RhoA Activation Mediates Phosphatidylinositol 3-Kinase–Dependent Proliferation of Human Vascular Endothelial Cells: An Alloimmune Mechanism of Chronic Allograft Nephropathy
Institut National de la Santé Et de la Recherche Médicale Unité 643 "Immunointervention en Allo et Xénotransplantation" and Institut de Transplantation et de Recherche en Transplantation, C.H.U. Hôtel-Dieu, Nantes, France
Correspondence to Dr. Béatrice Charreau, INSERM U643, 30 bd J. Monnet, 44093 Nantes Cedex 01, France. Phone: 33-2-40-08-74-16; Fax: 33-2-40-08-74-11; E-mail: charreau{at}nantes.inserm.fr
ABSTRACT. HLA class I ligation on graft endothelial cells (EC)has been shown to promote graft arteriosclerosis and chronicallograft nephropathy. This study investigated transcriptionaland functional changes mediated by anti-HLA antibodies (Ab),developed by transplant recipient, on vascular renal EC. Formimicking interactions that occur between alloantibodies andgraft endothelium, HLA-typed primary cultures of human EC wereincubated in vitro in the presence of monomorphic or polymorphicanti-HLA class I Ab. Gene expression analysis identified theupregulation of several molecules involved in cell signalingand proliferation, including the GTP-binding protein RhoA. Itwas demonstrated further that HLA class I ligation on EC induceda rapid translocation of RhoA to the cell membrane associatedwith F-actin stress fiber formation and cytoskeleton reorganization.Western blot analysis showed that anti-HLA class I Ab induced,in addition to RhoA, the activation of phosphatidylinositol3-kinase, reflected by the phosphorylation of Akt (Ser473) andGSK3 (Ser9), in EC. C3 exoenzyme, an inhibitor of RhoA, inhibitedRhoA translocation in response to HLA class I ligation and reducedphosphatidylinositol 3-kinase activation. EC proliferation andcell cycle progression, examined by 5,6-carboxyfluorescein diacetatesuccinimidyl ester staining, demonstrated that anti-HLA-inducedEC proliferation was efficiently prevented by the 3-hydroxy-3-methylglutarylCoA reductase inhibitor simvastatin (0.1 µmol/L) throughinhibition of RhoA geranylgeranylation. Taken together, thesefindings support the conclusion that RhoA is a key mediatorof signaling pathways that lead to cytoskeletal reorganizationand EC proliferation in response to alloantibodies that bindto HLA class I and demonstrate the specific and potent inhibitoryeffect of simvastatin on allostimulated EC growth.
Chronic allograft nephropathy is the major factor limiting long-termsurvival of renal allografts (1,2). The hallmark of chronicallograft nephropathy is transplant arteriosclerosis, whichis characterized by the intimal proliferation of endothelialcells (EC), smooth muscle cells (SMC), and fibroblasts, leadingto vessel obstruction and ischemia that causes late graft failure(3). Several risk factors have been identified, including bothimmune injury to transplant vessels and nonimmunological factors(e.g., ischemia/reperfusion, hypertension, hyperlipidemia).The immunologic mechanisms that induce chronic allograft nephropathyare poorly understood, but it is suspected that the associatedvascular changes are a result of early injury to the endotheliumof the graft mediated by allogeneic T cells and anti-HLA alloantibodies(2). The incidence of transplant arteriosclerosis is increasedin transplant recipients who produce anti-donor HLA antibodies(Ab) after transplantation, suggesting that anti-HLA Ab playa role in the pathogenesis of the disease (4,5). In previousstudies, it has been shown that anti-HLA Ab, developed by transplantrecipients after transplantation, are capable of transducingsignals via HLA class I molecules, which stimulate cell proliferation(6). Furthermore, ligation of class I molecules with Ab alsoresults in increased tyrosine phosphorylation of several intracellularproteins on EC (7,8). Treatment of cells with IFN- and TNF-upregulated MHC class I expression and potentiated anti-HLAAb-mediated proliferative responses (9). These findings supporta role for anti-HLA Ab in the transduction of proliferativesignals, which stimulate the development of intimal hyperplasiaassociated with chronic allograft nephropathy and renal transplantloss.
We previously demonstrated that natural, preformed, antidonorAb mediated changes in EC gene expression according to theirspecificity (10,11). In the present study, we investigated theeffect of HLA class I ligation mediated by anti-HLA Ab on endothelialgene expression. Primary cultures of HLA-typed human vascularEC, isolated from cadaveric transplant donors, were incubatedwith anti-HLA mAb directed to either monomorphic or polymorphicHLA class I regions. RNA differential display reverse transcription-PCR(RT-PCR) was used to compare gene expression between restingand anti-HLA-treated EC and to identify genes and molecularmechanisms upregulated upon HLA class I ligation. Among thecandidate genes found to be overexpressed, several cell-cycleregulators were identified, including the GTP-binding proteinRhoA. These changes in transcription suggested that anti-HLAclass I Ab could trigger EC proliferation via a Rho-dependentpathway. Thus, our study further examined the implication ofRhoA protein in signaling pathways that lead to EC proliferationand transplant arteriosclerosis. The fluorescent dye 5,6-carboxyfluoresceindiacetate succinimidyl ester (CFSE) has been used to monitorEC division and proliferation and therefore was used to demonstratethat control of Rho GTPase activation could modulate EC proliferation.
Reagents
Simvastatin was supplied by Merck, Sharp & Dohme. Thrombin,inhibitors of phosphatidylinositol 3-kinase (PI3-K) LY294002and wortmannin, and isoprenoid compounds farnesyl pyrophosphate(FPP) and geranylgeranyl pyrophosphate (GGPP) were obtainedfrom Sigma-Aldrich (Saint Quentin Fallavier, France). Clostridiumbotulinum C3 exoenzyme was purchased from Biomol Research Laboratories(Plymouth, PA). Basic fibroblast growth factor (bFGF) was fromR&D Systems (Abingdon, UK).
Primary EC Isolation and Culture
Human arterial EC were isolated from renal artery patches takenbefore kidney transplantation, as described previously (12).EC were grown in Endothelial Cell Growth Medium (ECGM) supplementedwith 10% FCS, 0.004 ml/ml ECGS/Heparin, 0.1 ng/ml human EGF,1 ng/ml human bFGF, 1 µg/ml hydrocortisone, 50 µg/mlgentamicin, and 50 ng/ml amphotericin B (C-22010; PromoCell,Heidelberg, Germany) at 37°C in a humidified 5% CO2 atmosphere.
Gene Expression Analysis RNA Differential Display RT-PCR.
EC were incubated for 2 h at 37°C in medium that contained100 U/ml recombinant human TNF- (provided by Prof. MüllerNeuman, Ludwigshafen, Germany) or 10 µg/ml anti-HLA classI mAb. Treatment of EC was performed using either monomorphic(anti-HLA-A,B,C mAb:W6/32, IgG2a) or polymorphic (anti-HLA-A2,anti-HLA-B51, and anti-HLA-Bw4) anti-HLA class I purified Ab(OneLambda, Canoga Park, CA). EC that were incubated with isotypiccontrol mouse IgG1 or IgG2a (10 µg/ml; Sigma-Aldrich)or medium alone were used as controls.
RNA isolation and RNA differential display RT-PCR were performedas described previously (10) using the RNA image kit (GenHunter,Brookline, MA). Selection, cloning, and sequencing of cDNA fragmentsand bioinformatic analysis were performed as previously reported(11,13).
Quantitative Real-Time RT-PCR.
The ABI PRISM 7700 sequence detection application program (PEApplied Biosystems, Foster City, CA) was used to measure fluorescenceemitted during PCR amplification of targeted sequences in a96-well reaction plate. Real-time detection of PCR productswas monitored by measuring the increase in fluorescence causedby the binding of SYBR Green (PE Applied Biosystems) to DNA.A standard curve using serial dilutions of the purified targetsequence (107, 106, 105, 104, 103, and 102 copies/well) allowedquantification. Normalization was obtained by the concomitantquantification of hypoxanthine-guanine phosphoribosyl transferasetranscripts in each sample. Each sample was analyzed in duplicate.
Oligonucleotide primers pairs for hypoxanthine-guanine phosphoribosyltransferase (sense, 5'-TGGAAAAGCAAAATACAAAGCC-3'; antisense,5'-CATGCAAAAAGCTCTACTAAGCAG-3') and for RhoA (sense, 5'-TTAGTCCACGGTCTGGTCTTCA-3';antisense, 5'-TATGAGCAAGCATGTCTTTCCA-3') generated PCR productsof 140 and 177 bp, respectively.
Semiquantitative RT-PCR.
Primer pairs were as follows: RhoA (sense, 5'-CAGTTCCCAGAGGTGTATGT-3';antisense, 5'-AGACAAGGCAACCAGATTTT-3') and -actin (sense, 5'-AATCTGGCACCACACCTTCTACA-3';antisense, 5'-CGACGTAGCACAGCTTCTCCTTA-3'). The PCR conditionswere 18 cycles for -actin and for RhoA, at a denaturation temperatureof 94°C for 30 s, annealing at 60°C for 30 s, and extensionat 72°C for 30 s. PCR products were run on 1.2.% agarosegels and stained with ethidium bromide.
Immunofluorescence Microscopy
EC were grown on four-well glass slides (Lab-Teck; Nunc, Naperville,IL), and confluent monolayers were treated with anti-HLA-A,B,CmAb (W6/32: 10 µg/ml), thrombin (1 U/ml), or TNF- (100U/ml) after a 24-h deprivation period. EC that were treatedwith culture medium alone or an isotypic mouse IgG2a were usedas controls. After treatment, EC were fixed in 4% paraformaldehyde-PBSfor 20 min and permeabilized in 0.1% Triton X-100 at room temperaturefor 15 min. For F-actin staining, cells were incubated with2 µg/ml TRITC-phalloidin (Sigma-Aldrich) for 20 min. Forimmunofluorescent detection of RhoA, assessed on resting ortreated confluent EC, cells were stained with 2 µg/mlFITC-labeled anti-RhoA mAb. The slides were examined with afluorescence microscope (Eclipse E600 Y-FL Epifluorescence,Nikon, Japan). Images were acquired using ACT-1 software (Nikon).
Western Blotting and Pull-Down Assays
Lysis of the EC membrane was performed on ice in 5 mmol/L MgCl2,100 mmol/L NaCl, 1% NP-40, 1 mmol/L PMSF, 0.2 U/ml aprotinin,and 100 µg/ml leupeptin. Total cell lysates were obtainedusing 10 mmol/L Tris-HCl (pH 7.4), 125 mmol/L NaCl, 1% SDS,1 mmol/L PMSF, 0.2 U/ml aprotinin, and 100 µg/ml leupeptin.Cell membrane fractions were obtained after ultracentrifugationand solubilization as described previously (14). For Westernblot analysis, proteins (10 to 20 µg per lane) were separatedby SDS-PAGE and transferred onto nitrocellulose membranes (ECLHybond; Amersham Biotech UK, Little Chalfont, England). Membraneswere washed for 30 min with TBS 0.1% Tween 20 (TBST) and preblockedfor 2 h in TBST that contained 5% BSA (blocking solution; Sigma).Incubation with primary Ab diluted in blocking solution wasperformed overnight at 4°C. After extensive washing withTBST, the bound antibody was detected by a peroxidase-conjugatedanti-mouse or anti-rabbit secondary antibody (CST). After 45min of washing, the blots were developed using the ECL Westernblotting detection system (Amersham, Les Ulis, France). Ab usedin this study were rabbit polyclonal IgG anti-Akt, anti-Phospho-Akt(Ser473), anti-Phospho-Akt (Thr308), anti-phospho GSK3 (Ser9),anti-PTEN, anti-IB, and anti-phospho-IB (Ser36), all were fromCell Signaling Technology (CST, Beverly, MA). Mouse anti-glyceraldehyde-3-phosphatedehydrogenase mAb was from Chemicon (Temecula, CA) and anti-RhoAspecific (26C4) from Santa Cruz Biotechnology (Santa Cruz, CA).Anti-rabbit and anti-mouse IgG, horseradish peroxidase-linkedAb (CST) were used as secondary Ab in chemiluminescent Westernblot assays. RhoA activation was determined by affinity precipitationof the active GTP-bound RhoA using a glutathione S-transferase(GST)-fusion protein of the Rho-binding domain of the Rho effectorrhotekin (GST-RBD) using the EZ-Detect Rho Activation kit (PierceBiotechnology, Rockford, IL). The active or GTP-Rho pulled downfrom lysate was detected by Western blot using a specific anti-RhoAantibody (26C4; Santa Cruz Biotechnology).
CFSE Staining and EC Proliferation Analysis
EC monolayers were grown to confluence and quiescent cells werecultured with 2% FCS in the absence of growth supplements for12 h before treatment. EC were then incubated for 24 h with10 µg/ml anti-HLA mAb (anti-HLA-A,B,C: W6/32 or anti-HLA-A2:HB-117), 10 µg/ml control IgG1(for HB-117) or IgG2a (forW6/32), 10 ng/ml bFGF, or medium alone. After treatment, cellswere harvested with trypsin/EDTA, washed twice in culture medium,and incubated in the presence of 5 µmol/L of CFSE (MolecularProbes, Eugene, OR) in PBS for 10 min at 37°C. After washing,labeled cells were plated onto six-well culture plates and culturedfor the indicated period of time (24 or 48 h) in ECGM supplementedwith 2% FCS. Cells were then harvested, washed three times inPBS, and fixed in PBS that contained 1% paraformaldehyde. Fluorescencewas measured on 10,000 cells/sample using a FACScalibur (BectonDickinson, Mountain View, CA). Data were analyzed using CellQuestProand ModFitLT software (Becton Dickinson Immunocytometry Systems,San Jose, CA). Cell proliferation was calculated using the ProliferationWizard Model. Parent cells correspond to cells labeled and fixedimmediately after the labeling step. The proliferation indexis the sum of the cells in all generations divided by the computednumber of original parent cells present at the start of theexperiment. Results are representative of at least three independentexperiments.
Statistical Analyses
Data are shown as mean ± SD. All data were evaluatedwith two-tailed, unpaired t test or compared by one-way ANOVA.P < 0.05 was considered significant for all tests.
HLA Class I Ligation on Vascular EC Increases Expression of Genes Related to Cell Proliferation
For mimicking the Ab-mediated allospecific interactions involvedin the chronic allograft nephropathy, cultured HLA-typed primaryEC were incubated with monomorphic (HLA-A,B,C: W6/32) or polymorphic(HLA-A2, HLA-Bw4, HLA-B51) anti-HLA class I Ab. EC that wereincubated with culture medium or stimulated with TNF- were usedas negative and positive controls, respectively. After a 2-htreatment, total RNA was extracted and subjected to RNA differentialdisplay RT-PCR analysis. Among the 45 cDNA fragments found tobe overexpressed in response to anti-HLA Ab binding, severalencoded proteins related to cell-cycle signaling, regulation,and proliferation, including the GTP-binding protein RhoA, thecytoplasmic microtubule motor protein dynein, the microtubule-associatedprotein 1B, the nibrin protein, the ribonucleoprotein A2/B1,and the syntaxin 3A (Table 1). For further exploring the signalingpathway involved in EC proliferation-mediated graft arteriosclerosis,a particular focus was given to determine the role of the GTP-bindingprotein RhoA. For providing additional evidence that RhoA couldbe overexpressed by EC in response to class I HLA ligation,primary cultures of these cells were assessed for RhoA expressionat the level of mRNA by using real-time quantitative RT-PCR.Upregulation of the transcript level for RhoA was first confirmedby quantitative RT-PCR analysis, performed independently onthree different primary EC cultures incubated with anti-HLAmAb, alone or cross-linked with anti-mouse IgG (Figure 1A).RhoA mRNA expression was observed further in response to bothmonomorphic and polymorphic anti-HLA binding with a maximalincrease obtained for anti-HLA-A,B,C (W6/32) and anti-HLA-A2(HB-117) mAb (Figure 1B), both directed to the largest numberof determinants on vascular EC. Concomitant to enhanced mRNAlevel for RhoA, mRNA for RhoB and RhoC but not Rac1 and Cdc42were also increased at 2 h after anti-HLA class I Ab bindingto vascular EC (data not shown).
Figure 1. Quantitative analysis of RhoA mRNA expression by reverse transcription-PCR (RT-PCR). (A) RhoA transcript levels were analyzed by real-time quantitative RT-PCR as described in Materials and Methods and expressed as arbitrary units after normalization to hypoxanthine-guanine phosphoribosyl transferase. *P < 0.05 versus controls. RT-PCR was performed on RNA from three independent, HLA-typed cultures of primary endothelial cells (EC; #1147, #11500, and #8186) that were treated for 2 h with an isotypic control IgG2a (IgG control), anti-HLA class I antibodies (Ab; W6/32, 10 µg/ml) alone or cross-linked with anti-mouse IgG (W6/32: CX, 10 µg/ml each). (B) RT-PCR was performed on RNA from EC (#8186) that were treated for 2 h with 10 µg/ml of different anti-HLA class I Ab (anti-HLA-A,B,C:W6/32 alone or cross-linked with anti-mouse IgG:W6/32:CX, anti-HLA-A2 or anti-HLA-Bw4). EC that were incubated with culture medium alone or TNF- (100 U/ml) were used as controls.
HLA Class I Ligation Induces RhoA Translocation on EC and Cytoskeleton Reorganization
To assess activation of RhoA in response to HLA class I ligation,we investigated RhoA translocation by immunofluorescence andimmunoblot detection. Intracellular localization of RhoA wasfirst analyzed by immunofluorescent labeling of RhoA in confluentEC monolayers (Figure 2A). Our data indicated that, in the restingstate, RhoA is mainly localized in the Golgi-ER (endoplasmicreticulum) system. Incubation of EC in the presence of anti-HLAAb for 8 min leads to the translocation of RhoA protein fromthe cytosolic part to the inner side of the cell membrane. Thetranslocation was also confirmed by Western blots performedon membrane fractions. EC that were stimulated with thrombinwere used as positive controls and with irrelevant mouse IgGas negative controls. Figure 2B shows the increase in proteinlevel for RhoA corresponding to the 23-kD band detected by immunoblottingon cell membrane lysates. In contrast, the overall amount ofRhoA did not change. RhoA plays an important role in temporaland spatial determination of the actin cytoskeletal control(15). Figure 3 shows that anti-HLA binding to EC surface inducedthe formation of cytoplasmic stress fibers and accumulationof a pronounced ring of F-actin. Time-course analyses indicatedthat cytoskeleton reorganization begins 15 min after HLA classI binding and is reflected by an increase in fluorescence intensityas compared with resting and control cells. EC that were stimulatedwith thrombin, used as controls, also exhibited a rapid increasein F-actin staining and stress fiber appearance (maximal at15 min).
Figure 2. RhoA translocation in response to HLA class I ligation on EC. (A) Representative immunofluorescence for RhoA localization was performed on confluent EC monolayers that were incubated for 8 min with 10 µg/ml irrelevant mouse IgG2a (a) or anti-HLA class I W6/32 mAb (b). Photographs are representative of three different experiments. (B) Western blot analysis performed on EC membrane fractions (top) and total lysates (bottom) after treatment for 10 min with medium alone, thrombin, an isotypic control IgG, or anti-HLA class I (10 µg/ml for both). Magnification, x600 in A.
Figure 3. F-actin staining and stress fiber formation. EC were grown to confluence in four-well glass slides and cultured without growth factors in the presence of 2% FCS (fetal calf serum) for 12 h. EC monolayers were then incubated with 1 U/ml thrombin or anti-HLA class I mAb (W6/32, 10 µg/ml) for the indicated period of time. After treatment, EC were fixed, permeabilized, and stained with 2 µg/ml TRITC-phalloidin. Photographs are representative of three different experiments. Magnification, x 600.
RhoA Activation in Response to HLA Class I Ligation Precedes RhoA Upregulation at mRNA and Protein Levels
The activation of RhoA subsequent to HLA class I cross-linkingwas characterized further by pull-down assays. As shown in Figure 4A,an increase in GTP-bound RhoA protein was observed in responseto HLA class I cross-linking. Time-course analysis showed thatactive (GTP-bound) RhoA requires 10 to 20 min to reach a maximumand then declined before returning to baseline at 60 min. Duringthe same period, the amount of RhoA in whole lysates did notchange significantly. Thus, concomitant to membrane translocation,RhoA activation, in response to HLA class I ligation, was confirmedby an increase in GTP-bound RhoA. In additional experiments,the effect of HLA ligation was examined on RhoA mRNA and proteinlevels after 1, 2, and 4 h. RT-PCR for 18 cycles indicates astrong increase in RhoA mRNA expression level at 1 and 2 h afterHLA class I cross-linking, which returns to basal level at 4h (Figure 4B). In parallel experiments, EC were harvested andlysed to measure RhoA protein by Western blots. It is interestingthat increase in mRNA level precedes a significant increasein RhoA total protein level in whole-cell lysates observed at4 h (Figure 4C). Taken together, these results suggest thatactivation of RhoA may feed back to promote an increase in bothRhoA mRNA and protein levels.
Figure 4. RhoA activation and upregulation at mRNA and protein levels. EC were incubated in the presence of anti-HLA class I mAb (10 µg/ml) for the indicated period of time. (A) Pull-down assay. RhoA activation was determined by affinity precipitation of the active GTP-bound RhoA using a glutathione S-transferase (GST)-fusion protein of the Rho-binding domain of the Rho effector rhotekin (GST-RBD). The GTP-Rho pulled down from lysate was detected by Western blot using a specific anti-RhoA antibody. The total amount of RhoA in cell lysates was used as a control for the comparison of RhoA activity. Blots were reprobed with anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Ab to ensure equal loading. (B) Semiquantitative RT-PCR analysis of RhoA mRNA levels. Total RNA was extracted from EC and subjected to RT-PCR (18 cycles of amplification). The specific PCR bands were separated in 1.2% agarose gels and stained with ethidium bromide. -Actin mRNA was amplified as a control. (C) Western blot analysis of RhoA protein level in cell lysates. Immunoblot analysis was performed with anti-RhoA Ab, detected with horseradish peroxidase (HRP)-conjugated anti-mouse Ab, and visualized with enhanced chemiluminescence. Bottom panel shows GAPDH levels in samples from a blot reprobed with anti-GAPDH Ab. Results shown are representative of three separate experiments.
RhoA Mediates PI3-K/Akt Pathway Activation in EC
Western blot analysis was performed to explore signaling eventsrelated to RhoA activation in response to alloantibodies bindingon human EC. EC that were stimulated with 1 U/ml thrombin or100 U/ml TNF- were used as controls. By Western blotting, wefurther examined PI3-K activity and found that, concomitantto RhoA activation, HLA class I ligation promotes Akt activationthrough phosphorylation at Ser473 (Figure 5A) and at Thr308(data not shown). In contrast, the total amount of Akt proteinshowed no difference. Immunoblots also indicated that anti-HLAclass I Ab mediate the induction of PTEN and the phosphorylationof GSK3 at Ser9 in EC, both maximal 20 min after treatment.IB phosphorylation at Ser36 was also observed but, in contrastwith TNF--induced phosphorylation of IB, was not correlatedwith a decreased level of constitutive IB. For addressing moredirectly the role of RhoA protein in anti-HLA-mediated PI3-Kactivation, Rho GTPases were selectively inhibited by clostridialcytotoxin. Pretreatment of EC with C3 exoenzyme, which selectivelyinactivates RhoA/B/C proteins (16), efficiently reduced RhoAtranslocation to the cell membrane and reduces Akt phosphorylation(Figure 5B) as well as PTEN expression and GSK3 phosphorylation(data not shown). However, the specific PI3-K inhibitors wortmanninand LY294002 also prevent Akt without decreasing RhoA translocation.Taken together, these data strongly suggest that PI3-K/Akt isa downstream target of Rho GTPases in this process.
Figure 5. Activation of phosphatidylinositol 3-kinase (PI3-K) pathway in response to HLA class I ligation on EC. (A) Representative Western blots for PI3-K and NF-B activation. EC were incubated in the presence of anti-HLA class I mAb (10 µg/ml), 1 U/ml thrombin, or 100 U/ml TNF- for the indicated period. Cell lysates were resolved by SDS-PAGE (10 to 15%) and subjected to Western immunoblot analysis by using the following antibodies: rabbit polyclonal IgG anti-Phospho-Akt (Ser473), anti-Akt, anti-phospho GSK3 (Ser9), anti-PTEN, anti-IB, and anti-phospho-IB (Ser36). Anti-rabbit and anti-mouse IgG, HRP-linked Ab were used as secondary Ab in chemiluminescent Western blot assays. For verifying the amount of loaded proteins, blots were reprobed with anti-GAPDH mAb. (B) Quantitative analysis of immunoblots showing RhoA translocation and Akt phosphorylation (Ser 473) after HLA class I ligation for 15 min on EC with or without a pretreatment (60 min) with C3 exoenzyme (2 µg/ml), wortmannin (10 nmol/L), or LY294002 (1 µmol/L). Results from three independent experiments are expressed as a mean of relative fold increase in protein (± SEM) as compared with medium-treated cells (*P < 0.01 versus cells treated in the absence of inhibitors).
CFSE Analysis of Anti-HLA-Induced EC Proliferation
Increased proliferation of EC is the major functional consequenceattributed to anti-HLA class I Ab (7,9,17). For further linkingthe signaling pathway to the cell proliferation, CFSE staininghas been used to document further EC division and proliferationpromoted by anti-HLA class I alloantibodies. In contrast withcell proliferation assays that measure bulk cell division overa narrow window of time or can identify cells that have recentlydivided without determining how many generations have occurred,CFSE staining allows the clear resolution and quantitative analysisof eight to 10 sequential cell divisions (18). The data in Figure 6show the CFSE staining profiles of EC over a 24-h period.Cells that were treated with culture medium or 10 ng/ml bFGFwere used as negative and positive controls, respectively. Theproliferative effect of HLA ligation on EC was reflected bya higher proliferation index (8.97 and 7.55 for anti-HLA-A,B,Cand -A2, respectively, versus 6.4 for medium). Moreover, a higherpercentage of EC committed to proliferate was observed in responseto both polymorphic (HLA-A2) and monomorphic (HLA-A,B,C) anti-HLAclass I mAb compared with medium-treated cells.
Figure 6. 5,6-Carboxyfluorescein diacetate succinimidyl ester (CFSE) profiles of anti-HLA class I-induced EC cycle progression and proliferation. CFSE profiles of EC that were analyzed 24 h after CFSE labeling. Before labeling, EC were incubated for 24 h with 10 µg/ml anti-HLA (anti-HLA-A,B,C:W6/32, anti-HLA-A2:HB117) mAb, 10 µg/ml of an isotype control IgG, 10 ng/ml basic fibroblast growth factor (bFGF), or medium alone. CFSE staining was measured by flow cytometry, and data were analyzed using CellQuestPro and ModFitLT software. Cell proliferation was calculated using the Proliferation Wizard Model. PI, proliferation index.
Simvastatin Prevents HLA Class I-Induced Proliferation of EC by Inhibiting Geranylgeranylation of RhoA
To provide a functional link between anti-HLA-induced proliferationand signaling pathways, we compared the effect of RhoA and PI3-Kinhibitors. On the basis of our results, we examined the possibilitythat clinically relevant inhibitors of RhoA, such as 3-hydroxy-3-methylglutarylCoA (HMG-CoA) reductases inhibitors or statins (14,19,20), mayprevent RhoA-mediated EC proliferation in response to anti-HLAAb. CFSE staining showed that a low, clinically relevant dose(0.1 µmol/L) of simvastatin efficiently abbrogates theproliferative effect of HLA class I ligation on vascular EC(Figure 7). Similar inhibition was achieved at 0.5 µmol/Lwithout affecting cell viability. As shown in Figure 7A, pretreatmentof EC with wortmannin before treatment with anti-HLA class ImAb also reduced EC proliferation assessed by CFSE staining.The inhibitory effect was reflected by both a decreased proliferationindex and a lower percentage of the last cell generation.
Figure 7. Inhibitory effect of simvastatin on EC proliferation induced by HLA class I ligation. CFSE staining of EC that were incubated for 24 h with 10 µg/ml anti-HLA Ab (W6/32), 10 µg/ml of an isotype control IgG, or medium alone. Pretreatment with inhibitors (50 and 100 nmol/L wortmannin, 10 µmol/L geranylgeranyl pyrophosphate [GGPP] or farnesyl pyrophosphate [FPP]) was performed for 2 h. Pretreatment with simvastatin (0.1 and 0.5 µmol/L) was performed for 18 h. (A) CFSE staining was measured by flow cytometry, and data were analyzed using CellQuestPro and ModFitLT software. Cell proliferation was calculated using the Proliferation Wizard Model. Results are representative of at least three independent experiments. (B) Respective effects of the isoprenoid compounds FPP and GGPP on cell proliferation in the presence of anti-HLA-class I Ab and simvastatin. Results from three independent experiments are expressed as mean ± SEM of relative PI calculated as a ratio between treated and culture medium-treated cells. (a)P < 0.01 versus culture medium-treated cells; (b)P < 0.01 versus cells treated with anti-HLA Ab alone; (c)P > 0.1 versus cells treated with anti-HLA Ab alone.
HMG-CoA reductase is a proximal rate-limiting enzyme in thecholesterol synthesis pathway from acetyl CoA. This pathwayalso generates several metabolic compounds that have importantcellular functions, including FPP and GGPP that contribute tolipid modifications of various proteins. To determine whetherthe inhibitory effect of simvastatin on EC proliferation inour model results from a reduction in lipid intermediates, wemade attempts to reverse simvastatin’s effect by the additionof the downstream byproducts FPP and GGPP at the onset of simvastatintreatment. As shown in Figure 7, A and B, GGPP abrogates theinhibitory effect of simvastatin (relative proliferation index:1.25 ± 0.1 versus 1.3 ± 0.1 [P > 0.1] and 0.87± 0.15 [P < 0.05] for EC treated with anti-HLA inthe absence or presence of simvastatin, respectively). In contrast,FPP was not able to reverse the inhibitory effect of simvastatinon cell division, suggesting that simvastatin prevents anti-HLA-mediatedEC proliferation through inhibition of RhoA geranylgeranylation.To explore further the impact of RhoA activation on the subsequentRhoA mRNA upregulation, we treated EC with or without simvastatin(0.1 µmol/L) before cross-linking of HLA class I withspecific Ab. Cells that were incubated with irrelevant IgG wereused as controls. As expected, simvastatin completely preventsthe induction of active GTP-bound RhoA in response to HLA classI ligation (Figure 8A). Effect of simvastatin on RhoA transcriptionwas then examined at later time points by RT-PCR. In the presenceof simvastatin, no increase of RhoA mRNA was observed as comparedwith resting levels (Figure 8B). These results demonstrate thatblocking RhoA activation with simvastatin also prevents upregulationof RhoA transcription.
Figure 8. Inhibitory effect of simvastatin on RhoA activation and transcription induced by HLA class I cross-linking. EC were pretreated for 18 h with or without simvastatin (0.1 µmol/L) before incubation with anti-HLA class I Ab (W6/32, 10 µg/ml) or irrelevant isotype control IgG for the indicated period. (A) Pull-down assays. The GTP-Rho pulled down from lysate was detected by Western blot using a specific anti-RhoA antibody. (B) Semiquantitative RT-PCR analysis of mRNA expression for RhoA. Total RNA was extracted from EC and subjected to RT-PCR (18 cycles of PCR amplification). The specific PCR bands were separated in 1.2% agarose gels and stained with ethidium bromide. -Actin mRNA was amplified as a control. Results are from a representative experiment of three performed.
HLA class I signaling pathways have been shown to be involvedin the proliferation of vascular EC and SMC associated withtransplant arteriosclerosis (6,7,9). However, molecular mechanismsand signaling pathways implicated in HLA class I-mediated ECproliferation remain unclear. Allospecific Ab-mediated ligationof class I molecules expressed on the surface of EC have beenshown to trigger intracellular signaling events reflected byphosphorylation of Src, paxillin, and focal adhesion kinase(8). Activation of the PI3-K/Akt pathway also occurs in responseto class I ligation on EC (21). Together, these findings indicatethat multiple pathways may act in concert to promote EC proliferation.However, upstream signaling events and the hierarchical activationcascade remain to be established. The present work demonstratesthat HLA class I ligation on vascular EC induces transcriptionalchanges, including the upregulation of genes implicated in thecell cycle and proliferation, and identified RhoA as a key targetfor this angiogenic process.
Differential gene expression analysis is a powerful tool toinvestigate transcriptional changes in cells or tissues. However,the change in the transcription level of a gene is not alwayscorrelated with the causal role of that gene. Moreover, changesin gene expression are not invariably associated with changesin protein synthesis. Thus, gene expression analysis may beviewed as a guiding tool to initiate functional investigations.Rho GTPases, including RhoA, play a central role in eukaryoticcells, coordinately controlling the organization of the actincytoskeleton with other cellular activities such as gene transcription,cell-cycle progression, and migration (22). The Rho GTPasesalso function as key regulators of important signaling pathways.This study demonstrated that RhoA was rapidly activated andthen upregulated at the transcriptional level in response toHLA class I ligation on vascular EC. To our knowledge and asreported in recent publications (23,24), regulation of RhoAat a transcription or protein level is almost unknown. Thesestudies showed an increase of RhoA mRNA associated with enhancedRhoA protein level. It is extremely interesting that, in EC,intercellular adhesion molecule-1 cross-linking also inducedupregulation of RhoA at both mRNA and protein levels (25). Consistentwith these data, our results may suggest that mRNA upregulationmay reflect of RhoA activation and consumption and may constitutea feedback response to RhoA activation, providing more RhoAfor subsequent activation.
Although other GTPases can be upregulated, our attention wasfocused on determining the role of the signaling protein RhoAin class I-mediated signaling leading to angiogenesis in humanEC. Consistent with previous results reported on PI3-K implicationon EC angiogenesis after alloantibodies binding (21), we demonstratedthat concomitant with RhoA activation, PI3-K activation occurredin human EC because of HLA class I ligation. In our study, usingC3 exoenzyme, an inhibitor of RhoA/B/C activation, indicatingthat PI3K/Akt is a downstream target of RhoA in this process,efficiently blocked PI3-K activation. These data are consistentwith previous findings showing that RhoA may regulate PI3-Kactivity, suggesting that PI3-K is downstream of RhoA, suchas in fibroblasts, platelets (26), and SMC (27).
Our results indicate that inhibition of RhoA using C3 exoenzymeor a clinically relevant dose of simvastatin efficiently preventsanti-HLA-induced EC growth. Indeed, simvastatin reproduced theinhibitory effect of C3 exoenzyme that selectively inactivatesRho GTPases, the processing of which involves geranylgeranylation.The ability of GGPP but not FPP to reverse the inhibitory effectof simvastatin on allospecific EC proliferation highlights theinvolvement of geranylgeranylation of proteins and further suggeststhe contribution of RhoA to this signaling pathway. Furthermore,similar results were achieved by the inhibition of PI3-K activitywith the synthetic inhibitors wortmannin or LY294002, suggestinga key role of this pathway in our model. Mechanistically, ourdata also provide evidence that simvastatin prevents both RhoAactivation and the further upregulation of RhoA at the mRNAlevel.
The PI3-K/Akt signaling pathway is a key regulator of the angiogenicphenotype in EC (28). Activated PI3-K and Akt have been shownto stimulate angiogenesis through increased expression of vascularendothelial growth factor mRNA in EC (29). Akt has several downstreamtargets that are involved in the regulation of the cell cycle,including E2F, forkhead transcription factor, S6 protein kinase,and GSK3 (30). A recent study documented the signaling pathwaysinvolved in angiogenesis by demonstrating that phosphorylationof GSK3, one of the many substrates for Akt, is essential forEC survival and migration in vitro and angiogenesis in vivo(31). Akt downregulates GSK3 through site-specific phosphorylationat Ser9. GSK3 is a cyclin D1 protein kinase (32). Inactivationof GSK3 through Akt-mediated phosphorylation, as observed inresponse to HLA class I ligation on EC, has been shown to decreaseturnover and to stabilize cyclin D1 (32). Consequently, we canhypothesize that modulation of cyclin D1 through GSK3 inactivationcould provide a second target for PI3-K in the regulation ofthe cell cycle. Supporting this hypothesis, we observed by flowcytometer analysis that a preincubation (18 h) with simvastatin,which in our study efficiently inhibits EC proliferation, significantlyreduced cyclin D1 expression in EC (data not shown).
PTEN and PI3-K have opposing functions in the control of cell-cycleprogression (30). Indeed, PTEN overexpression inhibits cellgrowth in a variety of normal and transformed cells. PTEN isprimarily expressed, in parallel to Akt, in the mid to lateG1 phase during cell-cycle progression before pRb hyperphosphorylation(33). However, coexpression of PTEN with activated PI3-K orAkt, as reported in our study, efficiently antagonizes PTEN-mediatedgrowth suppression (33). Therefore, PTEN induction may suggestthe existence of a negative feedback loop that occurs afterRho-dependent PI3-K activation. Nevertheless, the respectiverole and downstream targets of GSK3 and PTEN have to be investigated.
Inhibitors of HMG-CoA reductase, or statins, have been shownto be useful in the reversal of endothelial dysfunction, aneffect that may be independent of the reduction in cholesterollevels. Although their contribution to angiogenesis could varyaccording to the type of statin and dose levels (34), statinshave been showed to have direct beneficial effects, includinginhibition of SMC and EC proliferation (14) and preproendothelin-1gene expression (19). Most of these actions resulted from RhoAinactivation (35,36). Recently, it was shown that simvastatinprevents thrombin-induced translocation of RhoA to the plasmamembrane in EC (36). In accordance, we now demonstrate thatsimvastatin also inhibits activation of RhoA and further regulationmediated by HLA class I cross-linking. In repressing allogeneic-inducedangiogenesis through RhoA and subsequently PI3-K inhibition,statins therefore provide a new type of immunomodulation thatcould prevent chronic transplant nephropathy. The clinical relevanceof statins is supported by recent data showing the improvementin long-term graft survival in heart (37) and kidney (38) transplantrecipients under statin therapy.
In this study, RNA differential display RT-PCR also identifiedseveral other molecules involved in cell-cycle progression andregulation (Table 1). Although their precise regulation at mRNAand protein levels remains to be analyzed, the functions ofthese proteins further indicate that anti-HLA alloantibodiesalter EC cell cycle and proliferation. In addition, our datado not exclude roles for other Rho GTPases, most notably RhoBand RhoC.
Collectively, our data suggest that RhoA-dependent activationof the PI3-K/Akt signaling pathway promotes cell-cycle progressionand proliferation of vascular EC that are treated with anti-HLAalloantibodies. Our data established a specific effect of theHMG-CoA reductase inhibitor simvastatin on HLA-induced vascularEC proliferation. Consistent with previous reports (14,19,36),this antiangiogenic effect is dependent on the interferenceof simvastatin with geranylgeranylation and the membrane localizationof RhoA.
Acknowledgments
This work was supported by grants from la Fondation de France"Recherche Cardiovasculaire" and from L’ÉtablissementFrançais des Greffes.
This study was presented, in part, at the American TransplantCongress, Washington, DC, May 30–June 4, 2003.
We thank Merck, Sharp & Dohme for providing simvastatin.
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Received for publication June 25, 2003.
Accepted for publication June 2, 2004.
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Top
Abstract
Introduction
(Ser9), in EC. C3 exoenzyme, an inhibitor of RhoA, inhibited RhoA translocation in response to HLA class I ligation and reduced phosphatidylinositol 3-kinase activation. EC proliferation and cell cycle progression, examined by 5,6-carboxyfluorescein diacetate succinimidyl ester staining, demonstrated that anti-HLA-induced EC proliferation was efficiently prevented by the 3-hydroxy-3-methylglutaryl CoA reductase inhibitor simvastatin (0.1 µmol/L) through inhibition of RhoA geranylgeranylation. Taken together, these findings support the conclusion that RhoA is a key mediator of signaling pathways that lead to cytoskeletal reorganization and EC proliferation in response to alloantibodies that bind to HLA class I and demonstrate the specific and potent inhibitory effect of simvastatin on allostimulated EC growth. 
and TNF-
upregulated MHC class I expression and potentiated anti-HLA Ab-mediated proliferative responses (9). These findings support a role for anti-HLA Ab in the transduction of proliferative signals, which stimulate the development of intimal hyperplasia associated with chronic allograft nephropathy and renal transplant loss. 
B
