p21-activated kinases) or GTPase activating protein, IQGAP1 (1, 29), it is likely that the choice of one or the other GTPase for the regulation of junctional integrity is dependent on the type of the activating agent and/or the cell source.
In summary, we show for the first time that the Rho GTPases respond to variations in oxygen levels in an NADPH- and PI3-kinase-dependent manner to mediate changes in endothelial barrier function, the actin cytoskeleton, and adherens junctions. Understanding the signaling pathways leading to endothelial remodeling upon hypoxia/reoxygenation will help establish therapeutic targets in the treatment of conditions associated with oxidative stress such as acute respiratory distress syndrome.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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