Cytoskeletal Activation and Altered Gene Expression in ... - CiteSeerX

Cytoskeletal Activation and Altered Gene Expression in Endothelial Barrier Regulation by Simvastatin Jeffrey R. Jacobson, Steven M. Dudek, Konstantin G. Birukov, Shui Q. Ye, Dmitry N. Grigoryev, Reda E. Girgis, and Joe G. N. Garcia Center for Translational Respiratory Medicine, Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland.

The statins, a class of HMG-CoA reductase inhibitors, directly affect multiple vascular processes via inhibition of geranylgeranylation, a covalent modification essential for Rho GTPase interaction with cell membrane–bound activators. We explored simvastatin effects on endothelial cell actomyosin contraction, gap formation, and barrier dysfunction produced by the edemagenic agent, thrombin. Human pulmonary artery endothelial cells exposed to prolonged simvastatin treatment (5 ␮M, 16 h) demonstrated significant reductions in thrombin-induced (1 U/ml) barrier dysfunction (ⵑ 70% inhibition) with accelerated barrier recovery, as measured by transendothelial resistance. Furthermore, simvastatin attenuated basal and thrombin-stimulated (1 U/ml, 5 min) myosin light chain diphosphorylation and stress fiber formation while dramatically increasing peripheral immunostaining of actin and cortactin, an actin-binding protein, in conjunction with increased Rac GTPase activity. As both simvastatininduced Rac activation and barrier protection were delayed (maximal after 16 h), we assessed the role of gene expression and protein translation in the simvastatin response. Simultaneous treatment with cycloheximide (10 ␮g/ml, 16 h) abolished simvastatin-mediated barrier protection. Robust alterations were noted in the expression of cytoskeletal proteins (caldesmon, integrin ␤4), thrombin regulatory elements (PAR-1, thrombomodulin), and signaling genes (guanine nucleotide exchange factors) in response to simvastatin by microarray analysis. These novel observations have broad clinical implications in numerous vascular pathobiologies characterized by alterations in vascular integrity including inflammation, angiogenesis, and acute lung injury.

The statins, inhibitors of HMG-CoA reductase, are used for their lipid-lowering properties and favorable effects on the morbidity and mortality associated with coronary artery disease (1). Their beneficial effects, however, cannot be entirely attributed to reductions in lipid levels, as recent evidence suggests statins may alter outcomes in a variety of diverse conditions including bacteremia (2), osteoporosis (3), and cancer metastases (4). Moreover, several studies have demonstrated direct effects of the statins on the endothelium (5, 6) implicating these drugs as significant effectors of vascular function. Although regulation of nitric oxide (NO) production has been suggested (6, 7), the mechanism of statin action remains to be fully defined. Our growing understanding of the effects of statins on endothelial cell (EC) function, however, suggests that this class of drugs may hold promise in clinical conditions for which they have

(Received in original form July 17, 2003 and in revised form October 27, 2003) Address correspondence to: Joe G. N. Garcia, M.D., Division of Pulmonary & Critical Care, Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224. E-mail: [email protected] Abbreviations: endothelial cell, EC; guanine nucleotide exchange factor, GEF; hepatocyte growth factor, HGF; human pulmonary artery endothelial cells, HPAEC; myosin light chain, MLC; MLC kinase, MLCK; nitric oxide, NO; NO synthase, NOS; sphingosine 1-phosphate, Sph 1-P; transendothelial electrical resistance, TER. Am. J. Respir. Cell Mol. Biol. Vol. 30, pp. 662–670, 2004 Originally Published in Press as DOI: 10.1165/rcmb.2003-0267OC on November 20, 2003 Internet address: www.atsjournals.org

yet to be considered, including pulmonary hypertension (8), a disease characterized by EC proliferation and vascular remodeling, and acute lung injury, a disease defined by excessive vascular permeability. Vascular EC regulate solute transport between vascular compartments and surrounding tissues functioning as a semipermeable cellular barrier dynamically regulated by the cytoskeleton (9). Several models of enhanced vascular permeability have underscored the critical role of the balance between complex tethering forces (cell–cell, cell–matrix interactions) and cellular contractile forces involving ratcheting of bonds between actin and myosin resulting in actin stress fiber formation and increased tension (10). Increased cellular contraction is catalyzed by the phosphorylation of myosin light chains (MLC), a consequence of the coordinate activation of Rho GTPase with subsequent activation of its target effector Rho kinase and Ca2⫹/calmodulin-dependent EC MLC kinase (MLCK) (11). Cell contraction ultimately results in cell rounding and paracellular gap formation, key determinants of barrier dysfunction and increased vascular permeability (9). The statins inhibit prenylation, a covalent modification involving the addition of either farnesyl (15-carbon) or geranylgeranyl (20-carbon) side chains. One product of the prenylation pathway is cholesterol, however, activation of the Rho family GTPases, such as Rho and Rac, is also dependent on this modification. These small GTPases act as molecular switches controlling various cellular events, including actomyosin rearrangement, by cycling between GTP-bound (active) and GDP-bound (inactive) states. GTPase activation is strongly reliant on geranylgeranylation and subsequent translocation to the cell membrane. Despite evidence of Rho inhibition (5, 12), statin effects on EC cytoskeletal rearrangement and barrier regulation remain poorly characterized. We hypothesized that simvastatin confers endothelial cell barrier protection via early inhibitory effects on Rho proteins with subsequent passive relaxation of the cytoskeleton. Our results now indicate, however, profound delayed effects of simvastatin on not only Rho, but active Rac-dependent actin rearrangement, barrier regulation, and cytoskeletal gene expression in human lung endothelium.

Materials and Methods Materials and Reagents Unless otherwise specified, reagents were obtained from Sigma (St. Louis, MO). Rabbit anti-diphosphorylated MLC antibody was produced as previously described (13). Rabbit anti-MLC antibody (Sigma), horseradish peroxidase–linked anti-mouse and anti-rabbit antibodies (Cell Signaling Technology, Beverly, MA), mouse anti-cortactin antibody (Upstate Biotechnology, Lake Placid, NY), mouse anti-integrin ␤4 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anticaldesmon antibody (Sigma), rabbit anti-HA tag antibody (Santa Cruz Biotechnology), and Texas Red–conjugated phalloidin (Molecular Probes, Eugene, OR) were commercially obtained.

Cell Culture Human pulmonary artery EC (HPAEC) were obtained from Clonetics (San Diego, CA), used at passages 3–8, and maintained in EGM-2

Jacobson, Dudek, Birukov, et al.: Simvastatin Alters EC Function and Gene Expression

complete medium (Clonetics). Cells were incubated at 37⬚C in 5% CO2 and 95% air.

Measurement of Transendothelial Electrical Resistance HPAEC were grown to confluence over evaporated gold microelectrodes connected to a phase-sensitive lock-in amplifier as previously described (14). Transendothelial electrical resistance (TER) was measured using an electrical cell-substrate impedance sensing system (ECIS; Applied BioPhysics Inc., Troy, NY). As cells adhere and spread out on the microelectrode, TER increases, whereas cell retraction, rounding, or loss of adhesion is reflected by a decrease in TER. These measurements provide a sensitive biophysical assay that indicates the state of cell shape and focal adhesion.

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medium with simvastatin (5 ␮M) exhibited TER values similar to vehicle-pretreated cells (ⵑ 1 ⍀). However, simvastatin-pretreated monolayers demonstrated a marked reduction in the magnitude of thrombin-stimulated (1 U/ml) declines in TER (ⵑ 70% reduction) and a more rapid recovery to baseline (ⵑ 66% reduction in time to recovery) (Figures 1A and 1B). The duration of simvastatin treatment was an important determinant of this response as shorter durations (1 and 6 h) at similar concentrations

MLC Phosphorylation in Intact Endothelium HPAEC were analyzed for MLC phosphorylation by sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by Western immunoblotting with antibody specific for diphosphorylated MLC as previously described (13). Blots were scanned and quantitatively analyzed using ImageQuant software (v5.2; Amersham Biosciences, Piscataway, NJ).

Immunofluorescent Microscopy Confluent HPAEC grown on coverslips were exposed to experimental conditions, fixed with 3.7% formaldehyde in phosphate-buffered saline at 4⬚C for 15 min, and permeabilized with 0.25% Triton X-100 for 15 min. After blocking with 2% bovine serum albumin in phosphatebuffered saline for 30 min, cells were exposed to primary antibodies of interest for 60 min. Fluorescently-tagged secondary antibodies were applied for 60 min in the dark. F-actin was detected by staining with Texas Red–conjugated phalloidin. Cells were imaged using a Nikon video-imaging system.

Rac GTPase Activity Assay Determination of Rac activation (Rac-GTP) was performed as we previously described using a commercially available kit (Upstate Biotechnology, Inc., Lake Placid, NY) (15).

Synthesis of cDNA and Hybridization Probes Confluent HPAEC were suspended in 1 ml TRIzol (Life Technologies, Frederick, MD). Chloroform (0.2 ml) was added and samples briefly centrifuged at 4⬚C. Supernatant containing RNA was preserved and 0.5 ml isopropanol added followed by repeat centrifugation. Supernatant was again preserved and 100 ␮l DEPC water and 200 ␮l cold EtOH added. Before cDNA synthesis, RNA was purified using RNeasy Mini Kit (Qiagen, Valencia, CA) and quantified by spectrophotometric analysis. Double-stranded cDNA was synthesized from total RNA and biotin-labeled cRNA prepared as previously described (16).

Microarray Analysis Expression profiling was performed (Affymetrix GeneChip system, Santa Clara, CA) in the Gene Expression Profiling Core of the JHU Center for Translational Respiratory Medicine as previously described (17). Samples hybridized (45⬚C, 16 h) to an Affymetrix HGU95Av2 Array (ⵑ 12,000 full-length genes), were stained with streptavidin–phycoerythrin conjugate, and then scanned with a Hewlett-Packard GeneArray Scanner. Affymetrix Microarray Suite software was used to determine relative gene expression. GeneSpring software (v5.0.2; Silicon Genetics, Redwood City, CA) and MAPPFinder (18) were used for microarray data analysis.

Statistical Analysis ANOVA with a Student-Neuman-Keuls test was used to compare the means of data from two or more different experimental groups. Results are expressed as means ⫾ SD. Differences between groups were considered statistically significant when P ⬍ 0.05.

Results Simvastatin Attenuates Thrombin-Induced Human Lung Endothelial Barrier Dysfunction

TER was used as a sensitive measure of EC monolayer integrity and barrier function. HPAEC incubated for 16 h in complete

Figure 1. Effect of simvastatin on thrombin-induced barrier dysfunction. HPAEC were grown on gold microelectrodes to measure TER. Simvastatin (5 ␮M, 16 h) did not alter basal TER. However, relative to vehicle controls, simvastatin-treated EC demonstrate a dramatic attenuation of TER decline after thrombin (1 U/ml) and a more rapid recovery to baseline (A ). Maximal simvastatin effect is observed at 20 min (B ) (n ⫽ 5). Simvastatin effects on thrombin-induced barrier dysfunction were unaltered by L-NAME (100 ␮M) 1 h before thrombin (1 U/ml) (C ).

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failed to attenuate the thrombin response. As a result, overnight incubations (16 h) were used for subsequent experiments. Recent literature suggests that the statins may produce vascular effects via increased NO synthase (NOS) activity and NO production (6, 7). To evaluate the role of NOS in the barrierprotective effects of simvastatin, TER measurements were obtained in HPAEC pretreated with both simvastatin (5 ␮M, 16 h) and L-NAME (100 ␮M, 1 h), an NOS inhibitor, before thrombin stimulation (1 U/ml). Similar simvastatin-induced attenuation of the thrombin response was observed despite L-NAME addition, indicating that NOS inhibition does not alter the barrier-protective effects of simvastatin (Figure 1C). Simvastatin Attenuates Thrombin-Induced EC MLC Phosphorylation

Thrombin-induced EC barrier disruption involves prominent rearrangement of the cytoskeleton with actin stress fiber formation and cellular contraction (9). Stress fiber formation is driven by phosphorylated MLC, coordinately regulated by Rho kinase and MLCK. Accordingly, measurements of MLC phosphorylation allow characterization of changes in cytoskeletal regulation and contractility. HPAEC pretreated with varying concentrations of simvastatin (0.1–5 ␮M, 16 h) were stimulated with thrombin (1 U/ml, 5 min) and MLC diphosphorylation determined by Western blotting using an antibody specific for diphosphorylated MLC (Figure 2). These experiments revealed a clear concentrationdependent effect, as concentrations of 5 ␮M or greater were required to significantly attenuate both basal and thrombin-induced MLC diphosphorylation (83% and 38% reductions, respectively).

Of note, shorter durations of pretreatment with simvastatin (5 min and 2 h) did not significantly alter basal or thrombin-induced MLC phosphorylation. Effect of Simvastatin on Actin Stress Fiber Formation and Rac Activation

Confluent EC under basal conditions exhibit few transcytoplasmic actin stress fibers, whereas stress fiber formation increases dramatically in response to thrombin in association with paracellular gaps (11). HPAEC treated with simvastatin (5 ␮M) demonstrated evidence of cytoskeletal rearrangement early (2 h), and prolonged treatment (16 h) resulted in significantly reduced stress fibers in association with markedly increased cortical actin. This relative paucity of stress fibers persisted following thrombin stimulation in simvastatin-treated cells with evidence of reduced numbers of paracellular gaps (Figure 3A). Our prior studies using diverse barrier-protective agents such as sphingosine 1-phosphate (Sph 1-P) (15), hepatocyte growth factor (HGF) (19), and shear stress (20) all demonstrate similar increases in cortical actin as we observed with simvastatin. As each of these stimuli are associated with Rac GTPase activation, we next assessed Rac activation in simvastatin-treated (5 ␮M) HPAEC. Unlike HGF and Sph 1-P, brief (5 min) simvastatin treatment did not alter basal levels of Rac activation; however, significant increases in Rac-GTP were appreciable at 16 h (ⵑ 82% increase) (Figure 3B). Role of Cortactin Translocation in Simvastatin-Induced Barrier Enhancement

Cortactin, an actin-binding protein that translocates to the cell periphery, is involved in cortical actin polymerization and potentially in promoting barrier integrity (20, 21). In some cell systems, including shear stress, cortactin translocation is mediated by the activation of Rac (22). Accordingly, because of the growing recognition of its role as a key effector of cortical actin dynamics, we sought to characterize cortactin responses in simvastatintreated HPAEC. Immunofluorescent studies confirmed evidence of cortactin translocation in simvastatin-treated (5 ␮M) HPAEC beginning at 2 h and persisting for 16 h (Figure 3C). These findings were accentuated within 5 min of thrombin stimulation (1 U/ml) and remained evident 2 h after thrombin, suggesting a prolonged simvastatin effect. Thus, simvastatin-mediated barrier protection occurs only after ⬎ 6 h of pretreatment, a time course entirely consistent with the alterations in Rac activity and cortactin. Differential Gene Expression and Protein Synthesis Induced by Simvastatin

Figure 2. Effects of simvastatin on thrombin-induced EC MLC phosphorylation. Confluent HPAEC were pretreated with simvastatin (0.1, 1, or 5 ␮M, 16 h) before thrombin stimulation (1 U/ml, 5 min). Cells were subjected to Western blotting and probed with diphospho-MLC antibody. Relative to vehicle, both basal and thrombin-induced MLC diphosphorylation was significantly attenuated in EC pretreated with 5 ␮M simvastatin (n ⫽ 3).

As noted, barrier protection was only observed following sustained simvastatin pretreatment (⬎ 6 h), suggesting the possibility that changes in gene expression and protein translation may contribute to this vascular-protective response. To explore this possibility, human EC were treated with either simvastatin (5 ␮M) or vehicle for 24 h and RNA isolated for Affymetrix microarray analysis as described. Using conventional Affymetrix MAS software, marked changes in expression (⭓ 2-fold) were noted in 74 upregulated genes and 151 downregulated genes after simvastatin. Several genes that encode for proteins reported by our lab and by others to be involved in thrombin-mediated cytoskeletal dynamics and barrier regulation, including caldesmon (23), and the thrombin receptor PAR-1, were dramatically downregulated (Table 1). In addition, integrin ␤4, a protein known to be involved in cell–cell adhesion (24), was dramatically upregulated. Rac1 was also upregulated, as were specific guanine nucleotide exchange factors (GEFs), potential regulators of preferential Rho GTPase activity. Consistent with the known


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Figure 3. Actin rearrangement, Rac activation, and cortactin translocation in simvastatin-treated EC. Confluent HPAEC treated with simvastatin (5 ␮M, 16 h) demonstrate a diminution of central stress fibers relative to vehicle controls, both under basal conditions and in response to thrombin (1 U/ml, 5 min) (A ), as well as cortical actin ring enhancement (small arrows). Further, after thrombin stimulation, paracellular gaps observed in controls (large arrows) were sparse in simvastatin-treated cells. Bar, 10 ␮M. Rac activation was measured in HPAEC grown to ⵑ 75% confluence and treated with vehicle, simvastatin (5 ␮M for 5 min, 2 h, or 16 h), or Sph 1-P (1 ␮M, 1 min). Positive and negative controls (Rac-GTP and Rac-GDP, respectively) were separately prepared. Rac-GTP was pulled down with a PAK1-GST fusion protein and samples subjected to Western blotting and detection with anti-Rac1 antibody (B ). Although not evident at early time points (⭐ 2 h), Rac activation is increased at 16 h in simvastatin-treated cells relative to vehicle (n ⫽ 3). To characterize cortactin translocation HPAEC were grown on coverslips, treated with simvastatin (5 ␮M, 16 h), and stained for cortactin (C ). Vehicle cells revealed a diffuse cortactin distribution that was markedly increased after thrombin. In contrast, simvastatin-treated EC demonstrated peripheral cortactin translocation (small arrows) that remained evident after thrombin stimulation (1 U/ml, 5 min), persisting for 2 h after thrombin (not shown). Bar, 10 ␮M.

inhibitory effects of simvastatin on Rho activity, RhoA and RhoC genes were upregulated and Rho GDP dissociation inhibitor was downregulated, which we interpret as a compensatory response. These data indicate that simvastatin directly affects expression of specific genes related to Rho GTPase signaling and cytoskeletal regulation. Select gene expression data were validated by Western blotting of HPAEC lysates treated with vehicle or simvastatin (5 ␮M, 16 h) for integrin ␤4 and caldesmon (Figure 4A). Finally, to further analyze our microarray data we employed MAPPFinder (18), a tool that incorporates annotations of the Gene Ontology (GO) project (25) with GenMAPP, a free software package that allows microarray data to be analyzed by microarray pathway profiles (MAPPs) (26) (Table 2). GO terms associated with increased (⭓ 30%) gene expression in response to simvastatin included transcriptional repressor genes and struc-

tural constituents of the cytoskeleton. Highly consistent with the known action of simvastatin, there were increases in protein prenylation pathways genes and various regulators of the small GTPases. Along with increases in transcription repressor genes, we also observed a decrease in mRNA processing genes suggesting an overall suppression in transcription by simvastatin, including the thrombin receptor and Rho GDP-dissociation inhibitor pathway genes which exhibited decreased expression (⭓ 30%), findings that corroborate and expand on our initial analysis with respect to individual genes (Table 1). To establish the role of differential gene expression and downstream effects on protein synthesis in simvastatin-induced EC barrier protection, we used cycloheximide, a protein synthesis inhibitor. TER measurements of HPAEC grown to confluence and then incubated in complete media with both simvastatin (5 ␮M) and cycloheximide (10 ␮g/ml) simultaneously (16 h)

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TABLE 1. Effect of simvastatin on selected gene expression Gene Integrin ␤4 subunit hCOX-2 Thrombomodulin Rho GAP 4 RhoA Rho/Rac GEF 2 RhoC Rac1 PAR-1 Rho GDP dissociation inhibitor Caldesmon

Fold Change after Simvastatin 7.57 3.3 2.73 2.02 1.69 1.56 1.44 1.29 0.73 0.69 0.47

Microarray analysis was employed to identify changes in gene expression induced by human EC treatment with simvastatin (5 ␮M) for 24 h (n ⫽ 2). Data were pooled and normalized. Genes related to regulation of the cytoskeleton were specifically queried and those with significant changes in expression identified.

demonstrated a loss of barrier protection after thrombin stimulation (1 U/ml) relative to HPAEC treated with simvastatin alone (5 ␮M, 16 h) (Figure 4B). Next, we measured Rac activation in this setting. As total Rac was notably decreased in response to cycloheximide treatment, our results were normalized to total Rac. Compared with HPAEC treated with simvastatin alone (5 ␮M, 16 h), Rac activation was significantly attenuated (ⵑ 60% decrease) by treatment with simvastatin (5 ␮M, 16 h) in the presence of protein translation inhibition by cycloheximide (10

␮g/ml, 16 h) (Figure 4C). Although there are clear limitations of prolonged cycloheximide treatment, these data implicate a requirement for protein synthesis for full expression of simvastatin EC barrier-protective properties.

Discussion Although direct beneficial effects of statins on the endothelium have previously been reported, the mechanisms involved remain poorly characterized. In this study, we have focused on the EC cytoskeleton and barrier regulation and determined that prolonged simvastatin treatment dramatically attenuates EC barrier disruption evoked by thrombin (as measured by TER). These physiologic effects occur in concert with cytoskeletal modulation resulting in the abolishment of actin stress fibers and attenuation of paracellular gap formation. Of note, significant reductions in MLC phosphorylation by simvastatin were most pronounced under basal conditions, possibly reflecting the prominent contribution of MLCK to these events in response to thrombin stimulation. Nonetheless, these results are consistent with the increasing literature that the statins are potent inhibitors of Rho GTPase. In this regard, inhibition of prenylation of the Rho family GTPases has been proposed as an important mechanism to account for the effects of simvastatin on the endothelium. Specifically, inhibition of Rho by simvastatin attenuates actin stress fiber formation promoted by Rho kinase via its capacity to inhibit MLC phosphatase activity. Indeed, simvastatin effects on MLC phosphorylation and stress fiber formation are extraordinarily

Figure 4. Differential gene expression and protein synthesis induced by simvastatin. Genes demonstrating significant changes in expression in response to simvastatin were identified and validated by Western blotting. Consistent with microarray data, expression of caldesmon is significantly decreased (ⵑ 2-fold change) in simvastatin-treated (5 ␮M, 16 h) HPAEC, whereas integrin ␤4 is dramatically increased (⬎ 10-fold change) (A ). To confirm the role of simvastatininduced protein synthesis in EC barrier protection, HPAEC were grown on gold microelectrodes to measure TER. In response to thrombin stimulation (1 U/ml), simvastatin-induced (5 ␮M, 16 h) barrier protection was abrogated by the simultaneous treatment with cycloheximide (CHX, 10 ␮g/ml, 16 h) (B ) (n ⫽ 3). To characterize the role of Rac activation in this setting, HPAEC were grown to ⵑ 75% confluence and then treated with vehicle, simvastatin (5 ␮M, 16 h), cycloheximide (CHX, 10 ␮g/ml, 16 h), or both simvastatin and cycloheximide. Activated Rac (Rac GTP) was measured as described. Rac activation, normalized for total Rac, was significantly decreased in cells simultaneously treated with simvastatin and cycloheximide relative to cells treated with simvastatin alone (n ⫽ 2) (C ).


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Figure 5. Paradoxical effects of simvastatin on EC Rho family GTPases. Simvastatin inhibits HMG CoA reductase, thus blocking the prenylation pathway (A ) and downstream products including the farnesylated proteins such as cholesterol (B ), and the geranylgeranylated proteins which include the Rho family GTPases (C ). As would be predicted, simvastatin inhibits Rho. However, Rac is paradoxically activated. Although the mechanisms are unclear, specific GEFs upregulated by simvastatin are potential candidates.

similar to our prior results with botulinum toxin C3, a Rho kinase inhibitor (27). Aside from Rho kinase effects on MLC phosphatase, however, Rho is also known to regulate additional intracellular enzymatic targets including endothelial NOS (28). Several investigators have reported a direct effect of simvastatin on NOS and NO production, presumably via Rho inhibition. However, we failed to demonstrate that inhibition of NOS by L-NAME altered either thrombin-induced TER changes in EC or the simvastatin protective response. Thus, our findings implicate a unique mechanism of action and further elucidate the pleiotropic properties of this class of drugs. A highly novel and unexpected finding in the present study was prominent cortical actin thickening induced by simvastatin, results which we have previously noted with diverse barrierprotective agonists and biophysical stimuli (15, 19, 20). This observation suggested potential Rac GTPase activation as a mechanism for the cortical rearrangement and barrier protection. Rho inhibition by statins has previously been demonstrated;

however, the effect of statins on Rac remains unclear. Whereas Rac inhibition by statins has been reported in cardiomyocytes (29) and smooth muscle cells (30), a recent study described increased Rac-GTP in EC after prolonged statin treatment (31). However, these investigators observed that despite increased Rac-GTP, Rac-dependent signaling was inhibited. The apparent discordance between the role for Rac in cortical actin rearrangement, which we have previously observed, and the predicted inhibition of Rho GTPases including Rac by statins, led us to investigate the direct effect of simvastatin on human lung EC Rac activity. Our results now strongly support simvastatinmediated Rac activation with profound effects on cytoskeletal rearrangements. These results are consistent with our prior observations that Rac activation is commonly linked to other EC barrier promoting agents such as Sph 1-P (15), HGF (19), and mechanical shear stress (20), and is associated with translocation of cortactin to the cell membrane. Simvastatin induced prominent time-dependent Rac activation and cortactin translocation, leading us to speculate

TABLE 2. Gene Ontology (MAPPFinder) results for genes significantly altered in response to simvastatin No. Changed

No. Measured

No. in GO

% Changed

% Present

z Score

⭓ 1.3-fold increase Transcriptional repressor Rho small monomeric GTPase RAS guan-nuceotide exch factor Receptor signaling protein Nitric oxide biosynthesis Cytoskeleton structural constituent RAS GTPase activator Protein amino acid prenylation

5 5 2 5 4 16 2 3

20 20 4 36 12 90 5 9

28 29 4 49 13 121 7 10

25 25 50 13.9 33.3 17.8 40 33.3

71.4 69 100 73.5 92.3 74.4 71.4 90

2.99 2.68 2.67 2.63 2.51 2.49 2.24 2.24

⭓ 1.3-fold decrease mRNA processing Protein serine/threonine kinase Rho GDP-dissociation inhibitor Thrombin receptor Actin cross-linking

9 67 2 3 3

22 300 3 6 6

32 460 3 6 8

40.9 22.3 66.7 50 50

68.8 65.2 100 100 75

6.36 5.59 2.78 2.71 2.71

Analysis of microarray data with MAPPFinder identified Gene Ontology (GO) terms with high levels of gene-expression changes. The z score represents the relative degree of change for a given GO term with a greater z score indicative of results less likely to be due to chance. GO terms of interest with a fold change of at least 1.3 and a z score ⭓ 2 were identified.

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that this may be an important determinant of enhancement of barrier function. That these events are common to each of these otherwise distinct biophysical and bioactive agonists suggests that they are critical determinants of EC barrier enhancement. Our findings suggest for the first time that Rac-dependent signaling (peripheral actin polymerization) and cortactin translocation is evoked by simvastatin. Cortactin, a multidomain actin-binding protein that stimulates and stabilizes actin polymerization, is found in peripheral areas of dynamic cytoskeletal rearrangement, where it participates in cell migration and tumor invasion (32, 33). Although the functional contribution of cortactin to the barrier-promoting effects of simvastatin is still being characterized, our current concepts of barrier enhancement involve a key role for cortactin in barrier-enhancing cytoskeletal rearrangement (9). We now report that cortactin is concentrated at the cell periphery in response to simvastatin, a finding that mirrors Sph 1-P– stimulated effects and strongly suggests that cortactin may play a similar regulatory role in simvastatin-induced barrier regulation. Ongoing studies are exploring the potential mechanisms through which cortactin modulates these effects. A unique feature of simvastatin-induced EC barrier protection, however, is the absence of a significant effect on basal EC integrity as measured by TER. This is in contrast to Sph 1-P and HGF, which also induce Rac activation and cortactin translocation, but dramatically increase basal TER values. The mechanisms underlying these different characteristics are unknown, but suggest an important distinction between specific receptor agonists (Sph 1-P, HGF) and simvastatin. Additionally, the unique effects of simvastatin are further evidenced by the marked delay in Rac activation (16 h) compared with shear stress, Sph 1-P or HGF (within 5 min). Simvastatin-induced Rac activation was unexpected as, given statin inhibition of the prenylation pathway, the downstream inhibition of Rac would actually be predicted (Figure 5). One potential explanation for this paradoxical observation is that the inhibition of prenylation preferentially inhibits Rho and, via a normally tonic inhibitory effect on Rac, effectively increases Rac activation. This would be consistent with previous observations of a direct interaction between Rho and Rac in fibroblasts (34). A second possibility is that specific effectors of Rac activation, independent of the prenylation pathway, are induced by simvastatin either directly or in response to transcription-regulated

gene expression. Rac and Rho cycling between GTP- (active) and GDP-bound (inactive) states is regulated by numerous exchange factor proteins classified as GEFs, which facilitate the exchange of GDP for GTP, GAPs (GTPase-activating proteins, which increase the rate of GTP hydrolysis by Rho GTPases), and RhoGDI (guanine nucleotide dissociation inhibitors, which associate with inactivated Rho and Rac) (Figure 6) (35, 36). A number of GEFs are known to target multiple GTPases, whereas others specifically target Rac (Tiam1, Vav2, ␤PIX, GEF-H1) or Rho (p115RhoGEF, p190RhoGEF, LARG) (36, 37). We speculate that simvastatin treatment results in the activation or upregulation of Rac-specific GEF(s) that promote the exchange of GDP for GTP in the Rac guanine nucleotide binding site. This hypothesis is supported by the finding that simvastatininduced Rac activation and EC barrier protection were inhibited by the protein synthesis inhibitor, cycloheximide. Further studies elucidating Rac-specific GEFs in simvastatin-induced Rac activation are underway. Finally, consistent with the time course of the EC-protective effects of simvastatin, microarray analysis revealed Rac1 gene upregulation as well as upregulation of RhoA, RhoC, and Rho GAP 4, and downregulation of RhoGDI. The effect of simvastatin on Rho and Rho-related proteins likely represents compensatory responses to the inhibition of Rho activation. The increase in Rac1, however, is unexpected given the demonstrable Rac activation after simvastatin. The significance of these changes is unclear, but we have demonstrated significant and verifiable changes in the expression of specific proteins in response to simvastatin (integrin ␤4, caldesmon). That simvastatin is able to induce marked gene expression and is able to activate Rac, which is notably inhibited by cycloheximide, suggests that specific effectors of Rac activation exist but are yet to be identified. One particular protein of interest is integrin ␤4, as we have identified its expression (gene and protein) to be dramatically upregulated by simvastatin. This finding is consistent with the observed EC simvastatin response given evidence of a role for integrin ␤4 in cell–cell adhesion (24) and reports of the differential regulation of Rho GTPases by other integrin ␤ subunits, potentially via GEF intermediates (38). Indeed, the possibility that Rac activation may be mediated by integrin ␤4 would also account for the unique time course of this event as simvastatin-induced protein synthesis is inherently a delayed effect. Further explora-

Figure 6. Differential expression of cytoskeletal genes in response to simvastatin. Simvastatin (5 ␮M, 24 h) induced the differential expression of specific EC cytoskeletal-regulatory genes identified by cDNA microarray experiments. Consistent with the barrier-protective properties of simvastatin, integrin ␤4 and thrombomodulin were dramatically upregulated, whereas PAR-1 and caldesmon were downregulated. RhoA, RhoC, and Rho GAP 4 were upregulated, possibly as a compensatory response to the inhibition of Rho activation by simvastatin, as was Rac1. Several genes of interest, including cortactin, remained unchanged.


tion of a possible correlation between integrin ␤4 upregulation and subsequent Rac activation by simvastatin is warranted. We identified several other EC barrier-regulatory genes differentially expressed in response to simvastatin (Figure 6). In particular, the downregulation of the thrombin receptor, PAR-1, and the significant upregulation of thrombomodulin, a transmembrane EC surface glycoprotein that promotes thrombin inhibition and increases activated protein C (39), a potent acute-coagulant, are consistent with a simvastatin-induced EC thromboresistant phenotype, characteristic of a tight vascular barrier. We also noted significant downregulation of caldesmon, an actin-, myosin-, and calmodulin-binding cytoskeletal protein that serves as regulator of contractile activity in response to barrier-disruptive agonists, including thrombin (23). Additional temporal studies of simvastatin-induced gene expression may identify potential targets for further investigation. As noted, barrier protection as measured by TER was evident only after prolonged simvastatin treatment (16 h), leading us to focus on the biochemical events associated with this time course. However, we did appreciate evidence of cytoskeletal rearrangement after only 2 h of simvastatin that did not correspond to changes in MLC phosphorylation or Rac activation and did not confer barrier protection. This suggests induction of distinct signaling events outside of our focus and underscores the overall intricacy of simvastatin effects on the endothelium that remain to be fully characterized. In summary, our findings are consistent with the concept that statins have potent beneficial effects on vascular function via modification of intracellular events that drive cellular contraction, paracellular gap formation, and increased vascular permeability. Although regulation of the endothelial cytoskeleton is complex, we have identified Rho kinase inhibition, Rac activation, and cortactin translocation in response to simvastatin, and speculate that these events are critical determinants of its effects on EC barrier function. Given the availability, affordability, and relatively favorable safety profile of this class of drugs, the statins warrant consideration as possible therapeutic options for various vascular pathobiologies including acute lung injury as well as other syndromes of organ dysfunction characterized by vascular leak. Acknowledgments: The authors acknowledge the support of the Center for Translational Respiratory Medicine, the HopGene Program in Genomic Applications, grants from the NHLBI (HL 71411, HL 58064, HL 69340, and HL 66583), and the Dr. David Marine Endowment. The authors gratefully acknowledge John W. Fenton II for helpful discussions and Lakshmi Natarajan for expert cell culture preparation.

References 1. Scandinavian Simvastatin Survival Study Group. 1994. Randomised trial of cholesterol lowering in 4444 patients with coronary heart disease: the Scandinavian Simvastatin Survival Study (4S). Lancet. 344:1383–1389. 2. Liappis, A. P., V. L. Kan, C. G. Rochester, and G. L. Simon. 2001. The effect of statins on mortality in patients with bacteremia. Clin. Infect. Dis. 33:1352–1357. 3. Rejnmark, L., N. H. Buus, P. Vestergaard, F. Andreasen, M. L. Larsen, and L. Mosekilde. 2002. Statins decrease bone turnover in postmenopausal women: a cross-sectional study. Eur. J. Clin. Invest. 32:581–589. 4. Kusama, T., M. Mukai, T. Iwasaki, M. Tatsuta, Y. Matsumoto, H. Akedo, M. Inoue, and H. Nakamura. 2002. 3-hydroxy-3-methylglutaryl-coenzyme a reductase inhibitors reduce human pancreatic cancer cell invasion and metastasis. Gastroenterology 122:308–317. 5. van Nieuw Amerongen, G. P., M. A. Vermeer, P. Negre-Aminou, J. Lankelma, J. J. Emeis, and V. W. van Hinsbergh. 2000. Simvastatin improves disturbed endothelial barrier function. Circulation 102:2803–2809. 6. Mueck, A. O., H. Seeger, and D. Wallwiener. 2001. Further evidence for direct vascular actions of statins: effect on endothelial nitric oxide synthase and adhesion molecules. Exp. Clin. Endocrinol. Diabetes 109:181–183. 7. Mital, S., X. Zhang, G. Zhao, R. D. Bernstein, C. J. Smith, D. L. Fulton, W. C. Sessa, J. K. Liao, and T. H. Hintze. 2000. Simvastatin upregulates coronary vascular endothelial nitric oxide production in conscious dogs. Am. J. Physiol. Heart Circ. Physiol. 279:H2649–H2657.

669

8. Girgis, R. E., D. Li, X. Zhan, J. G. Garcia, R. M. Tuder, P. M. Hassoun, and R. A. Johns. 2003. Attenuation of chronic hypoxic pulmonary hypertension by simvastatin. Am. J. Physiol. Heart Circ. Physiol. 285:H938–H945. 9. Dudek, S. M., and J. G. Garcia. 2001. Cytoskeletal regulation of pulmonary vascular permeability. J. Appl. Physiol. 91:1487–1500. 10. Hirata, A., P. Baluk, T. Fujiwara, and D. M. McDonald. 1995. Location of focal silver staining at endothelial gaps in inflamed venules examined by scanning electron microscopy. Am. J. Physiol. 269:L403–L418. 11. Garcia, J. G., and K. L. Schaphorst. 1995. Regulation of endothelial cell gap formation and paracellular permeability. J. Investig. Med. 43:117–126. 12. Hernandez-Perera, O., D. Perez-Sala, E. Soria, and S. Lamas. 2000. Involvement of Rho GTPases in the transcriptional inhibition of preproendothelin-1 gene expression by simvastatin in vascular endothelial cells. Circ. Res. 87:616–622. 13. Petrache, I., A. D. Verin, M. T. Crow, A. Birukova, F. Liu, and J. G. Garcia. 2001. Differential effect of MLC kinase in TNF-alpha-induced endothelial cell apoptosis and barrier dysfunction. Am. J. Physiol. Lung Cell. Mol. Physiol. 280:L1168–L1178. 14. Garcia, J. G., K. L. Schaphorst, S. Shi, A. D. Verin, C. M. Hart, K. S. Callahan, and C. E. Patterson. 1997. Mechanisms of ionomycin-induced endothelial cell barrier dysfunction. Am. J. Physiol. 273:L172–L184. 15. Garcia, J. G., F. Liu, A. D. Verin, A. Birukova, M. A. Dechert, W. T. Gerthoffer, J. R. Bamberg, and D. English. 2001. Sphingosine 1-phosphate promotes endothelial cell barrier integrity by Edg-dependent cytoskeletal rearrangement. J. Clin. Invest. 108:689–701. 16. Mahadevappa, M., and J. A. Warrington. 1999. A high-density probe array sample preparation method using 10- to 100-fold fewer cells. Nat. Biotechnol. 17:1134–1136. 17. Cappola, T. P., L. Cope, A. Cernetich, L. A. Barouch, K. Minhas, R. A. Irizarry, G. Parmigiani, S. Durrani, T. Lavoie, E. P. Hoffman, S. Q. Ye, J. G. Garcia, and J. M. Hare. 2003. Deficiency of different nitric oxide synthase isoforms activates divergent transcriptional programs in cardiac hypertrophy. Physiol. Genomics 14:25–34. 18. Doniger, S. W., N. Salomonis, K. D. Dahlquist, K. Vranizan, S. C. Lawlor, and B. R. Conklin. 2003. MAPPFinder: using Gene Ontology and GenMAPP to create a global gene-expression profile from microarray data. Genome Biol. 4:R7. 1–12. 19. Liu, F., K. L. Schaphorst, A. D. Verin, K. Jacobs, A. Birukova, R. M. Day, N. Bogatcheva, D. P. Bottaro, and J. G. Garcia. 2002. Hepatocyte growth factor enhances endothelial cell barrier function and cortical cytoskeletal rearrangement: potential role of glycogen synthase kinase-3beta. FASEB J. 16:950–962. 20. Birukov, K. G., A. A. Birukova, S. M. Dudek, A. D. Verin, M. T. Crow, X. Zhan, N. DePaola, and J. G. Garcia. 2002. Shear stress–mediated cytoskeletal remodeling and cortactin translocation in pulmonary endothelial cells. Am. J. Respir. Cell Mol. Biol. 26:453–464. 21. Weed, S. A., A. V. Karginov, D. A. Schafer, A. M. Weaver, A. W. Kinley, J. A. Cooper, and J. T. Parsons. 2000. Cortactin localization to sites of actin assembly in lamellipodia requires interactions with F-actin and the Arp2/ 3 complex. J. Cell Biol. 151:29–40. 22. Weed, S. A., Y. Du, and J. T. Parsons. 1998. Translocation of cortactin to the cell periphery is mediated by the small GTPase Rac1. J. Cell Sci. 111:2433–2443. 23. Stasek, J. E., Jr., C. E. Patterson, and J. G. Garcia. 1992. Protein kinase C phosphorylates caldesmon77 and vimentin and enhances albumin permeability across cultured bovine pulmonary artery endothelial cell monolayers. J. Cell. Physiol. 153:62–75. 24. Abdel-Ghany, M., H. C. Cheng, R. C. Elble, and B. U. Pauli. 2001. The breast cancer beta 4 integrin and endothelial human CLCA2 mediate lung metastasis. J. Biol. Chem. 276:25438–25446. 25. Ashburner, M., C. A. Ball, J. A. Blake, D. Botstein, H. Butler, J. M. Cherry, A. P. Davis, K. Dolinski, S. S. Dwight, J. T. Eppig, M. A. Harris, D. P. Hill, L. Issel-Tarver, A. Kasarskis, S. Lewis, J. C. Matese, J. E. Richardson, M. Ringwald, G. M. Rubin, and G. Sherlock. 2000. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat. Genet. 25:25–29. 26. Dahlquist, K. D., N. Salomonis, K. Vranizan, S. C. Lawlor, and B. R. Conklin. 2002. GenMAPP, a new tool for viewing and analyzing microarray data on biological pathways. Nat. Genet. 31:19–20. 27. Garcia, J. G., A. D. Verin, K. Schaphorst, R. Siddiqui, C. E. Patterson, C. Csortos, and V. Natarajan. 1999. Regulation of endothelial cell myosin light chain kinase by Rho, cortactin, and p60(src). Am. J. Physiol. 276:L989–L998. 28. Laufs, U., and J. K. Liao. 1998. Post-transcriptional regulation of endothelial nitric oxide synthase mRNA stability by Rho GTPase. J. Biol. Chem. 273:24266–24271. 29. Laufs, U., H. Kilter, C. Konkol, S. Wassmann, M. Bohm, and G. Nickenig. 2002. Impact of HMG CoA reductase inhibition on small GTPases in the heart. Cardiovasc. Res. 53:911–920. 30. Negre-Aminou, P., R. E. van Leeuwen, G. C. van Thiel, P. van den IJssel, W. W. de Jong, R. A. Quinlan, and L. H. Cohen. 2002. Differential effect of simvastatin on activation of Rac(1) vs. activation of the heat shock protein 27-mediated pathway upon oxidative stress, in human smooth muscle cells. Biochem. Pharmacol. 64:1483–1491.

670


31. Vecchione, C., and R. P. Brandes. 2002. Withdrawal of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors elicits oxidative stress and induces endothelial dysfunction in mice. Circ. Res. 91:173–179. 32. Huang, C., J. Liu, C. C. Haudenschild, and X. Zhan. 1998. The role of tyrosine phosphorylation of cortactin in the locomotion of endothelial cells. J. Biol. Chem. 273:25770–25776. 33. Bowden, E. T., M. Barth, D. Thomas, R. I. Glazer, and S. C. Mueller. 1999. An invasion-related complex of cortactin, paxillin and PKCmu associates with invadopodia at sites of extracellular matrix degradation. Oncogene 18:4440–4449. 34. Ridley, A. J., H. F. Paterson, C. L. Johnston, D. Diekmann, and A. Hall. 1992. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell 70:401–410. 35. Bishop, A. L., and A. Hall. 2000. Rho GTPases and their effector proteins. Biochem. J. 348:241–255.

36. Zheng, Y. 2001. Dbl family guanine nucleotide exchange factors. Trends Biochem. Sci. 26:724–732. 37. Schmidt, A., and A. Hall. 2002. Guanine nucleotide exchange factors for Rho GTPases: turning on the switch. Genes Dev. 16:1587–1609. 38. Miao, H., S. Li, Y. L. Hu, S. Yuan, Y. Zhao, B. P. Chen, W. Puzon-McLaughlin, T. Tarui, J. Y. Shyy, Y. Takada, S. Usami, and S. Chien. 2002. Differential regulation of Rho GTPases by beta1 and beta3 integrins: the role of an extracellular domain of integrin in intracellular signaling. J. Cell Sci. 115:2199–2206. 39. Aritomi, M., N. Watanabe, R. Ohishi, K. Gomi, T. Kiyota, S. Yamamoto, T. Ishida, and I. Maruyama. 1993. Recombinant human soluble thrombomodulin delivers bounded thrombin to antithrombin III: thrombomodulin associates with free thrombin and is recycled to activate protein c. Thromb. Haemost. 70:418–422.