Cysteinyl leukotriene 2 receptor andprotease-activated receptor 1 activate stronglycorrelated early genes in human endothelial cellsBarbara Uzonyi*†, Katharina Lotzer*†, Steffen Jahn*, Cornelia Kramer*, Markus Hildner*, Ellen Bretschneider*,Dorte Radke*‡, Michael Beer*, Rudiger Vollandt§, Jilly F. Evans¶, Colin D. Funk�, and Andreas J. R. Habenicht*,**

*Institute for Vascular Medicine aud §Institute for Medical Statistics, Computer Sciences, and Documentation, Friedrich-Schiller University, Bachstrasse 18,07743 Jena, Germany; ‡Leibniz Institute for Natural Product Research and Infection Biology, e. V. Hans-Knoll-Institute (HKI), Beutenbergstrasse 11a, 07745Jena, Germany; ¶Amira Pharmaceuticals, 9535 Waples Drive, San Diego, CA 92121; and �Departments of Physiology and Biochemistry, Queens University,99 University Avenue, Kingston, ON, Canada K7L 3N6

Communicated by K. Frank Austen, Harvard Medical School, Boston, MA, February 14, 2006 (received for review December 14, 2005)

Cysteinyl leukotrienes (cysLT), i.e., LTC4, LTD4, and LTE4, are lipidmediators derived from the 5-lipoxygenase pathway, and the cysLTreceptors cysLT1-R�cysLT2-R mediate inflammatory tissue reac-tions. Although endothelial cells (ECs) predominantly expresscysLT2-Rs, their role in vascular biology remains to be fully under-stood. To delineate cysLT2-R actions, we stimulated human umbil-ical vein EC with LTD4 and determined early induced genes. We alsocompared LTD4 effects with those induced by thrombin that bindsto protease-activated receptor (PAR)-1. Stringent filters yielded 37cysLT2-R- and 34 PAR-1-up-regulated genes (>2.5-fold stimulation).Most LTD4-regulated genes were also induced by thrombin. More-over, LTD4 plus thrombin augmented gene expression when com-pared with each agonist alone. Strongly induced genes werestudied in detail: Early growth response (EGR) and nuclear receptorsubfamily 4 group A transcription factors; E-selectin; CXC ligand 2;IL-8; a disintegrin-like and metalloprotease (reprolysin type) withthrombospondin type 1 motif 1 (ADAMTS1); Down syndromecritical region gene 1 (DSCR1); tissue factor (TF); and cyclooxygen-ase 2. Transcripts peaked at �60 min, were unaffected by acysLT1-R antagonist, and were superinduced by cycloheximide. TheEC phenotype was markedly altered: LTD4 induced de novo syn-thesis of EGR1 protein and EGR1 localized in the nucleus; LTD4

up-regulated IL-8 formation and secretion; and LTD4 raised TFprotein and TF-dependent EC procoagulant activity. These datashow that cysLT2-R activation results in a proinflammatory ECphenotype. Because LTD4 and thrombin are likely to be formedconcomitantly in vivo, cysLT2-R and PAR-1 may cooperate to aug-ment vascular injury.

cysteinyl leukotriene 2 receptor gene signature � protease-activatedreceptor 1 gene signature � vascular inflammation

Leukotrienes (LTs), i.e., LTB4 and the cysteinyl LTs (cysLT)LTC4, LTD4, and LTE4 constitute a group of lipid mediators

derived from the 5-lipoxygenase (5-LO) pathway (1, 2). LTs areeither produced by leukocytes at sites of inflammation or formedthrough transcellular metabolism after uptake and metabolismof leukocyte-derived LTA4 by downstream enzymes of the 5-LOpathway (LTA4 hydrolase and LTC4 synthase) in cells thatnormally do not express 5-LO, such as endothelial cells (ECs) (3,4). LTs act through G protein-coupled surface receptors(GPCRs), i.e., the LTB4 receptors and the cysLT receptors(LT-Rs) (cysLT1-R and cysLT2-R) (5–10). LT-Rs are expressedon multiple target cells, including leukocytes, smooth musclecells, and ECs (1). Recent studies implicate the 5-LO pathway incardiovascular disease (11–17).

Considerable information is available on cysLT1-R, whereaslittle is known about cysLT2-R. We have used human umibilicalvein (HUV)ECs as a model of vascular cells to study cysLT2-Ractivation by demonstrating that cysLTs exclusively signalthrough cysLT2-R in this cell type (18): In fact, HUVECs are the

first primary cell type that selectively expresses cysLT2-R. In thestudy detailed below, we determined HUVEC gene signatures inresponse to LTD4 and characterized the resulting phenotypes.LTD4 responses were compared with those of the prototypevasoactive agonist thrombin, which, in HUVEC, acts throughprotease-activated receptor (PAR)-1 (19). Thrombin was used asa positive control for LTD4 because it shares many acute effectswith LTD4 on ECs, both act through GPCRs, and thrombin andLTD4 may be formed concomitantly during vascular injury (seebelow). Our data show that cysLT2-R activation induces earlygene signatures that resemble those after PAR-1 activation andthat the combined actions of LTD4 and thrombin further stim-ulate gene expression. Thus, if LTD4 and thrombin are producedat the same time and site, they may cooperate in EC activationduring states of vascular perturbation in vivo.

ResultsEarly LTD4-Induced Genes Resemble Those Induced by Thrombin.HUVECs were stimulated with 100 nM LTD4 or 10 nM throm-bin for 60 min in cultures from four umbilical cords, andmicroarrays were analyzed [Gene Expression Omnibus (GEO)accession no. GSE3589]. To identify regulated genes, stringentfilter criteria were applied. LTD4 induced 37 genes �2.5-fold at60 min (Fig. 1 A and B; and see Supporting Text and Table 1,which are published as supporting information on the PNAS website). LTD4-induced genes were the result of cysLT2-R activa-tion, because quantitative (Q)RT-PCR analyses showed barelydetectable cysLT1-R transcripts but significant numbers ofcysLT2-R transcripts, and induction of all 13 genes studied wasresistant toward a specific cysLT1-R antagonist (See Fig. 6, whichis published as supporting information on the PNAS web site;and data not shown). In addition, similar to Ca2� responses (18),LTC4 and LTD4 were equipotent in gene activation (data notshown) and cysLT2-R transcripts exceeded cysLT1-R transcriptsby a factor of 1,000 (see Table 2, which is published as supportinginformation on the PNAS web site). We next compared LTD4and thrombin responses in parallel cultures using 10 nM throm-

Conflict of interest statement: No conflicts declared.

Abbreviations: ADAMTS1, a disintegrin-like and metalloprotease (reprolysin type) withthrombospondin type 1 motif 1; COX, cyclooxygenase; cysLT, cysteinyl leukotriene; DSCR1,Down syndrome critical region gene 1; EC, endothelial cell; EGR, early growth response; LO,lipoxygenase; LT, leukotriene; LT-R, LT receptor; GPCR, G protein-coupled receptor; HUVEC,human umbilical vein EC; NFAT, nuclear factor of activated T cells; NR4A, nuclear receptorsubfamily 4 group A; SELE, E-selectin; QRT-PCR, quantitative RT-PCR; TF, tissue factor; PAR,protease-activated receptor.

Data deposition: The data reported in this paper have been deposited in the GeneExpression Omnibus (GEO) database (accession no. GSE3589).

†B.U. and K.L. contributed equally to this work.

**To whom correspondence should be addressed. E-mail: andreas.habenicht@mti.uni-jena.de.

© 2006 by The National Academy of Sciences of the USA

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bin. Thrombin induced 34 genes �2.5-fold at 60 min (Fig. 1 A andC). Most of the 37 LTD4-induced genes were also significantlyinduced by thrombin if not, in few instances, by a factor of�2.5-fold (Fig. 1 A). Thus, when LTD4-regulated early geneswere examined regarding their induction by thrombin, a signif-

icant correlation was apparent (Pearson correlation coefficient,r � 0.90). These data revealed that activation of cysLT2-Rgenerates early gene signatures that resemble those after PAR-1stimulation by thrombin (Fig. 1 A–D; and see Tables 3–6, whichare published as supporting information on the PNAS web site).

Fig. 1. Early LTD4- and thrombin-regulated genes in HUVECs. HUVECs were stimulated with 100 nM LTD4 or 10 nM thrombin. (A) Heatmap of genes up-regulated�2.5-fold by LTD4; thrombin was used for comparison. (B and C) Scatterplots of LTD4- and thrombin-stimulated cells versus control; lines depict 2.5-fold change.(D) Scatterplot of LTD4- versus thrombin-stimulated cells. (E and F) Comparison of gene expression of LTD4-plus-thrombin-treated cells with cells stimulated withLTD4 or thrombin alone. Probe sets were selected as described in Supporting Text. Line separates up- from down-regulated probe sets. Columns in A indicateumbilical cord preparations; dots in scatterplots indicate means of signal intensities of four (B–D) or three (E and F) umbilical cords.

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In addition to the 60-min time point, genes at 6 and 24 h afterstimulation with LTD4 were determined in a single array exper-iment (see Tables 7–14, which are published as supportinginformation on the PNAS web site). We next focused on genesthat were up-regulated at 60 min.

LTD4 and Thrombin Cooperate to Enhance Gene Expression. LTD4 andthrombin act through distinct members of GPCR subfamilies,cysLT2-R and PAR-1 (5–10, 19–22), yet they share many acuteeffects on ECs in vitro and in vivo and may be formed concom-itantly during vascular injury in vivo (see Discussion). Weexamined whether concomitant activation of cysLT2-R andPAR-1 by addition of LTD4 plus thrombin enhances geneexpression when compared with each agonist alone. For thispurpose, gene signatures of HUVECs prepared from threeumbilical cords were determined in response to LTD4 plusthrombin parallel to LTD4 or thrombin alone (see Tables 15 and16, which are published as supporting information on the PNASweb site). Fifty genes were up-regulated by at least one of the twoagonists, and their overall expression was significantly aug-mented by the concomitant addition of both agonists whencompared with each agonist alone (Fig. 1 E and F; and see Table17, which is published as supporting information on the PNASweb site; Wilcoxon signed rank test, P � 0.01). Thus, LTD4 plusthrombin augments early gene expression when compared witheach agonist alone.

CysLT2-R Activation Generates a Comprehensive Immediate-EarlyGene Program. LTD4-regulated genes were found to encodetranscription factors, signaling molecules, an inhibitor of cal-

cineurin signaling, chemokines, angiogenic factors, extracellularmatrix-degrading molecules, adhesion molecules, and tissuefactor (TF) (Fig. 1 A and Table 1) as well as cyclooxygenase(COX)-2. To ascertain that array signal intensities representedup-regulated transcripts, induction of 13 genes (includingCOX-2; data not shown) was confirmed by QRT-PCR analysesat 1 h, and transcript kinetics were determined (Figs. 2–5; and seeFig. 7, which is published as supporting information on the PNASweb site; and data not shown). Transcript levels of all genespeaked at �60 min after addition of LTD4. Because many ofthem are immediate-early genes in other agonist-response sys-tems (23), we examined their sensitivity toward protein-synthesisinhibition. Each of the LTD4-induced genes examined (n � 13)was superinduced in the presence of the protein-synthesis in-hibitor cycloheximide (Fig. 3 Inset and data not shown). Thesedata show that many, if not all, of the LTD4-regulated genes atthe 60-min time point (Fig. 1 A) belong to the immediate-earlygene family (23).

LTD4 Stimulates Early Growth Response (EGR) and Nuclear ReceptorSubfamily 4 Group A (NR4A) Transcription Factors. LTD4 stimulatedtranscripts of 14 transcription factors (Fig. 1 A). The EGR and

Fig. 2. Induction of EGR transcription factor family members by LTD4. Cellswere stimulated with 100 nM LTD4, and EGR QRT-PCR analyses were per-formed at various time points thereafter. (A) Transcript kinetics of EGR1, -2,and -3. (B) EGR1 protein is up-regulated by LTD4 or TNF�. (C) EGR1 localizes inthe nucleus at 1 h.

Fig. 3. Induction of the NR4A transcription factor family members upon LTD4

stimulation. Cells were stimulated with 100 nM LTD4, and NR4A family mem-ber transcript kinetics were performed by QRT-PCR. (Inset) NR4A1 transcriptinduction by 100 nM LTD4 at 1 h in the absence and presence of 10 �g�mlcycloheximide (CHX).

Fig. 4. LTD4 induces IL-8 expression and secretion. Cells were stimulated with100 nM LTD4 for the indicated time points. Supernatants were collected forELISA, and cells were harvested for QRT-PCR analysis. (Inset) IL-8 proteincontent of control (C) and LTD4-stimulated (L) cultures at 2 h. Columns rep-resent means of quadruplicate dishes � SEM (P � 0.005; Student t test).

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NR4A families were chosen for detailed analyses because theymay contribute to inflammation, EGR1 can induce TF bytransactivating the TF promoter, and EGR1 has been shown tomediate oxidative stress-induced vascular reperfusion injury(24–27). Members of the EGR and NR4A families were amongthe most strongly up-regulated genes, as judged by the foldchange of signal intensities of the corresponding probe sets(Table 1). QRT-PCR analyses showed that transcripts of eachtranscription factor reached different absolute expression levels(Figs. 2 A and 3). Transcript levels of EGR1 and -3 differed bya factor of 10, that of EGR1 and -2 by a factor of �10 (Fig. 2 A),and EGR4 was not induced. EGR1 transcript up-regulation wasfollowed by a pronounced induction of EGR1 protein thatbecame visible at 1 h, appeared as a protein doublet thereafter,and had returned to near baseline levels at 4 h (Fig. 2B).Immunolocalization experiments showed that EGR1 waspresent in the nucleus at 1 h in LTD4-stimulated cells (Fig. 2C).The magnitude of LTD4’s action on EGR1 was comparable tothat of TNF� (Fig. 2 B and C). Of NR4A family members,NR4A1 was the most strongly affected member that was inducedto an �5-fold-higher level than that of NR4A2 or -3 (Fig. 3).These data show that LTD4 markedly stimulates transcriptionfactors implicated in EC activation and reperfusion injury andthat EGR1 translocates to the nucleus subsequent to its LTD4-induced de novo synthesis.

LTD4 Stimulates Expression of Genes Implicated in EC Activation andTwo Antiinflammatory Genes. We chose additional genes impli-cated in EC-activation regulation for detailed analyses, i.e.,E-selectin (SELE), a disintegrin-like and metalloprotease withthrombospondin type 1 motif 1 (ADAMTS1), Down syndromecritical region gene 1 (DSCR1), CXCL2 (MIP2), and CXCL8(IL-8) (28–32). SELE, ADAMTS1, and DSCR1 transcriptspeaked �60 min and then declined but remained elevated for 6 h(Fig. 7). By contrast, elevated transcript levels of CXCL2 andIL-8 had returned to near baseline levels at 4 h (Figs. 4 and 7).Up-regulation of IL-8 transcripts was followed by IL-8 secretioninto the culture medium, with a maximum at 2 h (Fig. 4 Inset).Furthermore, two LTD4-responsive genes have established an-tiinf lammatory actions, i.e., COX-2 (data not shown) andDSCR1 (Fig. 7): COX-2 is a key prostacyclin-synthesis-regulating enzyme in ECs (33), and DSCR1 has been shown tostrongly down-regulate proinflammatory calcineurin�nuclear

factor of activated T cells (NFAT)-transactivated genes bydirectly inhibiting calcineurin (31, 34–36). In preliminary stud-ies, we observed marked up-regulation of COX-2 protein andprostacyclin formation (K.L. and A.J.R.H., unpublished work).Moreover, the strong LTD4-triggered DSCR1 (target of cal-cineurin in HUVECs) transcript up-regulation (Fig. 7) wasmarkedly attenuated by the immunosuppressive agent cyclo-sporin (data not shown). These data provide preliminary evi-dence that the Gq-coupled Ca2��calcineurin�NFAT pathwaymay be involved in gene activation of HUVECs by cysLT2-Rsignaling.

LTD4 Up-Regulates TF- and HUVEC-Associated Procoagulant Activity.TF is the key regulator of the extrinsic blood coagulation cascadeand has been shown to play multiple roles in vascular biology(37). Up-regulation of TF transcripts by LTD4 peaked at �60min and then declined but revealed biphasic kinetics with asecond peak at 2–3 h, and elevated TF transcripts were main-tained for �4 h (Fig. 5A), followed by induction of TF proteinwith a maximum at 6 h (Fig. 5B). Large differences of TF antigenconcentrations and TF-dependent procoagulant activities wererecently noticed in blood (38, 39). To explore whether theLTD4-mediated increase in TF antigen was accompanied byconcomitant EC-associated TF-dependent procoagulant activ-ity, formation of active Factor Xa after formation of theTF�Factor VIIa complex was determined (see Materials andMethods). LTD4 induced cell-associated procoagulant activity bya factor of �4-fold (Fig. 5C).

DiscussionThese studies support three major conclusions: (i) The diversenature of LTD4-induced early genes suggests that cysLT2-R signal-ing gives rise to a comprehensive early proinflammatory andprothrombotic EC phenotype and to two antiinflammatory genes;(ii) simultaneous LTD4�cysLT2-R and thrombin�PAR-1 signalingenhances early gene expression when compared with each agonistalone; and (iii) early LTD4- and thrombin-induced gene signaturesare similar. Because LTD4 and thrombin may be formed concom-itantly during vascular injury (3, 4, 20, 22, 39), our data areconsistent with the possibility that two rather distinct GPCRsubfamily members (5–10, 20–22, 40–42), cysLT2-R and PAR-1,cooperate to promote EC activation in vivo.

Fig. 5. LTD4 induces TF expression and procoagulant activity. Cells were stimulated with 100 nM LTD4 for increasing periods of time. (A) TF transcript kineticsin response to LTD4 determined by QRT-PCR. (B) TF protein expression at 6 h after addition of LTD4 or TNF�. (C) TF-dependent procoagulant activity. Cells werestimulated with LTD4 for 6 h, the culture medium was removed, and TF-dependent procoagulant activity was determined in cell lysates as described in Materialsand Methods. Data represent means of eight cell lysates � SEM derived from three umbilical cords (P � 0.01; Student t test).

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Binding of LTD4 to cysLT2-R activates genes that have notpreviously been associated with the action of cysLTs. Theseinclude transcription factors that participate in ischemic stressand reperfusion injury in mice (24–27) (EGR1 and NR4A);chemokines and cell-adhesion molecules involved in blood leu-kocyte recruitment to inflamed tissues (28–30, 43) (CXCL2,IL-8, and SELE); an extracellular matrix-degrading metal-loprotease implicated in carotid artery remodeling (44) anddegradation of the collagen-binding proteoglycans versican andaggrecan (32) (ADAMTS1), a potent endogenous inhibitor ofcalcineurin signaling (31) (DSCR1), the key regulator of theextrinsic blood coagulation cascade (42) (TF), and a key pros-taglandin synthesis-regulating enzyme (33) (COX-2). These datashow that cysLTs initiate activities through robust induction ofproinflammatory genes. These gene activations follow previ-ously noticed activities of LTD4 that occur within seconds as adirect result of cysLT2-R signaling (1–4, 7, 11, 14, 16, 18, 40, 41,45), and they also indicate that cysLT2-R signaling activates atleast two genes that may limit inflammation and, therefore, maybe involved in attenuation of the otherwise potent proinflam-matory actions of cysLTs (COX-2 and DSCR1). Moreover,cysLT2-R may signal through the Ca2��calmodulin�calcineurin�NFAT pathway, as evidenced by the potent inhibitory action ofthe immunosuppressant, i.e., cyclosporin, on LTD4-triggeredDSCR1 transcript up-regulation that we observed in threeindependent experiments (data not shown). It is noteworthy thatDSCR1 may provide a major endogenous feedback inhibitor ofNFAT signaling of inflammation in T cells and HUVECs byother GPCR and non-GPCR activation events (31, 34–36).Several genes up-regulated in response to cysLT2-R activationand studied here in detail are up-regulated in HUVECs by otherGPCRs, such as the major HUVEC thrombin receptor (PAR-1)or the histamine receptor (46) in a cyclosporin-sensitive manner(31, 34, 46). Thus, several of the early genes may be the result ofGq protein coupling, but additional G proteins may also beinvolved (6). The observation that the cysLT2-R in human mastcells was shown to up-regulate IL-8 in a Ca2�-insensitive manner(47) indicates, however, that cysLT2-R may couple G proteins ina cell-type-specific way (Gi versus Gq). Alternatively, cysLT2-Rmay couple several G proteins similar to other GPCRs. Inrecombinant systems, cysLT2-R and thrombin receptors appearto couple predominantly through Gq-mediated phospholipase C�-triggered Ca2� elevation, but coupling through Gi has alsobeen reported for thrombin in ECs (6, 48). That Gi mayparticipate in cysLT2-R signaling in HUVECs was indicated bya partial inhibition of LTD4-dependent ERK1�2 phosphoryla-tion by pertussis toxin (K.L. and A.J.R.H., unpublished work).More work is required to identify HUVEC cysLT2-R-couplingof G proteins, downstream signaling components, and the pre-cise role of NFAT-regulated gene activation. The potent LTD4induction of the EGR1 gene and the subsequent nuclear local-ization of EGR1 protein deserves special attention, becauseEGR1 transactivates secondary genes in response to ischemiaand reperfusion injury in mice (26), whereas high levels ofEGR1-inducible genes have been observed in mouse and humanatherosclerosis (25). We are presently attempting to identifycysLT2-R-dependent secondary genes in response to the imme-diate-early genes identified here, although the TF promoter andother gene promoters have already been shown to be transac-tivated by EGR1 (27). Identification of these secondary genesmay also uncover differences between thrombin�PAR-1- andLTD4�LTC4�cysLT2-R-dependent modulation of EC activation,because these GPCRs may conceivably serve distinct biologicalroles in vivo.

The similarities in LTD4’s and thrombin’s immediate-earlygene signatures (Fig. 1) add a new dimension to previouslynoticed shared responses of LTD4 and thrombin. The latterinclude reduction of blood pressure, initiation of platelet-

activating factor synthesis, secretion of von Willebrand factor,and surface expression of P-selectin, all of which occur withinseconds and can be assumed to result from acute cysLT2-Rsignaling independent of gene transcription (11, 42). By contrast,elaboration of immediate-early genes by a single LTD4 and�orthrombin challenge renders HUVECs to an apparent activatedphenotype that may persist for hours, if not longer (42). How-ever, these proinflammatory activities may be attenuated byCOX-2 and DSCR1 up-regulation, as noted above. That thecysLT2-R mediates subacute types of fibroproliferative lunginjury in vivo in response to bleomycin has recently beenreported in cysLT2-R-deficient mice (40, 41). Our data areconsistent with the possibility that biological activities of the5-LO pathway may result in lasting alterations of the ECphenotype, such as increased platelet–EC interactions, promo-tion of blood monocyte adhesion to the endothelium, participa-tion in extracellular matrix remodeling, and reperfusion injury ofthe heart (40, 41). The shared activities of LTD4 and thrombininvite examination of the possibility that still further actions ofthrombin in vivo may also be relevant for LTD4 actions on ECcysLT2-R (20, 42). It is likely that thrombin and LTD4 are formedconcomitantly (3, 4, 20, 42). It is tempting to speculate thatcysLTs may participate in the formation of complex inflamma-tory circuits (14) in diseased blood vessels through cysLT2-R-mediated inflammatory EC gene targeting. This finding posesthe interesting suggestion that cysLT2-R-specific antagonistsmay be of benefit in cardiovascular disease.

Materials and MethodsMaterials. LTD4 was obtained from Cayman Chemical (Ham-burg, Germany), TNF� from R & D Systems (Wiesbaden-Nordenstadt, Germany), and thrombin and all other reagentsfrom Sigma-Aldrich (Munich), unless otherwise noted; anti-EGR1 was from Santa Cruz Biotechnology, Cy3-conjugateddonkey anti-rabbit antibody from Dianova (Hamburg, Ger-many), and anti-TF antibody from American Diagnostica (Pfun-gstadt, Germany). Montelukast was a gift of R. N. Young, MerckFrosst (Quebec, Canada). Reagents for QRT-PCR analyses werefrom Invitrogen (Karsruhe, Germany).

Microarray Analysis. RNA was extracted and purified by TRIzol andRNeasy Micro kit (Qiagen, Hilden, Germany). RNA integrity wasdetermined on RNA 6000 Nano LabChips (Agilent Technologies,Palo Alto, CA). Preparation of cRNA and hybridization on U133Aarrays were performed according to the manufacturer’s protocols(Affymetrix, Santa Clara, CA). Additional information is publishedin Supporting Text. Microarray data of LTD4- and thrombin-stimulated HUVECs are published in National Center for Bio-technology Information’s Gene Expression Omnibus at www.ncbi.nlm.nih.gov�geo�(GEO accession no. GSE3589).

Cell Culture and ELISA of IL-8. HUVECs were maintained inserum-free medium (Invitrogen) and used within the first twopassages. Cells were stimulated with 100 nM LTD4, 10 nMthrombin, and 100 units�ml TNF� or solvent for varying timeperiods. For IL-8 measurements, the medium, including LTD4,was replaced, supernatants were collected, and IL-8 was deter-mined by ELISA (BD Biosciences, Heidelberg).

QRT-PCR. RNA was reverse-transcribed, and external standardswere prepared and sequence confirmed as described in ref. 16.QRT-PCR parameters were established by using SYBR green asa detection dye as described in ref. 16, with primers reported inTable 18, which is published as supporting information on thePNAS web site.

Western Blot. Cells were lysed for analysis of EGR1 in Laemmlibuffer or for TF in buffer containing 10 mM Tris�HCl, pH 7.5,

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10 mM MgCl2, 1% Triton X-100, and the protease-inhibitormixture Complete (Roche Diagnostics, Mannheim, Germany).Proteins were separated by 10% SDS�PAGE and blotted ontonitrocellulose membrane (GE Healthcare, Freiburg, Germany).Membranes were blocked and incubated with EGR1- (1:6,000)or TF- (1:4,000) specific antisera. Immunoreactive bands weredetected by chemiluminescence and CDP-Star reagent (AppliedBiosystems, Darmstadt, Germany).

Immunofluorescence. HUVECs were cultured on coverslips (Nunc,Wiesbaden, Germany). After stimulation, the cells were fixed with4% formaldehyde and permeabilized with 0.5% Triton X-100. Thecover slips were incubated with anti-EGR1 (1:100) and, afterwashing, with the secondary antibody (Cy3-conjugated anti-rabbitantibody, 1:600). DNA was stained with DAPI. Confocal laserscanning microscopy was performed with a Zeiss Axiovert 200microscope equipped with a LSM 510 laser scanner head.

TF-Dependent Procoagulant Activity. Cells were lysed in a buffer of50 mM Tris�HCl, pH � 7.4, 100 mM NaCl, and 0.1% TritonX-100, sonicated, and assayed by using a TF chromogenicactivity assay according to the manufacturer’s protocol (Amer-ican Diagnostica, Greenwich, CT).

We thank Katarina Ludajic and Christine Skerka [Leibniz Institute forNatural Product Research and Infection Biology, e. V. Hans-Knoll-Institute (HKI)] for help with the EGR1 immunoblot. This work wassupported by German Research Council Grants Ha 1083�13-1, 13-2,13-3, 12-5, and 12-6; Interdisciplinary Center for Clinical Research(IZKF) Jena Grant TP 4.4�4.9 (to A.J.R.H. and E.B.); German Ministryof Research and Development Grant BMBF No. 0312704 D (to D.R.);a Merck & Co. grant (to A.J.R.H.); and Canadian Institutes of HealthResearch Grants MOP-68930 and MOP-67146 (to C.D.F.). C.D.F. holdsa Tier 1 Canada Research Chair in Molecular, Cellular, and PhysiologicalMedicine.

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