HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY

A permissive role for tumor necrosis factor in vascular endothelial growthfactor–induced vascular permeabilityMatthias Clauss, Cord Sunderkotter, Baldur Sveinbjornsson, Stefan Hippenstiel, Antje Willuweit, Michael Marino, Elvira Haas,Rolf Seljelid, Peter Scheurich, Norbert Suttorp, Matthias Grell, and Werner Risau

Vascular endothelial growth factor (VEGF)induces both angiogenesis and an increasein vascular permeability, 2 processes thatare considered to be important for bothtumor growth and the delivery of drugs tothe site of tumors. This study demonstratesthat transmembrane expression of tumornecrosis factor (tmTNF) is up-regulated inthe endothelium of a murine methylcholan-threne (meth A)-induced sarcoma in com-parison to the adjacent normal dermal vas-culature and is also present on cultivatedhuman endothelial cells. It is further shownthat tmTNF is required for VEGF-mediated

endothelial hyperpermeability in vitro and invivo. This permissive activity of TNF ap-pears to be selective, because anti-TNF anti-bodiesablated theVEGF-inducedpermeabil-ity but not proliferation of cultivated humanendothelial cells. Furthermore, tnf gene–deficient mice show no obvious defects invascularization and develop normally butfailed to respond to administration of VEGFwith an increase in vascular permeability.Subsequent studies indicated that thetmTNF and VEGF signaling pathways con-verge at the level of a secondary messenger,the “stress-activated protein kinase-2”

(SAPK-2)/p38: (1) up-regulated endothelialexpression of tmTNF resulted in the continu-ous activation of SAPK-2/p38 in vitro, and(2) an inhibitor of SAPK-2/p38 activationabolished the vascular permeability activityof VEGF in vivo. In conclusion, the study’sfinding that continuous autocrine signalingby tmTNF sensitizes endothelial cells torespond to VEGF by increasing their vascu-lar permeability provides new therapeuticconcepts for manipulating vascular hyper-permeability. (Blood. 2001;97:1321-1329)

© 2001 by The American Society of Hematology

Introduction

Vascular endothelial growth factor (VEGF) is essential for bothvasculogenesis and angiogenesis1 but was first identified as atumor-produced vascular permeability factor (VPF) because of itspotent activity in the Miles assay of vascular permeability.2

Accordingly, VEGF causes the accumulation of fluids in dermaland ascitic tumors.3,4 Furthermore, VEGF-induced vascular perme-ability has been suggested to be involved in vasculopathies ofdiseases different than cancer, including rheumatoid arthritis,acquired immunodeficiency syndrome, and allergic inflamma-tion.5-7 In animal experiments it has been shown that the rapidinduction of VEGF-mediated vascular permeability correlates withthe formation of endothelial vesiculovacuolar organelles (VVOs)and fenestrations.8 An increase of vascular permeability has 2important consequences for potential therapeutic interventions intumors: First, it facilitates drug delivery9 and, secondly, it contrib-utes to the formation of hemorrhagic necroses, which are inducedby the administration of the soluble form of tumor necrosis factor-a(TNF), both in experimental tumor models and in isolated limbperfusions in patients.10-12

In addition to its ability to induce tumor necrosis, TNF is apotent inflammatory cytokine that is involved in various immuno-logic and pathophysiologic reactions.13 It is predominantly pro-duced by activated macrophages and lymphocytes as a 26-kd

transmembrane protein, which can be proteolytically cleaved to thesoluble 17-kd form.14 Several reports describe pathophysiologicchanges in transgenic animals in which transmembrane TNF(tmTNF) expression was artificially up-regulated.15 However, littleis known about the role of endogenously expressed tmTNF.

This study demonstrates that tmTNF is present in the endothe-lium of methylcholanthrene (meth A) sarcomas and that theexpression of tmTNF in endothelial cells in vitro is linked to anautocrine continuous activation of these cells. We also show thatneutralization of TNF ablates vascular permeability, induced byVEGF in endothelial cells, in vitro and in vivo, which suggests thatthe ability of VEGF to induce vascular permeability is facilitatedby endogenously expressed TNF.

Material and methods

Immunohistochemistry

Tumor tissue from mouse bearing meth A sarcoma16 was perfusion-fixedwith 4% paraformaldehyde and embedded in paraffin. Sections of tumorsand other tissues were stained with antibody using indirect immunohisto-chemistry. Polyclonal antimouse TNF (Genzyme Virotech, Russelheim,Germany) antibodies (1:100 diluted serum) were visualized with Cy5-conjugated goat antirabbit antibodies (diluted 1:1000; Dianova, Hamburg,

From the Department of Molecular Cell Biology, Max-Planck-Institute, BadNauheim, Germany; Institute of Experimental Dermatology and Department ofDermatology, Wilhelms-University of Munster, Munster, Germany; Departmentof Experimental Pathology, University of Tromsø, Tromsø, Norway;Department of Internal Medicine, Charite, Humboldt-University, Berlin,Germany; Ludwig Institute for Cancer Research, New York, NY; Institute of CellBiology and Immunology, University of Stuttgart, Stuttgart, Germany; andMerck KGaA, Biomed Fo/ONC, Darmstadt, Germany.

Submitted May 1, 2000; accepted October 11, 2000.

Supported by the European Community (Biomed 2: BMH4-CT98-3277 to

M.C.), the Deutsche Forschungsgemeinschaft (SFB 547/B2 to N.S., 547/C5 toM.C. and W.R.), and the Engelhorn Stiftung (fellowship to M.G.).

M.G. and W.R. contributed equally to this work.

Reprints: Matthias Clauss, Max-Planck-Institute, Park Str 1, 61231 BadNauheim, FRG; e-mail: m.clauss@kerckhoff.mpg.de.

The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.

© 2001 by The American Society of Hematology

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Germany). Cryostat sections from the same type of tumor were fixed withcold acetone and stained with TNF receptor-2–human immunoglobin G(TNFR2-huIgG) fusion protein17,18(10 mg/mL) and visualized using rabbitantihuman IgG antibody (diluted 1:100; Cappel, Durham, NC), biotinylatedgoat antirabbit IgG antibody (diluted 1:250; Boehringer, Mannheim,Germany), and fluorescein isothiocynate (FITC)-labeled streptavidin (di-luted 1:1000; Zymed, San Francisco, CA). Rat antimouse CD31 (1mg/mL;Pharmingen, San Diego, CA) was visualized with tetrarhodamine isothio-cyanate–conjugated goat antirat IgG (diluted 1:25; Zymed).

Cell culture and transfection of endothelial cells

Human umbilical cord vein endothelial cells (HUVECs) were prepared andcultivated as described previously.19 Spontaneously immortalized endothe-lial cells were characterized by the presence of the VEGF receptor-2(VEGFR-2) and von Willebrand factor using antibodies and immunofluores-cence (data not shown) as described recently.20,21 To stably introducetmTNF in endothelial cells, immortalized HUVECs (HUEs) were trans-fected with a noncleavable tmTNF mutant (kindly provided by T. Calarco)huTNF (1-12) in pcDNA3ZEO or with empty vector as control using theSuperFect reagent (Qiagen, Hilden, Germany). Cells were selected forresistance to zeocin, and clones were isolated, transferred into 96-wellplates, and analyzed by fluorescence-activated cell sorter (FACS) analysiswith the TNF-specific antibody T1.17

Cytofluorometric analysis and acid wash

For FACS analysis, confluent HUVECs were detached with 5 mMethylenediaminetetraacetic acid in Hank’s balanced salt solution in 25 mMHEPES and incubated with primary unconjugated monoclonal antibody(mAb) T1 for 30 minutes at 4°C and 10mg/mL as final concentration,washed twice with FACS buffer (phosphate-buffered saline [PBS] supple-mented with 1% bovine serum albumin [BSA] and 0.1% NaN3), and thenincubated with PE-conjugated goat antimouse IgG (Dianova) for 30minutes at 4°C. Flow cytometry analysis was performed on a FACStar(Becton Dickinson Immunocytochemistry Systems, Mountain View, CA).Isotype-matched control antibodies were used to determine the backgroundstaining. To distinguish tmTNF from receptor-bound TNF, cells wereincubated for 5 minutes on ice with isotonic solution with the pH adjusted to2.3 by the addition of hydrochloric acid to glycine (100 mM) buffer.22 Cellstreated or untreated were detached, and preparation and flow cytometry wasperformed as described above.

IL-6 determination

For interleukin (IL)-6 assays, cells were seeded in 96-well microtiter platesovernight. After washing the cells with PBS, they were cultured for anadditional 24 hours in the absence or presence of anti-TNF mAb T1 andcontrol IgG, respectively. Supernatants were removed and cleared bycentrifugation for 10 minutes at 15 000 rpm. IL-6 concentrations weredetermined using a commercially available IL-6 enzyme-linked immunosor-bent assay (ELISA) kit according the manufacturer’s recommendations(Pharmingen).

Tissue factor activity test

Tissue factor activity was assessed as described previously.23 Briefly, clonesof immortalized HUVECs, transfected with either tmTNF or control vector,were grown to confluence in 35-mm dishes. Assays were carried out withwhole cells obtained in suspension following scraping. Clotting times weremeasured after addition of human plasma and recalcification. Tissue factoractivity equivalents were determined by the use of a standard curve withrecombinant tissue factor.

NF-kB and p38 MAP kinase determination

To determine nuclear factor (NF)-kB activity by the luciferase reporterassay, cells were transfected with a minimal promoter containing a3–NF-kB element-driven firefly luciferase reporter plasmid using theSuperFect reagent. After one day of recovery, the cells were treated as

indicated, washed, and harvested in lysis buffer (Promega, Madison, WI);45-mL aliquots of cell lysates were mixed with 100mL luciferase assayreagent (Promega), and the luciferase activity was determined using aLumat LB9501 (Berthold, Wildbad, Germany). For normalization oftransfection efficacy, a second,b-galactosidase–encoding cytomegaloviruspromoter was used. For analysis of p38 mitogen-activated protein (MAP)kinase phosphorylation cells were treated as indicated, lysed in 120mLsodium dodecyl sulfate loading buffer, boiled, and electrophoresed underreducing conditions. After transfer onto nitrocellulose membranes, Westernblots were performed according the instructions of the manufacturer by theuse of specific antibodies capable of recognizing the phosphorylated (pp38)or unphosphorylated (p38) p38 MAP kinase forms (BioLabs, Boston, MA).

Endothelial permeability and proliferation

Hydraulic conductivity by VEGF was determined according to Suttorp etal.24 Briefly, 3H2O was applied together with recombinant human VEGF(rhVEGF; 1 ng/mL, produced as described19 from baculovirus-infectedinsect cells) to the upper chamber, and the transflux of tracer wasdetermined by continuously sampling from the lower chamber. Forexperiments with neutralizing antibodies, cells were preincubated for 20minutes at 37°C prior to the addition of reagents. For determination ofendothelial proliferation, HUVECs were seeded in 24-well plates (6000cells per well) and subsequently cultivated for 3 days with or without VEGF(10 ng/mL) in the presence or absence of antibodies. Cells were thendetached by treatment with trypsin, and cell numbers were counted in aCasy cell counter (Scharfe-System, Reutlingen, Germany).

Modified Miles assay

Miles assay was modified according to Senger et al2: Briefly, mice wereinjected intravenously with 2.5 mg FITC-labeled BSA (Sigma, Munich,Germany) in 100mL PBS. After 5 minutes, mice were anesthetized andVEGF (0.2mg in 20 mL PBS) or solvent (PBS) was injected into the left orright ear, respectively. Six minutes later, the animals were killed, and theextravasated fluorescent dye was measured using a fluorescence photom-eter. In some experiments mice were pretreated for 30 minutes (prior toVEGF administration) intraperitoneally with polyclonal anti-TNF serum(100 mL; Genzyme), the TNF-aproteinase inhibitor (TAPI; 500mg peranimal), the stress-activated protein kinase-2 (SAPK-2)/p38 inhibitor SB203580 (10mg per mouse; Upstate Biotechnology, Lake Placid, NY), orsolvent (PBS or dimethyl sulfoxide). Alternatively, Evans blue dye (1.2 mgin 100mL PBS) was used instead of FITC-labeled BSA, and extravasationof the dye was documented by photography.

Results

TNF is up-regulated in the endothelial lining of tumorblood vessels

Based on the reasoning that endothelial cells produce TNFfollowing stimulation25 and that the tumor vasculature is in apreactivated state,26 we analyzed sections from transplanted meth Asarcomas for endogenous TNF expression. We found stronglabeling for TNF in the endothelial lining of the tumor vasculatureusing either anti-TNF antibodies (Figure 1A, arrow) or a TNFR2-IgG fusion protein (Figure 1C). Control antibodies and an unrelatedIgG fusion protein did not stain the tumor vasculature (data notshown). Specific staining of the endothelium was demonstrated bycostaining with an anti–platelet endothelial cell adhesion moleculeantibody (Figure 1D). Normal dermal vasculature, distal from thetumor, also reacted weakly with TNF-specific antibodies (Figure1B, arrow), whereas vessels from other organs (lung and liver)showed no detectable staining (data not shown). Of note, theTNFR2-IgG fusion protein recognizes the transmembrane form ofTNF (tmTNF) but not receptor-bound soluble TNF.17

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Human endothelial cells can express the transmembrane formof TNF leading to activation

Because tmTNF is strongly up-regulated on the tumor vasculaturebut also expressed on normal dermal vasculature, we askedwhether TNF is expressed and active on cultivated endothelialcells. We found a low constitutive expression of TNF on the surfaceof some, but not all, preparations of endothelial cells derived fromHUVECs, as demonstrated by FACS analysis (Figure 2A) using aspecific anti-TNF mAb T1. This observation was also made withhuman microvascular and human aortic endothelial cells (data notshown). In several preparations of HUVECs (6 of 17) we found alow constitutive TNF-dependent production of IL-6, as demon-strated using TNF-specific neutralizing antibodies (Figure 2B,hatched bars). This finding indicates a constitutive TNF-dependentautocrine stimulation of these cells, similar to what we have foundin TNF-transfected cell lines.27

The observed endothelial TNF expression in vitro apparentlycomprises tmTNF because the T1 antibody does not recognizesoluble TNF receptor–bound TNF.17 Furthermore, no soluble TNFwas detectable in the supernatants of VEGF-stimulated HUVECswhen assessed either by the use of an ELISA (detection limit 10pg/mL; R&D Systems, Minneapolis, MN) or a standard cytotoxic-ity assay using L929 cells (detection limit 1 pg/mL28). In addition,we performed acid washes of endothelial cells to confirm that theantigen detected is not soluble TNF bound to the receptors. Afteracid wash, a method described to remove membrane- and receptor-bound soluble proteins,22 the antibody could still detect TNF on thesurface of endothelial cells, indicating that this TNF is membrane-integrated and not simply attached to the cells (Figure 3A,B).

TNF is required for VEGF-induced permeability of endothelialcell monolayers but not for VEGF-induced endothelialcell proliferation

In light of the herein provided evidence for constitutive endothelialpreactivation by endogenous tmTNF and the previously demon-strated synergistic interaction between soluble TNF and VEGF,29

we assessed whether endothelial TNF is involved in VEGF-

induced permeability and proliferation of HUVECs. A neutralizingantibody against TNF completely inhibited the VEGF-inducedincrease in permeability of an endothelial monolayer (Figure 4A)as determined by measuring the hydraulic conductivity of HUVECmonolayers grown on polycarbonate filter membranes.30 We didnot find a change of the constitutive, low expression of tmTNF onHUVECs upon VEGF treatment in the course of the experiment.Furthermore, HUVECs stimulated with VEGF in the presence ofneutralizing TNF-specific antibodies have not become refractory tothe induction of permeability, because addition ofStaphylococcusaureusa toxin, a well-established permeability-increasing agent,24

resulted in a rapid increase in the water filtration rate (datanot shown).

In contrast to the effects on endothelial permeability, theneutralizing antibodies against TNF did not affect the stimulationof endothelial proliferation by VEGF (Figure 4B). This indicatesthat in endothelial cells, VEGF-mediated permeability, but notVEGF-mediated proliferation, is dependent on the permissive roleof TNF.

Figure 2. Autocrine activation of TNF-expressing HUVECs. (A) TNF expressionof HUVECs revealed by FACS analysis using TNF-specific mAb T1 (aTNF, solid line)versus an isotype-matched control antibody (Co, dotted line). (B) IL-6 production inthe supernatants of HUVECs as determined by ELISA in the absence (empty bars) orpresence (hatched bars) of the TNF-specific mAb T1 (30 mg/mL each). Shown are 5examples of IL-6–expressing HUVECs generated from different cord donors (No. 21-36).

Figure 1. Immunohistologic analysis of endothelial TNF expression. (A) TNFstaining on the endothelial lining (arrow) of the tumor-associated vasculature oftransplanted meth A sarcomas. A strong autofluorescent reaction of some trapped redblood cells is also visible. (B) Weak staining of blood vessels (arrow) for TNF can beobserved in paraffin-embedded dermal tissue distant from the tumor. (C,D) Doublestaining of tmTNF and CD31 as an endothelial cell marker in frozen sections oftransplanted meth A sarcomas using a TNFR2-IgG fusion protein (C) and a ratantimouse CD31-specific mAb (D).

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TNF is permissive for VEGF-induced vascularpermeability in vivo

To examine whether VEGF-induced endothelial permeability invivo depends on the activity of TNF, we used a modified Milesassay of vascular permeability.2 In this assay an intravenousinjection of FITC-conjugated albumin is followed by an intrader-mal injection of 0.2mg VEGF into the ear. After 6 minutes theextravasation of the fluorophore is quantitatively assessed by dyeextraction and measurement in a fluorescence reader. The ratiosbetween the values derived from the VEGF- versus the solvent-(PBS) treated ears correspond to the permeability effect of VEGF.Pretreatment with TNF-neutralizing antibodies blocked the abilityof VEGF to induce permeability (Figure 5A), whereas controlantibodies had no effect (Figure 5A). Intriguingly, TNF-deficientmice31 also showed a significant defect in the ability to respond tothe permeability-increasing activity of VEGF in comparison towild-type mice of the same strain (Figure 5B). These results werealso directly illustrated using intravenous injection of Evans blueinstead of FITC-albumin prior to VEGF treatment of TNF-deficientversus wild-type mice (Figure 6A,B).

TAPI, an inhibitor of TNF-release, does not abolishVEGF-induced vascular permeability in vivo

To test the possibility that VEGF-induced permeability in vivo ismediated by the generation of soluble TNF, we applied TAPI, ahydroxamic acid–based inhibitor of the TNF-converting enzyme.32

Pretreatment of the animals with this inhibitor at concentrationsthat have been shown to prevent release of TNF in vivo32 did notreduce but rather increased the effect of VEGF on vascular

permeability (Figure 7A). These data indicate that the generation ofthe soluble form of TNF is not essential for this VEGF effect butthat the transmembrane form of TNF has an obligatory role inVEGF-induced vascular permeability in vivo.

A specific inhibitor of SAPK-2/p38 activation inhibitsVEGF-induced vascular permeability

Our in vitro data demonstrated that tmTNF expression in endothe-lial cells leads to continuous activation as assessed by IL-6.Because the IL-6 induction is NF-kB– and SAPK-2/p38–dependent,27 we have investigated the possibility that activatedSAPK-2/p38 is required for the increase of permeability by VEGF.Therefore, we pretreated mice with an inhibitor of SAPK-2/p38

Figure 4. Effects of TNF-neutralizing antibodies on VEGF-induced endothelialcell permeability and proliferation. (A) HUVECs were preincubated in the pres-ence (closed diamonds) or absence (closed triangles) of the neutralizing TNF-specific mAb T1 (10 mg/mL) prior to treatment with VEGF (1 ng/mL) or left untreated(open diamonds), and hydraulic conductivity was determined as described in the text.(B) HUVECs were cultivated for 3 days with or without VEGF (10 ng/mL) in thepresence (hatched bars) or absence (empty bars) of the neutralizing TNF-specificantibody T1 (30 mg/mL), and cells were counted. Data are presented as means(6 SD) of 3 separate experiments.

Figure 3. Endothelial surface TNF is in the transmembrane form. Cytofluorimet-ric analysis of immortalized HUVECs without (A) or with (B) acid wash was performedas described in the text. FACS analysis was performed using TNF-specific mAb T1(aTNF, solid line) versus an isotype-matched control antibody (Co, dotted line).

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prior to assessment in the Miles assay. As shown in Figure 7B,inhibition of SAPK-2/p38 abolished VEGF-mediated increase invascular permeability to the same extent as did neutralization ofTNF or the use oftnf2/2 animals.

The transmembrane form of TNF causes continuous activationof intracellular signaling in endothelial cells

Our observations that endothelial cells in vitro can secrete IL-6 andthat this production can be reduced by pretreatment with anti-TNFantibody suggests that endothelial transmembrane expression leadsto continuous activation of signaling molecules such as SAPK-2/p38 and thus primes the endothelium to respond to VEGF as a VPFin vivo. To test this hypothesis, we analyzed the principal capacity

of endothelial tmTNF to cause continuous activation of intracellu-lar signaling molecules. Because activation of NF-kB and SAPK-2/p38—2 signaling molecules that mediate TNF-induced IL-6 produc-tion in HeLa cells27—was difficult to assess in primary HUVECsbecause of their given heterogeneity, we employed spontaneouslyimmortalized HUVECs (HUEs) and introduced an uncleavablemutant of tmTNF in these cells (HUEtmTNF). Testing several clonesof these cells for NF-kB activation, we found a continuousactivation of NF-kB in tmTNF-overexpressing but not in controlHUE clones, as shown by transient reporter gene assays (Figure 8,empty bars). In addition, control clones but not the tmTNF-expressing clones could be further stimulated by application ofsoluble TNF (Figure 8A, shaded bars). Furthermore, Western blotanalysis revealed that in tmTNF-overexpressing clones SAPK-2/p38 is activated to an extent comparable to soluble TNF-treatedcontrol clones (Figure 8). This activation was blocked by preincu-bation of the tmTNF-expressing cells with the neutralizing anti-TNF antibody T1 or the specific SAPK-2/p38 inhibitor SB203580(Figure 8B). Together, these data demonstrate that endogenousoverexpression of tmTNF continuously activates endothelial cells.

The transmembrane form of TNF causes increasedproduction of tissue factor

Next, we wanted to know whether the continuous activation ofsignaling molecules in tmTNF-overexpressing cells also causesincreased production of TNF-inducible genes. Because the immor-talized HUVECs have lost the ability to synthesize IL-6, weassessed tissue factor, which is also induced by TNF in endothelialcells.33 We found increased tissue factor production in TNF-transfected clones versus control-transfected clones (Figure 9),suggesting that tmTNF overexpression in endothelial cells canaccount for an activated and procoagulant state.

Figure 6. Ablation of vascular permeability in TNF gene–deficient ( tnf 2/2) mice.VEGF-induced vascular permeability was determined in wild-type (A) and TNF-deficient mice (B) after intravenous injection of Evans blue and subsequent dermalinjection of VEGF into the left ear (arrows) and of PBS into the right ear.

Figure 5. TNF is essential for VEGF-induced vascular permeability in vivo.VEGF-induced vascular permeability in BALB/c mice with or without pretreatmentwith anti-TNF antibodies (A) in TNF gene–deficient (tnf2/2) and the correspondingwild-type mice (SV129) (B). Shown are scatter diagrams with each symbol represent-ing the ratio between the VEGF- and PBS-treated ear of one tested animal. Depictedare the mean (line) and the significance (P values) as calculated from an unpaired ttest using a commercial program (InStat 2.01, GraphPad, San Diego, CA).

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Discussion

This study demonstrates a novel principle for a permissive cytokinecrosstalk because the presence of endothelial tmTNF evokes acellular response by cooperating with a second, unrelated solublecytokine, VEGF. Our data support the hypothesis that TNFpreactivates endothelial cells to a “VEGF responsive status” bycontinuous signaling, leading to constitutive and continuous SAPK-2/p38 and NF-kB activation (Figure 8A,B). Furthermore, ourfinding that inhibition of the SAPK-2/p38 ablated a VEGF-mediated increase in vascular permeability—to the same extent asdid neutralizing of TNF or the use oftnf2/2 animals—suggests thatalso in endothelial cells in vivo there is continuous SAPK-2/p38

activation and that this activation is a prerequisite for VEGF to actas a VPF.

Although our observation of continuous SAPK-2/p38 activationis derived from TNF overexpression studies in vitro, it suggests aplausible mechanism to explain the TNF-dependent IL-6 produc-tion34 in endothelial cells in vitro and the VEGF-induced permeabil-ity in vitro and in vivo. Because we have demonstrated that in vivoextremely short pretreatment (30 minutes) with either SAPK-2/p38inhibitor (SB203580) or TNF antibodies is sufficient to abolishinduction of vascular permeability in response to a further shorttreatment (6 minutes) with VEGF, a direct cytosolic effect is morelikely to be involved than transcriptional activity. In this context,SAPK-2/p38 activation has been described to induce transcription-independent effects such as the induction of actin reorganizationand cellular motility.35 However, additional signaling pathwaysmay be involved in VEGF-induced permeability changes. Forexample, other TNF signaling molecules such as SAPK-1/JNK

Figure 8. Constitutive NF-kB and SAPK-2/p38 activation by immortalizedHUVECs expressing tmTNF. (A) Constitutive NF-kB activation in immortalizedHUVECs expressing tmTNF (HUEtmTNF) but not in control transfected cells (HUECo)as assessed by NF-kB reporter assays (empty bars). The effect of soluble TNF (10ng/mL) on NF-kB activation is also depicted (shaded bars). Shown are the meansand the SD of a minimum of 3 independent experiments (with 5 clones in case of theHUEtmTNF and 4 clones in case of the HUECo). (B) Continuous SAPK-2/p38 activationin tmTNF-expressing HUE cells as determined by Western blot analysis. The effectsof pretreatment (6 hours) with either 30 mg/mL TNF-specific mAb T1 (T1) or theSAPK-2/p38–specific inhibitor SB203580 (SB) on HUEtmTNF cells and of 10 ng/mLsoluble TNF (20 minutes) on HUECo cells on phosphorylation (pp38, upper panel) andon protein expression (p38, lower panel) of SAPK-2/p38 using the appropriateantibodies (as described in the text) are shown.

Figure 7. Analysis of VEGF-induced vascular permeability in vivo. VEGF-induced vascular permeability in BALB/c mice pretreated (30 minutes) with TAPI orsolvent (A) or the SAPK-2/p38 specific inhibitor SB203580 (VEGF plus SB) or solvent(VEGF) (B). Shown are scatter diagrams with each symbol representing the ratiobetween the VEGF- and PBS-treated ear of one tested animal. Depicted are themean (line) and the significance (P values) as calculated from an unpaired t test usinga commercial program (InStat 2.01). Note the different y-axis scales. Althoughvariations between the values of individual animals were observed, probably due toinjuries of small capillaries during injections, the differences between the individualgroups were highly significant.

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have not been analyzed in this context. Further, an essential role ofnitric oxide (NO) signaling for VEGF-mediated vascular permeabil-ity, both in vitro and in vivo, is well documented.36-39 We supposehere that VEGF-induced NO formation, leading to further down-stream signaling events such as the activation of extracellularsignal–regulated kinase 1/2 (ERK1/2), cooperates with continuousSAPK-2/p38 and NF-kB signaling in TNF-expressing endotheliumto initiate cellular events leading to hyperpermeability (Figure 10).

Our study is the first to demonstrate that the induction ofendothelial permeability, an important (patho)physiologic processattributed to VEGF, essentially relies on the function of TNF.Although administration of soluble TNF by itself can induceendothelial permeability in vivo, this occurs with slower kineticsand is dependent on the recruitment of leukocytes.40 It has beensuggested that the VEGF-induced formation of VVOs and fenestra-tion in the endothelium contribute to the rapid induction ofVEGF-mediated vascular permeability in vivo.8 In vitro, we haveshown recently that VEGF-mediated endothelial permeabilityoccurs with a delay (150 minutes) as compared with the responsein vivo, although the same cells were able to secrete P-selectinwithin 15 minutes.30 This different kinetic of in vivo and in vitropermeability induction can be explained by the delay in VVOformation observed in vitro41 (unpublished observation, 1998).Again, the molecular mechanisms of VEGF- and TNF-inducedpermeability seem to be different. Soluble TNF had no consistenteffect on endothelial permeability within the time frame studied(maximum 3 hours) in our test system. This may be due to the factthat TNF decreases endothelial monolayer barrier function byrearranging the cytoskeleton and forming intracellular gaps,42

whereas VEGF-induced permeability was observed in the absenceof gap formation.30

The delineation of the molecular mechanisms leading to

vascular permeability in vivo is difficult. For example, stimulus-dependent changes in the endothelium surface as well as floweffects are confounding parameters that cannot to be separatedfrom barrier function in the Miles assay.43 The principal ability ofVEGF to induce vascular permeability, however, has been demon-strated in a more sophisticated system using isolated perfusedmicrovessels.44 In fact, flow changes appear to be importantbecause, in a rabbit skin model of vascular permeability, VEGFwould only increase plasma leakage when prostaglandin E2 wascoapplied.45Although we cannot totally exclude the possibility thatthe role of TNF in vivo as measured by the Miles assay may alsoimply additional parameters, this assay was a helpful tool toestablish the role of several mediators and modulators in VEGF-induced permeability. These mediators include the known vasoac-tive substances prostacyclin and NO,46 but also family members ofSrc kinases because mice deficient in pp60c-src or pp62c-yesdisplayed refractory to VEGF-induced vascular permeability.47

Most recently, angiopoietin-1 emerged as a molecule that, whenoverexpressed in vivo, protects the vasculature against plasmaleakage induced by VEGF.48,49In conclusion, our data, demonstrat-ing an essential role for endothelial TNF expression and continuousSAPK-2/p38 activation in vascular permeability evoked by VEGF,provide the basis for further studies in the light of the findingslisted above.

Our suggestion that vascular permeability is caused by 2pathways, one dependent on the rapid induction of NO formationand the other one on constitutive SAPK-2/p38 activation, may helpto identify mechanisms that dissect VEGF activities leading toangiogenesis from those initiating vascular hyperpermeability. NOformation has been demonstrated to be essential for VEGF-mediated angiogenesis in vivo. In vitro, NO-dependent ERK1/2phosphorylation has been delineated as an essential element inVEGF-induced endothelial proliferation.38,50-54In contrast, SAPK-2/p38 activation causes inhibition rather than stimulation ofendothelial proliferation.55 Furthermore, our own results demon-strate that only VEGF-induced permeability, but not increasedendothelial cell proliferation, essentially depends on TNF (Figure4B). The notion that TNF-dependent signaling events, such aspermanent SAPK-2/p38 activation, are permissive for VEGF-induced vascular permeability but not for angiogenesis is in linewith results from mice that lack the genes for VEGF or TNF. Miceheterozygous forvegfgene deficiency suffer from a lethal impair-ment of embryonic vessel formation and capillary sprouting,whereas homozygoustnf gene–deficient mice develop normallywith no obvious vascular afflictions, indicating that physiologicangiogenesis is independent of TNF.31,56

Our finding that in unstimulated HUVECs low levels of TNFsurface protein are expressed is supported by mRNA expressiondata from other groups,57,58 which demonstrate low endothelial

Figure 10. Convergence of continuous TNF and VEGF signaling pathwaysleading to vascular permeability. Shown are candidate pathways for tmTNFsignaling (as shown in this study) and for VEGF signaling (as described in the text)leading to vascular permeability.

Figure 9. Constitutive tissue factor production in tmTNF-expressing endothe-lial cells. Tissue factor production in immortalized HUVECs expressing tmTNF(HUEtmTNF) versus control transfected cells (HUECo) was assessed as described inthe text. Shown are the means and the SD of a minimum of 3 independentexperiments (with at least 5 clones in case of the HUEtmTNF and 3 clones in case of theHUECo).

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TNF expression in comparison to induced TNF expression instimulated cells. Furthermore, in vivo, additional evidence forup-regulated endothelial TNF expression in pathologic situationssuch as atheroma59 and multiple sclerosis60,61 was provided.Unstimulated HUVECs express the tmTNF as demonstrated byWestern blot analysis62 and by ELISA of membrane fractions.63

Until now, it was unclear whether these observations were of anyphysiologic significance. This study provides several lines ofevidence for the hypothesis that the transmembrane form of TNFrather than soluble TNF mediates the VEGF-initiated induction ofpermeability in endothelial cells. For instance, expression of thetransmembrane form of TNF can be demonstrated in endothelialcells both in vitro (Figure 2A) and in vivo (Figure 1). The reagents,which we used to detect TNF expression (TNF receptor–IgG fusionprotein and human TNF-specific mAb T1) do not interact withsoluble TNF when bound to cellular TNF receptors.17 In addition,no soluble TNF was detectable in the supernatants of VEGF-stimulated and unstimulated HUVECs by the use of immunologicor functional assays. Finally, pretreatment of the animals with theinhibitor of the TNF-converting enzyme, TAPI, did not reduce theeffect of VEGF on vascular permeability (Figure 7). Although thisinhibitor is not specific for the cleavage of TNF in vitro and invivo,14 it was shown to block the effects of soluble TNF in themodel of lethal endotoxemia in D-galactosamine–sensitized ani-mals.32 The finding that the inhibition of TNF cleavage can lead toan accumulation of tmTNF molecules on the cell membrane32

would be in line with our observation that TAPI pretreatmentdid not reduce but rather increased the VEGF-induced vascularpermeability.

Based on our findings that TNF is up-regulated in the endothe-lium of the meth A sarcoma and that VEGF-induced permeabilityin vivo can be inhibited by treatment with anti-TNF antibodies, we

hypothesize that hyperpermeability of tumor vessels is dependenton the coordinate overexpression of endothelial tmTNF andtumor-secreted soluble VEGF (Figure 10). The permissive functionof tmTNF for VEGF-induced permeability but not for VEGF-mediated angiogenesis is of potential significance for the treatmentof solid tumors, perhaps by combining antiangiogenesis andchemotherapy. Such therapy would require conditions in whichangiogenesis is reduced, but vascular permeability is maintained oreven increased, to achieve optimal drug delivery. Therefore, ourconclusion that only the VEGF-induced signaling, which causespermeability but not proliferation, is dependent on TNF providesthe basis for the development of novel drugs, which either canincrease selectivity of antiangiogenic processes or can enhance thelocal delivery of cytostatic drugs to the tumor. Such drugs may bedeveloped based on the herein reported evidence that endogenousTNF-mediated signaling is essential for VEGF-mediated permeabil-ity but not for angiogenesis. Alternatively, therapeutic angiogenesiswith VEGF, which is limited by VEGF-induced vascular permeabil-ity,64 should benefit from the application of either anti-TNFantibodies or SAPK-2/p38 inhibitors.

Acknowledgments

We are grateful to R. Black (Immunex, Seattle, WA) for a generousgift of TAPI, D. Moosmayer for TNF receptor–IgG fusion protein,and T. Calarco (Chiron, Emeryville, CA) for providing us with theclone for uncleavable human TNF. We greatly appreciate theexcellent technical assistance of C. Fangmann, S. Merfeld, G.Zimmermann, and E. Richards. We would also like to thank C.Mitchell for critically reading the manuscript and giving valuablesuggestions.

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A PERMISSIVE ROLE FOR TNF 1329BLOOD, 1 MARCH 2001 z VOLUME 97, NUMBER 5

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doi:10.1182/blood.V97.5.13212001 97: 1321-1329  

Werner RisauMichael Marino, Elvira Haas, Rolf Seljelid, Peter Scheurich, Norbert Suttorp, Matthias Grell and Matthias Clauss, Cord Sunderkötter, Baldur Sveinbjörnsson, Stefan Hippenstiel, Antje Willuweit, 

induced vascular permeability−growth factor A permissive role for tumor necrosis factor in vascular endothelial

 

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