The FASEB Journal • FJ Express Full-Length Article

The extracellular adherence protein from Staphylococcusaureus abrogates angiogenic responses of endothelialcells by blocking Ras activation

Astrid C. S. Sobke,*,1 Dennis Selimovic,† Valeria Orlova,‡,2 Mohamed Hassan,#

Triantafyllos Chavakis,‡,2 Athanasios N. Athanasopoulos,‡ Uwe Schubert,§

Muzaffar Hussain,� Gerald Thiel,¶ Klaus T. Preissner,§ and Mathias Herrmann**Institute of Medical Microbiology and Hygiene, University of Saarland Hospital, Homburg/Saar,Germany; †Clinic of Operative Dentistry and Periodontology, University of Saarland Hospital,Homburg/Saar, Germany; ‡Department of Internal Medicine I, University Heidelberg, Heidelberg,Germany; §Institute of Biochemistry, Justus-Liebig-University, Giessen, Germany, �Institute of MedicalMicrobiology, University Hospital, Munster, Germany, ¶Department of Medical Biochemistry andMolecular Biology, University of Saarland Hospital, Homburg/Saar, Germany; and #Department ofDermatology, Heinrich-Heine-University, Dusseldorf, Germany

ABSTRACT The extracellular adherence protein(Eap), a broad-spectrum adhesin secreted by Staphylo-coccus aureus, was previously shown to curb acute in-flammatory responses, presumably through its bindingto endothelial cell (EC) ICAM-1. Examining the effectof Eap on endothelial function in more detail, we hereshow that, in addition, Eap functions as a potentangiostatic agent. Concomitant treatment of EC withpurified Eap resulted in the complete blockage of themitogenic and sprouting responses elicited by vascularendothelial growth factor (VEGF)165 or basic fibroblastgrowth factor (bFGF). Moreover, the induction oftissue factor and decay-accelerating factor were re-pressed by Eap, as determined by qRT-polymerasechain reaction (qRT-PCR), with a corresponding reduc-tion in Egr-1 protein up-regulation seen. This angiosta-tic activity was accompanied by a corresponding inhibi-tion in ERK1/2 phosphorylation, while activation ofp38 was not affected. Inhibition occurred downstreamof tyrosine kinase receptor activation, as comparableeffects were seen on TPA-induced ERK1/2 phosphor-ylation. Similar to previously described angiostaticagents like angiopoietin-1 or the 16-kDa prolactin frag-ment, Eap blockage of the Ras/Raf/MEK/ERK cas-cade was localized by pull-down assay at the level of Rasactivation. Eap’s combined anti-inflammmatory and an-tiangiogenic properties render this bacterial proteinnot only an important virulence factor during S. aureusinfection but open new perspectives for therapeuticapplications in pathological neovascularization.—Sobke, A. C. S., Selimovic, D., Orlova, V., Hassan, M.,Chavakis, T., Athanasopoulos, A. N., Schubert, U.,Hussain, M., Thiel, G., Preissner, K. T., Herrmann, M.The extracellular adherence protein from Staphylococ-cus aureus abrogates angiogenic responses of endothe-lial cells by blocking Ras activation. FASEB J. 20,E2156–E2165 (2006)

Key Words: proliferation � sprouting � tissue factor � ERK1/2� Akt/PKB

STAPHYLOCOCCUS AUREUS, a gram-positive bacteriumwhose natural habitats are the human skin and mucousmembranes, is the “classical” and most common caus-ative agent of abscess formation and surgical site infec-tions (SSIs), which today constitute the second mostfrequent group of nosocomial infections (1–3). SSI istypically associated with impaired wound healing, butthe basis of these healing disturbances are still poorlyunderstood. A typical feature of chronic wounds is theabsence of the so-called granulation tissue, a highlyvascularized tissue that is essential for the repair process(for a review, see ref. 4). Pathogens like S. aureus thusappear to have evolved efficient strategies to counteractthe host’s inflammatory and angiogenic responses.

In this context, a group of cationic, noncovalentlybound and partially secreted broad-spectrum adhesinsof S. aureus has lately raised particular attention, asthese proteins were found to have profound immuno-modulatory properties (5–8). Their significance asimportant virulence factors of S. aureus is supported bytheir ubiquitious distribution. In particular, expressionof the extracellular adherence protein (Eap), alsoknown as MHC class II analog protein (Map), wasfound in 97.9% of clinical isolates (9). Moreover, eaptranscription was recently reported to be mainly depen-dent on the regulator Sae, which has been connectedwith virulence gene expression during chronic S. aureus

1 Correspondence: Institute of Medical Microbiology andHygiene, University of Saarland Hospital, D-66421 Hom-burg/Saar, Germany. E-mail: astrid.sobke@gmx.de

2 Present address: Experimental Immunology Branch, NCI,Bethesda, MD, USA.

doi: 10.1096/fj.06-5764fje

E2156 0892-6638/06/0020-2156 © FASEB

infections in humans and device-associated infectionsin mice (10–12).

Previously, we demonstrated that binding of Eap tointercellular adhesion protein-1 (ICAM-1) severely im-pairs neutrophil recruitment in S. aureus-infected mice(7, 8). Because activated macrophages are the majorsource of angiogenic chemokines and growth factors,in particular, bFGF and vascular endothelial growthfactor (VEGF) (13, 14), Eap’s interference with leuko-cyte recruitment will consequently have a negativeeffect on granulation tissue formation and healing, aswas recently shown in an in vivo model of woundhealing and angiogenesis in mice (15). Currently,however, it is still unclear whether these effects of Eapare exclusively due to its anti-inflammatory activity orwhether Eap also has a more direct impact on angio-genesis. It has been known for some time that staphy-lococcal surface proteins can bind basic growth factorslike bFGF in a charge-dependent manner (16). More-over, it is becoming increasingly clear that endothelialresponses to bFGF/VEGF are not solely mediated bytheir specific tyrosine kinase receptors but rather re-quire the sequestration of a number of coreceptorstogether with FGF receptor 1 (FGFR-1) and VEGFreceptor 2 (VEGFR-2/ KDR/Flk-1) (17–22). Severalendogenous inhibitors of angiogenesis, including en-dostatin, thrombospondin, and platelet factor 4, werepreviously reported to mediate their activities via inter-action with some of these coreceptors, in particular,integrins and heparan sulfate proteoglycans (HSPG)(23–27). These receptors and/or their ligands werealso identified as possible targets of Eap and relatedproteins, which were shown to bind several extracellu-lar matrix (ECM) proteins, including fibronectin andvitronectin, immunoglobulins like IgG and ICAM-1,and heparan sulfate (7, 28–30).

In this study we thus addressed the question, whether1) Eap does interfere with angiogenic responses inde-pendent from its anti-inflammatory functions, 2) suchan effect would be restricted to its interference withVEGF/bFGF interactions with their specific receptors,and 3) such an effect would be explainable by theinduction of corresponding alterations in angiogenicsignal transduction.

MATERIALS AND METHODS

Cell culture

Human umbilical vein endothelial cells (HUVEC) were iso-lated as described previously (31). Bovine retinal endothelialcells (BREC) were kindly provided by Drs. S. Zink and P.Rosen (University of Dusseldorf, Germany) (32). Cells weremaintained on fibronectin-coated culture dishes in endothe-lial cell growth medium (PromoCell, Heidelberg, Germany)at 37°C under 5% CO2 in a humid atmosphere.

Eap purification

Eap was purified from Staphylococcus aureus strain Newman, asdescribed previously (15). In brief, cell wall proteins were

obtained by lithium chloride extraction, and the extractsubsequently adsorbed in batch onto SP Sepharose (Amer-sham-Pharmacia, Freiburg, Germany). After stepwise elutionwith increasing NaCl concentrations, the pooled eluted frac-tions (between 600 and 800 mM NaCl) were sterile filtered,and further purified by cation exchange chromatography ona Mono S 10/100 GL tricorn column and afterward by gelfiltration using an Superdex 75 HR 10/30 column operatedon the AKTA fast performance liquid chromatography system(Amersham-Pharmacia, Freiburg, Germany). Protein concen-tration and purity of the product were checked by SDS-PAGEand Bradford assay, respectively. Purified Eap was found to befree of detectable endotoxin.

Real-time polymerase chain reaction

HUVEC were serum-starved overnight in endothelial cellbasal medium (PromoCell) with 0.5% FCS and then stimu-lated as indicated in fresh medium. After 12 h, total RNA wasisolated using the Nucleospin RNAII kit from Machery-Nagel(Duren, Germany). Reverse transcription was performed withthe high-capacity cDNA Archive kit from Applied Biosystems(Weiterstadt, Germany). 75 ng of cDNA per reaction wereused in real-time polymerase chain reaction (PCR), per-formed on an ABI Prism 7000 Sequence Detection Systeminstrument using the SYBR Green Master Mix kit fromApplied Biosystems (Foster City, CA). Primers were designedwith the Primer Express Software version 2.0.0. and ampli-cons chosen to span an intron region. Primer sequencesfor the decay- accelerating factor (DAF) were 5�-CATGATT-GGAGAGCACTCTATTTATTG-3� forward and 5�-TTGGTGGGACCTTGGAAGTTAG-3� reverse, and for the “tissue factor”(TF) 5�-CCCCAGAGTTCACACCTTACCT-3� forward and 5�-CACTTTTGTTCCCACCTGTTCA-3� reverse. Data were ana-lyzed according to the comparative Ct method and werenormalized by �-actin expression in each sample. Primersequences for �-actin were 5�-TGAGGCACTCTTCCAGC-CTT-3� forward and 5�-CACTTCATGATGGAGTTGAAGG-TAGT-3� reverse. Melting curves for each PCR reaction weregenerated to ensure the purity of the amplification product.

Endothelial cell proliferation. [3H]thymidineincorporation assay

HUVEC were seeded on fibronectin-coated, 24-well platesand grown to 60–70% confluence. Plates were washed twicewith HBSS (Biochrom, Berlin, Germany), and cells wereincubated in serum-reduced basal medium (endothelial cellbasal medium with 28 mM HEPES, supplemented with 0.5%FCS, 100 U/ml penicillin, and 100 �g/ml streptomycin) for18 h. Cells were then transferred to medium containing 0.1%FCS and 0.5% BSA and stimulated with the factors indicatedfor 24 h. During the last 4 h, 1 �Ci [3H]thymidine was addedper well. Cells were washed twice with ice-cold PBS, and DNAwas acid-precipitated by incubation with 10% w/v TCA for 20min at 4°C. Wells were washed once with ice-cold 10% w/vTCA, the contents solubilized in 0.2 M NaOH with 1% SDS,and wells were rinsed once with 0.2 M HCl. [3H]thymidineincorporation was determined by liquid scintillation countingin an Wallac 1410 instrument (Perkin Elmer Wallac, Freiburg,Germany).

Cell number counts

HUVEC were plated onto 96-well plates and incubated for12 h, after which the medium was changed to MCDB-131(CC-Pro, Neustadt, Germany) containing 0.05% FCS. Cellswere then incubated for 72 h in the absence or presence of

E2157EAP ABROGATES ANGIOGENESIS BY BLOCKING RAS

mitogens or Eap as indicated. Cells were subsequently col-lected by trypsinization, and the total cell number was deter-mined with a Casy-Counter (Scharfe System, Reutlingen,Germany).

Sprouting assay

Sprouting assays were performed according to the methoddescribed by Nehls and Drenckhahn (33). In brief, BRECwere grown to confluence on Cytodex-3 microcarrier beads.Three hundred milliliters of a fibrinogen solution (1.8 mg/ml) with 200 U/ml aprotinin as protease inhibitor was placedtogether with 50–80 microcarrier beads per well into a24-well culture plate. Polymerization was started by additionof 0.65 U/ml thrombin into each well, and plates wereincubated for 20 min at room temperature. Medium wasexchanged against endothelial cell basal medium with 0.5%FCS containing either 2 ng/ml bFGF or 0.9% saline asnegative control. Eap was added at the concentrations indi-cated, and samples were assayed in triplicate. Plates were thenincubated for 48 h and subsequently fixed in 3% w/v form-aldehyde in PBS. Tube formation was assessed by counting allcapillaries, which were at least as long as the bead diameter(average 150 �m) at 100 � magnification in a light micro-scope.

Western blot analysis

Cells were seeded in endothelial cell basal medium contain-ing 0.5% FCS; they were transferred to basal medium with0.5% BSA for 4 h on the following day, and then stimulatedas indicated. Inhibitors or Eap were included at the concen-trations and for the times stated. GF109203X and manumycinA were purchased from Calbiochem (San Diego, CA). Cellswere washed twice with ice-cold PBS and lysed on ice by theaddition of 200 �l of radio-immunoprecipitation assay (RIPA)buffer (50 mM Tris-HCl, pH 7.4, 1% w/v Igepal™, 0.25% w/vdesoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM NaF, 0.5mM sodium orthovanadate, 1 mM sodium pyrophosphate, 2�g/ml aprotinin, 5 �g/ml leupeptin, 10 �g/ml pepstatin A,and 0.2 mg/ml PMSF). Cell debris was removed by centrifu-gation at 10,000 g for 5 min at 4°C. Protein concentration wasdetermined using the Bradford assay (Bio-Rad Laboratories,Munchen, Germany), and 50 �g of total cellular protein wassubjected to SDS-PAGE on 10% gels. Samples were electro-blotted onto nitrocellulose membrane and regular transferconfirmed by staining with Ponceau S. Blots were blocked andincubated with primary antibodies corresponding to therecommendations of the supplier of the respective antibody(Ab): antiphosphoThr180/Tyr182-p38, antiphosphoSer473-Akt, and antiphosphoSer217/221-MEK1/2 rabbit polyclonalantibodies, and antiphosphoThr202/Tyr204-ERK1/2 mousemonoclonal were purchased from Cell Signaling Technology(Beverly, MA), anti-ERK1/2 rabbit polyclonal from UpstateBiotechnology (Lake Placid, NY), and anti-Raf-1 rabbit, anti-PKC� goat polyclonal as well as anti-Egr-1 rabbit polyclonalfrom Santa-Cruz Biotechnology (Santa Cruz, CA). Immuno-reactive bands were visualized on X-ray films using respectivehorseradish peroxidase-conjugated secondary antibodies(Bio-Rad Laboratories, Munchen, Germany) and enhancedchemiluminescence (ECL, Amersham-Pharmacia, Freiburg,Germany).

Preparation of cytosol and membrane fraction

HUVEC were treated, as described above, but scraped intoice-cold homogenization buffer (10 mM HEPES pH 7.4, 1mM EDTA, 10 mM �-glycerol phosphate, 0.1 mM DTT, 1 mM

NaF, 0.5 mM sodium orthovanadate, 2 �g/ml aprotinin, 0.2mg/ml PMSF, 5 �g/ml leupeptin, 10 �g/ml pepstatin A) andlysed by sonication (3 times, 5-s bursts, 50 W) on ice. Wholecells and nuclei were removed by centrifugation at 500 g for2 min at 4°C. Membrane fraction was separated by centrifu-gation at 20,000 g for 30 min at 4°C. The supernatantcontaining the cytosolic fraction was preserved, and the pelletwas dissolved in homogenization buffer containing 1% TritonX-100 by short sonication. Western blot analysis was per-formed as described above.

Pull-down assay

HUVEC were grown to 80–90% confluence and serumstarved overnight prior to stimulation without or withVEGF165 (10 ng/ml, 5 min), bFGF (100 ng/ml, 15 min), inthe absence or presence of Eap (10 �g/ml). Cells werewashed with ice-cold PBS and lysed in MLB (25 mM HEPES,pH 7.5, 150 mM NaCl, 1% w/v Nonidet P-40, 10 mM MgCl2,1 mM EDTA, 25 mM NaF, 1 mM sodium orthovanadate, 10%w/v glycerol and protease inhibitors). Lysates were centri-fuged for 5 min at 13,000 g and active Ras was precipitatedwith 10 �g RBD-conjugated agarose (Upstate Biotechnology,Lake Placid, NY) for 30 min at 4°C. Precipitates were washedthrice with lysis buffer, resuspended in 2 � sample buffer, andanalyzed by SDS-PAGE on 15% gels. Ras was detected usingrespective rabbit polyclonal antibody from Cell SignalingTechnology.

RESULTS

Eap inhibits the angiogenic response of primaryendothelial cells

Real-time PCR was employed to study the transcrip-tional up-regulation of TF and DAF as one of the earlysteps during endothelial activation. In agreement withprevious reports (34, 35), we found mRNA levels ofDAF to be raised approximately twofold by eitherVEGF165 or bFGF in HUVEC. In contrast, expression ofTF was increased approximately fivefold by VEGF165,whereas bFGF exerted only an insignificant effect onTF expression (Fig. 1A). In comparison, HUVEC thatwere cotreated with 20 �g/ml of purified Eap, dis-played a strongly attenuated response, the inductionbeing reduced by about half.

The proliferation of endothelial cells is essential fornew vessel formation, and both bFGF and VEGF havebeen described as potent mitogens for cultured endo-thelial monolayers. Proliferation in the presence orabsence of Eap was assessed by measuring de novo DNAsynthesis, as well as by cell number counts. As shown inFig. 1B, [3H]thymidine incorporation was raised 4.0 �1.6 and 8.7 � 2.7 fold in HUVEC by VEGF165 and bFGF,respectively. In the presence of 20 �g/ml Eap, thismitogenic response was completely abrogated (1.28�0.66and 1.36�0.17). The antiproliferative activity of Eap wasconcentration dependent, with complete inhibition seenat 20 �g/ml (Fig. 1C).

An important aspect of neovascularization is thecapacity of endothelial cells to differentiate into abranched, vascular network. Primary endothelial

E2158 Vol. 20 December 2006 SOBKE ET AL.The FASEB Journal

cells can be coaxed to form capillary-like structures invitro, when they are grown in a 3D fibrin matrixcontaining an angiogenic stimulus. Analyzing thebFGF- and VEGF-induced capillary-like sprout forma-tion of BREC in the presence and absence of Eap, astrong inhibitory effect on sprouting was observed(Fig. 1D).

Eap inhibits VEGF165 and bFGF-induced ERKphosphorylation but has variable effects on Aktphosphorylation

Changes in endothelial cell functions are mediated byinduction of specific signaling cascades. We thus exam-ined the phosphorylation of key kinases in the absenceor presence of Eap. Lysates of HUVEC stimulated with100 ng/ml of either VEGF165 or bFGF were immuno-blotted with phosphospecific antibodies directedagainst the activated forms of ERK1/2 (phospho-Thr202/Tyr204), p38 (phospho-Thr180/Tyr182), andAkt/PKB (phospho-Ser-473). VEGF treatment induceda strong phosphorylation of ERK1 and 2, with a maxi-mal response seen at 5 min (Fig. 2A). There was also afast, albeit weaker phosphorylation of p38, whereasAkt/PKB phosphorylation progressed more slowly. Themaximal effect was not seen before 15 min of stimula-tion. By 60 min, the phosphorylation levels of all three

kinases had dropped to or below basal levels. One hourpreincubation with Eap led to a significant reduction inthe level of the subsequent VEGF165- triggered ERK1/2phosphorylation without altering the kinetics of theresponse. Under the same conditions, Eap did notaffect the VEGF165-induced p38 and Akt/PKB phos-phorylation. This specific suppression of the MAPKpathway through Eap was not restricted to the VEGF165elicited response, since similar results were obtained inbFGF-stimulated HUVEC. Here, the maximal respon-sein ERK1/2 phosphorylation was seen after 15 min,and this was again significantly lowered by pretreat-ment with Eap (Fig. 2B). In addition, there was anaugmental effect of Eap on the bFGF-induced Akt/PKBphosphorylation: while bFGF on its own caused only amarginal increase in Akt/PKB phosphorylation atserine 473, in the presence of Eap this was clearlyenhanced.

As depicted in Fig. 2C, the observed effects of Eapare concentration-dependent. Fig. 2E shows the den-sitometric evaluation of a typical experiment, takingthe level of ERK1/2-phosphorylation achieved in theabsence of Eap as 100%. In good agreement with arecent report, which found Eap’s impact on leuko-cyte-endothelial interactions to be maximal at �30�g/ml (8), inhibition with 20 �g/ml Eap reachedbetween 80 and 90% of the maximal effect observed.

A B C

D Figure 1. Eap interferes with angiogenic response of EC primary cultures.A) mRNA levels of DAF and TF were determined by real-time PCR inHUVEC stimulated with either 100 ng/ml VEGF or bFGF in the absence(open bars)or presence (solid bars) of 20 �g/ml Eap. Results are themeans � sd of 3 independent experiments and shown as a fold increaseover basal expression (basal � 1, indicated by dashed line); *P 0.05;**P 0.01; ***P 0.001. B) HUVEC were grown to subconfluence in24-well plates, serum-starved overnight (0.5% FCS), and then stimulatedwith either VEGF (25 ng/ml), bFGF (25 ng/ml), or medium alone (0.1%FCS and 0.5% BSA) for 20 h in the absence (solid bars) or presence (openbars) of 20 �g/ml isolated Eap. Cells were pulse-labeled with tritium-thymidine for another 4 h and analyzed for thymidine incorporation. Datarepresent mean counts per minute � sd with n � 5. *P 0.05; **P 0.01;***P 0.001. C) HUVEC were treated with VEGF or bFGF in serum-reduced medium (0.2% FCS), in the absence or presence of increasingconcentrations of Eap as indicated. Proliferation is expressed as relativechange in cell number after 72 h (basal cell number�1, indicated bydashed line). Data are mean � sd, n � 3. D) Eap inhibits capillary sproutformation. Capillary-like sprout formation of BREC was analyzed without(open bars) or in the presence (filled bars) of either 2 ng/ml bFGF or 25ng/ml VEGF and together with the indicated concentration of isolatedEap. Data represent mean � sd of 30 beads analyzed in triplicate each.

E2159EAP ABROGATES ANGIOGENESIS BY BLOCKING RAS

Whereas basal phosphorylation states were reducedto nondetectable levels by incubation with Eap, theinhibitory effects on the responses stimulated with100 ng/ml of either VEGF or bFGF were never com-plete. Intracellular signal transduction pathways forVEGF-induced activation of ERK1/2 were previouslyclaimed to be heterogeneous and to depend on theorigin of the EC (36). Consequently, we studied theeffect of Eap on signal transduction in BREC forcomparison. As shown in Fig. 2D and E, the resultsobtained with BREC during VEGF stimulation areindistinguishable from those obtained with HUVEC atlow concentrations of Eap; however, at higher concen-

trations, inhibition becomes almost complete. In con-trast to HUVEC, BREC responded to bFGF stimulationwith a strong activation of Akt, and this, too, waseffectively inhibited through Eap.

To test whether these Eap-induced changes in signaltransduction may be responsible for the disturbances inbiological function described above, we examined theeffect of Eap on the induction of the transcriptionfactor Egr-1. De novo synthesis of Egr-1 was previouslyreported to be necessary for transcriptional activationof TF expression (31). As shown in Fig. 3, induction ofEgr-1 was decreased by Eap to a similar degree astranscriptional up-regulation of TF.

Figure 2. Eap interferes with ERK1/2 activation. Serum-deprived HUVEC were left untreated or incubated with 100 �g/ml Eapfor 1 h before stimulation with 100 ng/ml of either VEGF (A) or bFGF (B) for the times indicated. After cell lysis, 50 �g of totalcell protein per lane were analyzed by SDS-PAGE on 10% gels and submitted to Western blot analysis with antibodies specificfor the phosphorylated forms of either ERK1/2, p38, or Akt. HUVEC (C) or BREC (D) were incubated with increasingconcentrations of Eap as indicated. Stimulation periods were chosen as 5 or 15 min, depending on time points of maximalactivation seen in (A) and (B). E) Densitometric evaluation of ERK1/2 (left) and Akt (right) phosphorylation levels, takingphosphorylation in the absence of Eap as 100% (n�3; *P0.05; **P0.01; ***P0.001).

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Inhibition of the MAPK cascade occurs upstream ofERK1/2, and downstream of PLC�

To delineate the target of Eap in more detail, westudied the effects of combinations of Eap, the PKCinhibitor GF109203X, and the farnesyl transferase in-hibitor manumycin A on the VEGF-induced MAPKcascade. As farnesylation of Ras is a prerequisite for itshomodimerization and localization to the plasma mem-

brane, preincubation of cells with manumycin A indi-rectly interferes with Ras activation. As shown in Fig. 4A,GF109203X and Eap had a substantial inhibitory effecton ERK1/2 phosphorylation, with the combined effectresulting in the complete blockage of the phosphory-lation response. In comparison, manumycin A treat-ment on its own had only a minor impact on ERK1/2phosphorylation, whereas concomitant treatment withEap resulted again in an additive effect. To further testwhether the impact of Eap would be independent ofreceptor activation, we examined the effects of Eap,GF109203X, and manumycin A on activation of theMAPK pathway during stimulation with the phorbolester TPA. As demonstrated in the right panel of Fig.4A the results are very similar to those seen underVEGF stimulation. The MAPKK MEK1/2 are placeddirectly upstream of ERK1/2 and downstream of Raf-1in the signal cascade. They are activated through Raf-1by phosphorylation at Ser-217/221 (37). Immunoblot-ting with a phospho-Ser-217/221 specific Ab showedEap to have similar effects on MEK activation as on ERK(Fig. 4A). The VEGF-induced MEK1/2 activation is

Figure 3. Induction of Egr-1 is inhibited by Eap. Egr-1synthesis in HUVEC was induced by VEGF treatment for thetimes indicated. Western blot analysis was performed againstEgr-1 in 50 �g of total cell protein.

Figure 4. Inhibition through Eap occurs downstream of PLC. A) Comparison of the Eap effect with known inhibitors andactivators of VEGF-induced MAPK signal cascade. Serum-deprived HUVEC were stimulated with VEGF (100 ng/ml; left) or TPA(100 nM; right) for 5 min. Where indicated, cells were pretreated with GF109203X (5 �M, 30 min), manumycin A (20 �M, 4 h)or Eap (40 �g/ml, 60 min). Control cells were pretreated with DMSO. MAPK pathway activation was analyzed byimmunoblotting with antiphospho-ERK1/2 and antiphospho-MEK1/2 antibodies. Total ERK1/2 was determined to confirmequal loading. B) Membrane translocation and activation of PKC� are not affected by Eap. HUVEC were stimulated with TPA(100 nM) for the times indicated and cytosolic and membrane fractions isolated. Activation of PKC� is accompanied byelectrophoretic mobility shift.

E2161EAP ABROGATES ANGIOGENESIS BY BLOCKING RAS

unusual in that it relies on PLC-dependent activationof novel PKC� (38). Activation of PKC requires itstranslocation to the plasma membrane and autophos-phorylation. As shown in Fig. 4B, Eap had no impact onthe membrane translocation or phosphorylation levelsof PKC�. Consequently, the inhibitory effect of Eap onMAPK cascade induction in EC has to occur down-stream of PLC and upstream of MEK1/2 phosphory-lation, which is at the level of either Ras or Raf-1.

Eap exerts its effect at the level of Ras but not at thelevel of Raf-1

Raf-1 constitutes an important relay point within theMAPK cascade, integrating positive and negative inputsfrom upstream pathways (for a review, see Ref. 37).Activation of Raf-1 is accompanied by a characteristicshift in electrophoretic mobility due to an increasedphosphorylation state (39). As expected, stimulationwith TPA induced the occurrence of an additional,retarded band in HUVEC (Fig. 5A). The intensity ofthis band was again reduced on treatment with Eap.Pull-down assays were used to investigate Ras activation.As shown in Fig. 5B, there was a profound reduction inactivated, GTP-bound Ras after VEGF, as well as bFGFstimulation in Eap-exposed HUVEC, indicating inter-ference of Ras activation as the earliest target of Eapblockage of MAPK pathway activation.

DISCUSSION

We reported previously that S. aureus Eap interferedwith leukocyte accumulation at inflammatory locations

in vivo, presumably by disturbing leukocyte-endothe-lium interactions, as demonstrated in vitro (7, 8). Inaddition, we recently showed that this versatile proteinseverely impairs wound healing and angiogenesis inmice and that this effect may go beyond its interferencewith the inflammatory response (15). The aim of thisstudy thus was to elucidate the basis of Eap’s potentialimpact on endothelial function. The data presentedhere demonstrate for the first time that Eap selectivelyinhibits Ras-dependent signaling pathways by an as yetunidentified mechanism and that this inhibition isassociated with a corresponding attenuation of angio-genic responses in isolated primary endothelial cells.

Angiogenesis involves the concerted activity of manycell types. To evaluate the specific role of Eap-endothe-lial interactions in this context, we tested its effect onthe behavior of isolated endothelial cells in vitro. Co-treatment with Eap was found to severely impair orcompletely abrogate essential angiogenic responseselicited by bFGF or VEGF165 in macrovascular as well asmicrovascular cells, that is, the induction of genetranscription, proliferation, and differentiation intotubular networks. At concentrations of 40 �g/ml, Eapblocked the transcriptional activation response by �50to 60%, whereas proliferation, as well as tubular mor-phogenesis, were completely abrogated at Eap concen-trations of 20 �g/ml or less. A common feature distin-guishing these last two responses from the short-termeffect of gene up-regulation is their need for sustainedand/or tightly regulated ERK and Ras signaling (20,40). Similarly acting endogenous inhibitors of angio-genesis, like platelet factor 4 (PF4), 16-kDa humanprolactin (16 K hPRL), thrombospondin (TSP), an-giostatin, or endostatin, were previously shown to havean impact on the signal transduction network of endo-thelial cells, activating proapoptotic pathways and/orinhibiting MAPK signal transduction (41–45). To elu-cidate the potent antiangiogenic activity of Eap in moredetail, we investigated its effect on the activation ofproangiogenic phosphorylation cascades.

Being the two major angiogenic growth factors inphysiological, as well as pathological neovasculariza-tion, the intracellular signaling cascades linked to themain endothelial receptors for VEGF165 and bFGF, i.e.,VEGF receptor 2/KDR/Flk-1 and FGF receptor 1,respectively, have been relatively well characterized(compare Fig. 6). In particular, was it possible toassociate certain angiogenic responses with the activa-tion of a specific pathway: so is the activation of p38MAPK essential to actin rearrangements, permeabilitychanges, and migration, ERK1/2 MAPK mediate tran-scriptional induction and cell proliferation, while Aktactivation is responsible for cell survival by inhibitingproapoptotic pathways, apart from its contribution toeNOS activation (for a review, see Ref. 46). Examiningthe activation of these three key kinases in HUVEC, wefound Eap to cause a significant impairment of theVEGF165- and bFGF-induced ERK1/2 phosphorylation,while, at the same time, Akt and p38 MAPK activationwere either not affected or, in the case of bFGF

Figure 5. Eap blocks Ras activation. A) Eap interferes withRaf-1 activation. HUVEC were serum-starved and then stimu-lated with 100 nM TPA for the times indicated. Activation ofRaf-1 was determined by phosphorylation-induced shift inelectrophoretic mobility. B) Eap blocks Ras activation. Pull-down assays were performed on HUVEC stimulated in thepresence or absence of Eap.

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stimulation, even augmented. With the high concentra-tions of stimulants employed here, the inhibitory effecton ERK1/2 phosphorylation was not complete: themaximal inhibition levels obtained were between 50and 60%, with an IC50 of �150 nM.

Some antiangiogenic factors have previously beenshown to act, at least in part, by interfering with VEGFand bFGF binding to endothelial cells (47, 48). Thispossibility was excluded here by demonstrating inter-ference of Eap with activation of the Raf/MEK/ERKcascade on TPA exposure. Phorbol esters such as TPAmimic the function of sn-1,2-diacylglycerol by inducingthe membrane translocation of PKC and the guanidinenucleotide exchange factor RasGRP3 and are able toinduce the proliferation and tube formation of cul-tured endothelial cells in a tyrosine kinase receptor-independent manner (49–51). The fact that the Eap-induced blockage of ERK1/2 activation does indeedoccur downstream of PKC, was confirmed by the dem-

onstrating that in the presence of Eap membranetranslocation and activation of PKC� was unaffected.

In the past, there has been some controversy over therelative contributions made by Ras-dependent vs. Ras-independent pathways during VEGF-triggered MAPKpathway activation (52, 53). Yashima et al., studying theVEGF-induced ERK1/2 activation in different endothe-lial cell types, came to the conclusion that in microvas-cular EC, the Ras-dependent branch is used preferen-tially, whereas PKC plays the dominant role inmacrovascular EC (36). Because the Eap blockage onstimulated ERK1/2 phosphorylation was incomplete inHUVEC, we investigated its effect on primary endothe-lial cells derived from a microvasuclar source, that is,BREC. Indeed, in these cells, a nearly complete inhibi-tion of the MAPK pathway could be achieved at highEap concentrations.

Within the MAPK cascade, Raf-1 has a special role inthe integration of positive and negative crosstalk fromother pathways (reviewed in Ref. 37). Raf-1 activationwas previously described to be accompanied by a char-acteristic electric mobility shift, which was slightly de-creased in the presence of Eap. Raf-1 activation isdependent on the small GTPase Ras, and Ras-GTPformation was finally identified as the first event in theMAPK pathway to be repressed by Eap.

In contrast to the Raf/MEK/ERK module, there isonly very little known about the signal transductioncomponents linking Akt activation to receptor auto-phosphorylation in EC. The effect of Eap on Akt serine473 phosphorylation was found to depend on theparticular cell type examined and the stimulant used.Because PI3-kinase induction upstream of Akt activa-tion may occur in a Ras-dependent or -independentmanner, the Eap-induced blockage of Akt phosphory-lation observed in bFGF-stimulated BREC seem toindicate the involvement of Ras in these cells. Insupport of this assumption, Zubilewicz et al. previouslydemonstrated bFGF-induced Ras-dependent PI3-kinaseactivation in bovine chorocapillary EC (54). Very muchin contrast to these findings, bFGF alone was only aweak inductor of Akt in HUVEC, but this minor activa-tion was increased �2.5-fold by concomitant treatmentwith Eap.

Taken together, Eap thus does not only exert angio-static activities comparable to those of established en-dogenous angiostatins, but there are also striking par-allels in the impact on the endothelial signaltransduction network bringing about these alterations.So was 16 K hPRL reported to affect VEGF signaling viainhibition of Ras activation, and PF4 was shown toinhibit VEGF-mediated ERK1/2 activation without af-fecting PI3-kinase induction (41, 42).

Clearly, elucidation of Eap activities at the membranelevel may also shed some light onto signaling in eukary-otic cells. From the bacterial viewpoint, Eap appears tobe the ideal adhesin: mediating staphylococcal bindingto the ECM, as well as to the eukaryotic surface, while atthe same time soothing the host’s response. The potentanti-inflammatory and antiangiogenic activities of Eap

Figure 6. Schematic illustration of bFGF- and VEGF- inducedsignal cascades and proposed model of the effect of Eap onendothelial function. Activation of FGF receptor 1 (FGFR-1)by bFGF leads to binding of adaptor protein FRS2, which, inturn, recruits Grb2 complexed to docking protein Gab1 andguanidine nucleotide exchange factor Sos. Sos-mediated ac-tivation of Ras results in induction of the Raf/MEK/ERK-cascade-promoting proliferation, gene expression, and differ-entiation, as well as in activation of the phosphatidylinositol3�-kinase (PI3K)/Akt pathway, which enhances cell survivalthrough inhibition of proapoptotic proteins (a). Preventionof Ras activation by externally added Eap will block both ofthese pathways, whereas an alternative Ras-independent path-way of PI3K/Akt activation is actually supported by Eap (b). Incontrast, Akt/PI3K induction on autotyrosinephosphoryla-tion of activated VEGF receptor may occur via direct interac-tion of the regulatory subunit of PI3K with the receptor andis not affected by Eap. Central to the induction of theRaf/MEK/ERK-pathway by VEGF is the generation of thesecond messenger sn-1,2-diacylglycerol by activated PLC.Diacylglycerol (DAG) is a physiological activator of proteinkinase C (PKC), which, in turn, will phosphorylate Raf-1directly, but, in addition, activates sphingosine kinase, thesubsequent increase in local membrane S1P levels leading toa decrease in Ras-growth-associated protein (GAP) activity.This, together with the induction of GRP3 promoted by DAGand PKC, will cause activation of Ras.

E2163EAP ABROGATES ANGIOGENESIS BY BLOCKING RAS

make this versatile protein a good candidate for futurepharmacological application, for instance, in inhibitingpathological neovascularization as occurs in tumorgrowth, or curbing the overshooting host responses insepsis.

The authors would like to thank Dr. Claudia Rubie fortechnical advice with qRT-polymerase chain reaction and Dr.Banhu Sinha for helpful discussions. This work was supportedby grants from the Deutsche Forschungsgemeinschaft to M.Herrmann (H3 1850/6–1), K. T. Preissner (PR327/18–2),and T. Chavakis (CH279/2–1 and CH279/2–2). M. Hussainwas supported by SFB492.

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Received for publication March 28, 2006.Accepted for publication July 24, 2006.

E2165EAP ABROGATES ANGIOGENESIS BY BLOCKING RAS

The FASEB Journal • FJ Express Summary

The extracellular adherence protein from Staphylococcusaureus abrogates angiogenic responses of endothelialcells by blocking Ras activation

Astrid C. S. Sobke,*,1 Dennis Selimovic,† Valeria Orlova,‡,2 Mohamed Hassan,#

Triantafyllos Chavakis,‡,2 Athanasios N. Athanasopoulos,‡ Uwe Schubert,§

Muzaffar Hussain,� Gerald Thiel,¶ Klaus T. Preissner,§ and Mathias Herrmann**Institute of Medical Microbiology and Hygiene, University of Saarland Hospital, Homburg/Saar,Germany; †Clinic of Operative Dentistry and Periodontology, University of Saarland Hospital,Homburg/Saar, Germany; ‡Department of Internal Medicine I, University Heidelberg, Heidelberg,Germany; §Institute of Biochemistry, Justus-Liebig-University, Giessen, Germany, �Institute of MedicalMicrobiology, University Hospital, Munster, Germany, ¶Department of Medical Biochemistry andMolecular Biology, University of Saarland Hospital, Homburg/Saar, Germany; and #Department ofDermatology, Heinrich-Heine-University, Dusseldorf, Germany

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-5764fje

SPECIFIC AIMS

The secreted extracellular adherence protein (Eap)from Staphylococcus aureus was previously reported toseverely impair neutrophil recruitment, repair angio-genesis, and wound healing in S. aureus-infected mice.Because the broad spectrum of Eap ligands includesangiogenic costimulators like fibrinogen, ICAM-1, anda range of extracellular matrix (ECM) proteins, the aimof this study was to decipher the influence of thisversatile adhesin on molecular signaling events and theangiogenic behavior of endothelial cells.

PRINCIPAL FINDINGS

1. Eap inhibits the angiogenic response of primaryendothelial cells

Endothelial activation by angiogenic growth factorsresults in transcriptional up-regulation of genes like the“decay- accelerating factor” and “tissue factor” (TF) asan early event. Here, we found mRNA levels of DAF tobe raised approximately twofold by either vascularendothelial growth factor (VEGF)165 or basic fibroblastgrowth factor (bFGF) in HUVEC, whereas the expres-sion of TF was increased approximately fivefold byVEGF165 (Fig. 1A). Cotreatment of HUVEC with 20�g/ml of purified Eap resulted in a strongly attenuatedresponse, the induction being reduced about half.Endothelial proliferation, as assessed by measuring denovo DNA synthesis (Fig. 1B), as well as by cell numbercounts, was even more severely impaired by Eap:[3H]thymidine incorporation was raised 4.0 � 1.6 and8.7 � 2.7 fold in HUVEC by VEGF165 and bFGF,

respectively. In the presence of 20 �g/ml Eap, thismitogenic response was completely abrogated(1.28�0.66 and 1.36�0.17). The antiproliferative activ-ity of Eap was concentration-dependent, with completeinhibition seen at 20 �g/ml. Similarly, the bFGF- andVEGF-induced capillary-like sprout formation of bovineretinal endothelial cells (BREC) grown in a fibrin 3Dmatrix was efficiently repressed by the inclusion of aslittle as 5 �g/ml Eap (Fig. 1C).

2. Eap inhibits VEGF165 and bFGF-induced ERKphosphorylation but has variable effects on Aktphosphorylation

We next immunoblotted lysates of stimulated HUVECwith phosphospecific antibodies directed against theactivated forms of key kinases in angiogenic trans-duction, i.e. ERK1/2 (phospho-Thr202/Tyr204), p38(phospho-Thr180/Tyr182), and Akt/PKB (phospho-Ser-473). Preincubation with Eap led to a significantreduction in the level of the subsequent VEGF165-triggered ERK1/2 phosphorylation without altering thekinetics of the response (Fig. 2A). Under the sameconditions, Eap did not affect the VEGF165-induced p38and Akt/PKB phosphorylation. This specific suppres-sion of the MAPK pathway through Eap was not re-stricted to the VEGF165 elicited response, as similarresults were obtained in bFGF- stimulated HUVEC (Fig.

1 Correspondence: Institute of Medical Microbiology andHygiene, University of Saarland Hospital, D-66421 Hom-burg/Saar, Germany. E-mail: astrid.sobke@gmx.de

2 Present address: Experimental Immunology Branch, NCI,Bethesda, MD, USA.

doi: 10.1096/fj.06-5764fje

26210892-6638/06/0020-2621 © FASEB

2B). In contrast, there was an augmental effect of Eapon the bFGF-induced Akt/PKB phosphorylation; whilebFGF on its own caused only a marginal increase inAkt/PKB phosphorylation at serine 473, in the pres-

ence of Eap, this was clearly enhanced. Similarly, therewas also a minor increase of p38 phosphorylation in thepresence of Eap. The observed effects of Eap wereagain concentration-dependent. Comparable resultswere also seen in BREC, except that these microvascu-lar cells responded to bFGF with a strong phosphoryla-tion of Akt/PKB, which was actually inhibited by Eap.

3. Inhibition of the MAPK cascade occurs upstreamof ERK1/2 and downstream of PLC�

To delineate the target of Eap in more detail, westudied the effects of combinations of Eap, the PKCinhibitor GF109203X, and the farnesyl transferase in-hibitor manumycin A (indirect inhibitor of Ras) on theVEGF-induced MAPK cascade. Both GF109203X andEap had a substantial inhibitory effect on ERK1/2phosphorylation, with the combined effect resulting inthe complete blockage of the phosphorylation re-sponse. In comparison, manumycin A treatment on itsown had a weaker impact on ERK1/2 phosphorylation,whereas concomitant treatment with Eap resulted againin an additive effect. To test whether the impact of Eapwas independent of receptor activation, we examinedthe effects of Eap, GF109203X, and manumycin A onactivation of the MAPK pathway during stimulationwith the phorbol ester TPA. The findings were compa-rable to those seen on VEGF stimulation. Immunoblot-ting with a phospho-Ser-217/221 specific antibody re-vealed Eap to have similar effects on MEK activation ason ERK. Activation of PKC requires its translocation tothe plasma membrane and autophosphorylation. Eaphad no impact on the membrane translocation orphosphorylation levels of PKC�.

4. Eap exerts its effect at the level of Ras but not atthe level of Raf-1

Raf-1 constitutes an important relay point within theMAPK cascade, integrating positive and negative inputsfrom upstream pathways. Activation of Raf-1 is accom-panied by a characteristic shift in electophoretic mobil-ity due to an increased phosphorylation state.

Thus, stimulation of HUVEC with TPA induced theoccurrence of an additional, retarded band on Raf-1immunoblots. The intensity of this band was againreduced on treatment with Eap. Pull-down assays wereused to investigate Ras activation. There was a profoundreduction in activated, GTP-bound Ras after VEGF, aswell as bFGF stimulation in Eap-exposed HUVEC, indi-cating interference of Ras activation as the earliesttarget of Eap blockage of MAPK pathway activation.

CONCLUSIONS AND SIGNIFICANCE

Staphylococcus aureus is the “classical” and most commoncausative agent of abscess formation and surgical siteinfections (SSI), which typically are accompanied byimpaired wound healing and defects in granulation

Figure 1. Eap interferes with angiogenic response of ECprimary cultures. (A) mRNA levels of DAF and TF weredetermined by real-time polymerase chain reaction (PCR) inHUVEC stimulated with either 100 ng/ml VEGF or bFGF inthe absence (open bars) or presence (solid bars) of 20 �g/mlEap. Results are shown as fold increase over basal expression(means�sd) of three independent experiments and shown as*P � 0.05; **P � 0.01; ***P � 0.001. (B) HUVEC were grownto subconfluence in 24-well plates, serum-starved overnight(0.5% FCS), and then stimulated with either VEGF (25ng/ml), bFGF (25 ng/ml), or medium alone (0.1% FCS and0.5% BSA) for 20 h in the absence (solid bars) or presence(open bars) of 20 �g/ml Eap. Cells were pulse-labeled withtritium-thymidine for another 4 h and analyzed for thymidineincorporation. Data represent mean counts per minute � sdwith n � 5; *P � 0.05; **P � 0.01; ***P � 0.001. (C) Eapinhibits capillary sprout formation. Capillary-like sprout for-mation of BREC was analyzed in the absence (open bars) orpresence (solid bars) of 2 ng/ml bFGF and with the indicatedconcentrations of isolated Eap. Data represent the mean � sdof 30 beads analyzed in triplicate each.

2622 Vol. 20 December 2006 SOBKE ET AL.The FASEB Journal

tissue formation. Previously, we reported Eap to se-verely impair angiogenesis and wound healing in S.aureus-infected mice. However, since Eap was shown tohave potent immunosuppressive activities, it was so farunclear to what extent its adverse effects on angiogen-esis are the consequence of interference with theinflammatory response. Examining isolated primaryEC, we here demonstrate that Eap directly blocks theangiogenic response at different stages of activation(transcriptional up-regulation, proliferation, differenti-ation into tubular networks) and in EC of variableorigin (microvascular bovine retinal vs. macrovascularhuman umbilical vein EC). At the molecular level,these effects are shown to be based on correspondingalterations in proangiogenic signal transduction, inparticular, ERK1/2 phosphorylation was found to beseverely disturbed in the presence of Eap. In formerstudies staphylococcal surface proteins were reportedto bind basic growth factors like bFGF in a charge-dependent manner and interference with VEGF andbFGF binding to endothelial cells is also part of theantiangiogenic activity of thrombospondins. By demon-strating comparable effects of Eap on TPA-inducedERK1/2 phosphorylation, we can exclude such compe-tition for binding sites as the basis of the effect de-scribed here and show that inhibition has to occurdownstream of tyrosine kinase receptor activation. Sim-ilar to endogenous inhibitors of angiogenesis, like the16-kDa human prolactin fragment (16 K hPRL) orangiopoietin-1 (Ang-1), Eap was found to interfere withERK1/2 activation by blocking Ras-GTP formation. InbFGF-stimulated BREC, Eap also caused the completeblockage of the strong Akt phosphorylation observed in

these cells. Because PI3-kinase induction upstream ofAkt activation may occur in a Ras-dependent or -inde-pendent manner, this implies the involvement of Ras inthis cell type. Contrary to its effect on Ras-dependentpathways, Eap had a positive influence on basal p38 andAkt activation levels, and there was also a synergisticeffect with bFGF on phospho-Ser-473- Akt formation inHUVEC (2.5-fold increase with Eap over bFGF alone).Akt phosphorylation at serine 473 leads to subsequentinhibition of proapoptotic factors and nuclear exclu-sion of the “forkhead box class-O winged helix tran-scription factor” (FoxO). This was previously reportedas an important mechanism through which Ang-1 mod-ulates endothelial function. An enhancement of Aktactivation may thus add to Eap’s antiangiogenic activity.Further studies will be needed to determine the exactnature of the Eap receptor(s). However, during recentyears, it has become increasingly clear that endothelialresponses to bFGF/VEGF are not solely mediated bytheir specific tyrosine kinase receptors but rather re-quire the sequestration of a number of coreceptorstogether with FGF receptor 1 (FGFR-1) and VEGFreceptor 2 (VEGFR-2/ KDR/Flk-1) at specific mem-brane subdomains. Elucidation of Eap’s interactionwith the endothelium may thus help to shed some lightinto these complex processes. The potent anti-inflam-matory and antiangiogenic activities of Eap make thisversatile protein not only an important virulence factorduring S. aureus infection but open new perspectivesfor future pharmacological application, for instance ininhibiting pathological neovascularization as occurs intumor growth, or curbing the overshooting host re-sponses in sepsis.

Figure 2. Eap interferes with ERK1/2 activation. Serum-deprived HUVEC were left untreated or incubated with 100 �g/ml Eapfor 1 h before stimulation with 100 ng/ml of either VEGF (A) for the times indicated. Each lane contains 50 �g of total cellprotein; blots were tested with antibodies specific for the phosphorylated forms of either ERK1/2, p38, or Akt. HUVEC or BREC(B) were incubated with increasing concentrations of Eap, as indicated.

2623EAP ABROGATES ANGIOGENESIS BY BLOCKING RAS