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An Inhibitory Role of the Phosphatidylinositol 3-Kinase-signaling Pathway in Vascular Endothelial Growth Factor-induced Tissue Factor Expression*

Open AccessPublished:September 07, 2001DOI:https://doi.org/10.1074/jbc.M105474200
      Vascular endothelial growth factor (VEGF) is not only essential for vasculogenesis and angiogenesis but is also capable of inducing tissue factor, the prime initiator of coagulation, in endothelial cells. In this study we have analyzed the VEGF-elicited pathways involved in the induction of tissue factor in human umbilical cord vein endothelial cells. Using specific low molecular weight inhibitors we could demonstrate a crucial role of the p38 and Erk-1/2 mitogen-activated protein (MAP) kinases. In contrast, treatment with wortmannin or LY294002, inhibitors of phosphatidylinositol 3 (PI3)-kinase, resulted in a strong enhancement of the VEGF-induced tissue factor production, indicating a negative regulatory role of the PI3-kinase on tissue factor-inducing pathways. Accordingly, transduction with constitutively active Akt led to a reduction of VEGF-induced tissue factor production. Western blot analyses using antibodies specific for phosphorylated p38 showed an enhanced activation of this MAP kinase in human umbilical cord vein endothelial cells when stimulated with VEGF in the presence of wortmannin in comparison to either agent alone. Thus, the negative regulation of the PI3-kinase pathway on endothelial tissue factor activity can be explained at least in part by a suppression of this MAP kinase-signaling pathway. This is the first demonstration of a reciprocal relationship between procoagulant activity and the PI3-kinase-Akt signaling pathway, and it reveals a novel mechanism by which tissue factor expression can be controlled in endothelial cells.
      VEGF
      vascular endothelial growth factor
      TNF
      tumor necrosis factor
      NFκB
      nuclear factor κB
      VEGFR
      VEGF receptor
      PI3-kinase
      phosphatidylinositol 3-kinase
      PKC
      protein kinase C
      MAP
      mitogen-activated protein
      PlGF
      placenta growth factor
      HUVEC
      human umbilical cord vein endothelial cell
      FACS
      fluorescence-activated cell sorter
      MEK
      MAP kinase/extracellular signal-regulated kinase kinase
      Vascular endothelial growth factor (VEGF)1 is the major inducer of angiogenesis and vasculogenesis (for review see Ref.
      • Risau W.
      ). It is distinct from other endothelial growth factors such as platelet-derived growth factor and the fibroblast growth factors by its ability to selectively stimulate endothelial cell growth and, in addition, to initiate endothelial procoagulant activity and vascular permeability (
      • Clauss M.
      • Gerlach M.
      • Gerlach H.
      • Brett J.
      • Wang F.
      • Familletti P.C.
      • Pan Y.-C.
      • Olander J.V.
      • Connolly D.T.
      • Stern D.
      ,
      • Senger D.
      • Galli S.
      • Dvorak A.
      • Peruzzi C.
      • Harvey V.
      • Dvorak H.F.
      ). These permeability and procoagulant activities have been suggested to contribute to the sensitization of the tumor vasculature to undergo tumor necrosis factor (TNF) α-mediated hemorrhagic necrosis in experimental models (
      • Shimomura K.
      • Manda S.
      • Mukomoto K.
      • Kobayashi S.
      • Nakano K.
      • Mori J.
      ,
      • Nawroth P.P.
      • Handley D.
      • Matsueda G.
      • de Waal R.
      • Gerlach H.
      • Blohm D.
      • Stern D.M.
      ,
      • Clauss M.
      • Ryan J.
      • Stern D.
      ). However, tumor endothelium, either without treatment or strongly enhanced after systemic TNF infusion, is the only example of endothelial tissue factor and procoagulant activity expression in vivo (
      • Contrino J.
      • Hair G.
      • Kreutzer D.L.
      • Rickles F.R.
      ,
      • Zhang Y.
      • Deng Y.
      • Wendt T.
      • Liliensiek B.
      • Bierhaus A.
      • Greten J.
      • He W.
      • Chen B.
      • Hach-Wunderle V.
      • Waldherr R.
      • Ziegler R.
      • Mannel D.
      • Stern D.M.
      • Nawroth P.P.
      ). In tissues others than tumors, tissue factor is not expressed (with the exception of the spleen) in the endothelial lining even after treatment with lethal endotoxin concentrations (
      • Drake T.A.
      • Cheng J.
      • Chang A.
      • Taylor Jr., F.B.
      ). What bans tissue factor from the endothelium under physiological conditions and what enables tissue factor expression in certain tumors are not understood.
      In contrast to the in vivo situation, in vitrotissue factor expression on the surface of endothelial cells can be induced by TNF, interleukin-1, and VEGF (
      • Nawroth P.P.
      • Stern D.M.
      ,
      • Bevilacqua M.P.
      • Pober J.S.
      • Majeu G.R.
      • Cotran R.S.
      • Gimbrone Jr., M.A.
      ). The ability of TNF to induce tissue factor in endothelial cells could be linked to the transcription factor nuclear factor κB (NFκB) (
      • Bierhaus A.
      • Zhang Y.
      • Deng Y.
      • Mackman N.
      • Quehenberger P.
      • Haase M.
      • Luther T.
      • Müller M.
      • Böhrer H.
      • Greten J.
      • Martin E.
      • Baeuerle P.A.
      • Waldherr R.
      • Kisiel W.
      • Zeigler R.
      • Stern D.M.
      • Nawroth P.P.
      ,
      • Moll T.
      • Czyz M.
      • Holzmuller H.
      • Hofer-Warbinek R.
      • Wagner E.
      • Winkler H.
      • Bach F.H.
      • Hofer E.
      ,
      • Parry G.C.
      • Mackman N.
      ). Recently, endothelial tissue factor induction by VEGF was found to be independent of NFκB but because of the induction or activation of the transcription factors Egr-1 and nuclear factor of activated T-cells (
      • Armesilla A.L.
      • Lorenzo E.
      • del Arco P.G.
      • Martinez-Martinez S.
      • Alfranca A.
      • Redondo J.M.
      ,
      • Mechtcheriakova D.
      • Wlachos A.
      • Holzmuller H.
      • Binder B.R.
      • Hofer E.
      ).
      Two VEGF receptors (VEGFR-1 and VEGFR-2) are present on vascular endothelial cells, and VEGF signaling is mediated by receptor dimerization, leading to autophosphorylation of the cytosolic domains of these receptors. Phosphorylated VEGF receptors serve as docking sites for adapter molecules or for signaling enzymes such as PI3-kinase. VEGF has been shown to activate PI3-kinase generating phosphatidylinositol 3,4,5-trisphosphates, which create docking sites for the protein kinase B better known as Akt (
      • Gerber H.P.
      • McMurtrey A.
      • Kowalski J.
      • Yan M.
      • Keyt B.A.
      • Dixit V.
      • Ferrara N.
      ,
      • Bos J.L.
      ). Subsequent serine/threonine phosphorylation on Akt has been demonstrated as a VEGF-dependent signaling pathway. Akt as the major downstream signal of PI3-kinase has been shown to induce various endothelial functions including survival, migration, and nitrogen monoxide (NO) production and was demonstrated as a potent proangiogenic signaling molecule (
      • Dimmeler S.
      • Fleming I.
      • Fisslthaler B.
      • Hermann C.
      • Busse R.
      • Zeiher A.M.
      ,
      • Kureishi Y.
      • Luo Z.
      • Shiojima I.
      • Bialik A.
      • Fulton D.
      • Lefer D.J.
      • Sessa W.C.
      • Walsh K.
      ,
      • Luo Z.
      • Fujio Y.
      • Kureishi Y.
      • Rudic R.D.
      • Daumerie G.
      • Fulton D.
      • Sessa W.C.
      • Walsh K.
      ,
      • Chavakis E.
      • Dernbach E.
      • Hermann C.
      • Mondorf U.F.
      • Zeiher A.M.
      • Dimmeler S.
      ). In addition to PI3-kinase activation, VEGFR-2 autophosphorylation has been demonstrated to recruit and activate phospholipase Cγ. Phospholipase Cγ cleaves membrane-bound phosphatidylinositol bisphosphate resulting in soluble inositoltrisphosphate, which causes the release of Ca2+from intracellular storage pools, and diacyglycerol formation, which is the main activator of protein kinases C (PKC). PKC activation is upstream of the Raf/MEK/Erk-signaling cascade and is involved in VEGF effects such as proliferation and vascular permeability (
      • Xia P.
      • Aiello L.P.
      • Ishii H.
      • Jiang Z.Y.
      • Park D.J.
      • Robinson G.S.
      • Takagi H.
      • Newsome W.P.
      • Jirousek M.R.
      • King G.L.
      ,
      • Wu H.M.
      • Yuan Y.
      • Zawieja D.C.
      • Tinsley J.
      • Granger H.J.
      ).
      The signaling events leading to VEGF-mediated tissue factor induction are only partially understood. Based on the findings that nuclear factor of activated T-cells and Egr-1 but not NFκB are essential transcription factors for VEGF-induced tissue factor production, evidence for an involvement of Ca2+ and MAP kinase-dependent pathways has been provided (
      • Armesilla A.L.
      • Lorenzo E.
      • del Arco P.G.
      • Martinez-Martinez S.
      • Alfranca A.
      • Redondo J.M.
      ,
      • Mechtcheriakova D.
      • Wlachos A.
      • Holzmuller H.
      • Binder B.R.
      • Hofer E.
      ,
      • Mechtcheriakova D.
      • Schabbauer G.
      • Lucerna M.
      • Clauss M.
      • De Martin R.
      • Binder B.R.
      • Hofer E.
      ). Using a test system for tissue factor production (
      • Clauss M.
      • Gerlach M.
      • Gerlach H.
      • Brett J.
      • Wang F.
      • Familletti P.C.
      • Pan Y.-C.
      • Olander J.V.
      • Connolly D.T.
      • Stern D.
      ,
      • Mechtcheriakova D.
      • Wlachos A.
      • Holzmuller H.
      • Binder B.R.
      • Hofer E.
      ,
      • Clauss M.
      • Grell M.
      • Fangmann C.
      • Fiers W.
      • Scheurich P.
      • Risau W.
      ), we show here that inhibition of PI3-kinase activity by wortmannin and LY294002 synergistically enhanced VEGF-induced tissue factor activity. This effect of PI3-kinase inhibitors on VEGF-induced endothelial tissue factor seemed to involve MAP kinases, because the inhibition of PI3-kinase signaling correlated with an enhanced phosphorylation of the p38 MAP kinase and to a certain degree of the Erk-1/2 MAP kinase, two essential mediators of VEGF-induced endothelial tissue factor activity.

      DISCUSSION

      This study demonstrates that VEGF can induce signal transduction pathways that regulate endothelial tissue factor production in a positive as well as a negative fashion. Positive regulation was shown to essentially depend on the two MAP kinases p38 and Erk-1/2 by the use of small chemical inhibitors. Negative regulation of tissue factor production occurred via the activation of the PI3-kinase-Akt-signaling pathway as demonstrated with two selective inhibitors of the PI3-kinase and transfection of a constitutively activated mutant of Akt. Although the use of chemical inhibitors is limited by the specificity of the inhibitors, there are several lines of evidence suggesting that the observed negative regulatory effect is indeed caused by the VEGF-induced PI3-kinase activity. First, the wortmannin concentration used (100 nm) is in the range described to be specific for the PI3-kinase (half-maximal at 5 nm), and a dose-response curve for tissue factor induction displayed a very similar response as measured with the PI3-kinase (data not shown). Furthermore, we could confirm our results with a second specific PI3-kinase inhibitor (LY294002). Finally, the use of a constitutively active Akt mutant strongly reduced VEGF-induced procoagulant activity, linking this kinase, which is directly downstream of PI3-kinase, to the negative regulation of tissue factor induction.
      To explain the negative regulation of tissue factor production by the PI3-kinase-Akt pathway several alternative explanations are possible. For example, in lipopolysaccharide-activated macrophages inhibition of the PI3-kinase was demonstrated to lead to prolonged NFκB activation (
      • Diaz-Guerra M.J.
      • Castrillo A.
      • Martin-Sanz P.
      • Bosca L.
      ). Although VEGF has also been reported to induce NFκB activation in endothelial cells (
      • Marumo T.
      • Schini-Kerth V.B.
      • Busse R.
      ), this induction is controversial and condition-dependent. For example, VEGF-induced NFκB activation was shown in bovine retina endothelial cells using gel shift assays (
      • Marumo T.
      • Schini-Kerth V.B.
      • Busse R.
      ), but no such effect could be observed in HUVECs by assessment of nuclear translocation (
      • Mechtcheriakova D.
      • Wlachos A.
      • Holzmuller H.
      • Binder B.R.
      • Hofer E.
      ). Furthermore, VEGF-induced tissue factor induction was demonstrated to be mediated largely by the transcription factor Egr-1. (
      • Bierhaus A.
      • Zhang Y.
      • Deng Y.
      • Mackman N.
      • Quehenberger P.
      • Haase M.
      • Luther T.
      • Müller M.
      • Böhrer H.
      • Greten J.
      • Martin E.
      • Baeuerle P.A.
      • Waldherr R.
      • Kisiel W.
      • Zeigler R.
      • Stern D.M.
      • Nawroth P.P.
      ,
      • Moll T.
      • Czyz M.
      • Holzmuller H.
      • Hofer-Warbinek R.
      • Wagner E.
      • Winkler H.
      • Bach F.H.
      • Hofer E.
      ,
      • Mechtcheriakova D.
      • Wlachos A.
      • Holzmuller H.
      • Binder B.R.
      • Hofer E.
      ). In fact, we failed to demonstrate any effect of VEGF treatment by the use of an NFκB reporter construct in HUVECs, and in addition, no regulatory role of PI3-kinase on NFκB activation was observed (data not shown). These data are in accordance to the recent observation that the PI3-kinase-Akt pathway has no influence on NFκB activation in endothelial cells (
      • Madge L.A.
      • Pober J.S.
      ). Therefore, the observed negative regulatory role of the PI3-kinase-Akt pathway on tissue factor production is very unlikely to occur via NFκB modulation.
      Another possible negative regulatory role of the PI3-kinase may be mediated by affecting endothelial NO-synthetase expression, because PI3-kinase inhibitors were described to enhance shear stress-induced transcription of this enzyme (
      • Malek A.M.
      • Jiang L.
      • Lee I.
      • Sessa W.C.
      • Izumo S.
      • Alper S.L.
      ). NO formation was shown previously to be downstream of phospholipase Cγ and PKC but upstream of the Erk-1/2 MAP kinase (
      • Wu H.M.
      • Yuan Y.
      • Zawieja D.C.
      • Tinsley J.
      • Granger H.J.
      ,
      • Parenti A.
      • Morbidelli L.
      • Cui X.L.
      • Douglas J.G.
      • Hood J.D.
      • Granger H.J.
      • Ledda F.
      • Ziche M.
      ), which is in line with our findings that PKC and MAP kinases are major signaling pathways for VEGF-induced tissue factor production (Fig. 1). However, our findings also show that VEGF-induced tissue factor production is independent on the generation of NO (Fig. 1 B). In conclusion, our observations are in favor of the hypothesis that activation of Raf by PKC occurs in HUVECs by a pathway that does not involve NO formation.
      Alternatively, the strong enhancement of tissue factor production by chemical inhibition of the PI3-kinase-Akt-signaling pathway can be explained by an inhibitory role of this pathway on p38 and/or Erk-1/2 activation, two kinases shown in this study to be essential for VEGF-induced tissue factor production (Fig. 1 B). According to this hypothesis VEGF elicits an anticoagulant pathway by PI3-kinase-mediated suppression of the p38 and/or Erk-1/2 MAP kinases and in parallel procoagulant pathways by p38 and Erk-1/2 MAP kinase activation. In fact, in this study we could demonstrate that the PI3-kinase-signaling pathway inhibits p38 MAP kinase phosphorylation. This is in confirmation of a very recent report showing that the blockade of PI3-kinase-Akt also led to enhanced VEGF activation of p38 and apoptosis (

      Gratton, J. P., Morales-Ruiz, M., Kureishi, Y., Fulton, D., Walsh, K., and Sessa, W. C. (2001) J. Biol. Chem., 276, in press

      ). In addition, negative regulatory roles of the PI3-kinase-Akt-signaling pathway on the activation of Erk-1/2 were demonstrated in various different cellular systems including differentiated myotubes, a human breast cancer cell line, and also in HUVECs (
      • Madge L.A.
      • Pober J.S.
      ,
      • Rommel C.
      • Clarke B.A.
      • Zimmermann S.
      • Nunez L.
      • Rossman R.
      • Reid K.
      • Moelling K.
      • Yancopoulos G.D.
      • Glass D.J.
      ,
      • Zimmermann S.
      • Moelling K.
      ). In contrast, a positive regulatory role of the PI3-kinase-mediated Erk-1/2 activation has been proposed in HUVECs because inhibition of the PI3-kinase led to a decrease in VEGF-induced Erk-1/2 phosphorylation (
      • Thakker G.D.
      • Hajjar D.P.
      • Muller W.A.
      • Rosengart T.K.
      ). This controversy observed for the same type of primary cells (HUVECs) is also reflected by our data. Seven experiments supported the hypothesis that PI3-kinase activation in HUVECs is inhibitory, whereas three experiments suggested a rather stimulatory role for the PI3-kinase on Erk-1/2 activation in response to VEGF. Obviously the PI3-kinase effect on this pathway is condition-dependent, which can be explained by genetic and individual (historical) differences of the donor cords from which the umbilical cord vein endothelial cells were isolated. In conclusion, the inhibitory effect of the PI3-kinase pathways on VEGF-induced tissue factor production can be explained at least in part by an inhibitory effect on the p38 MAP kinase.
      There are numerous reports suggesting that VEGFR-2 is the major if not exclusive signaling VEGF receptor for endothelial cell functions including angiogenesis. This hypothesis has been derived also from studies using receptor-specific homologues such as the PlGF that binds to VEGFR-1 but not to VEGFR-2 (
      • Park J.E.
      • Chen H.H.
      • Winer H.
      • Houck K.A.
      • Ferrara N.
      ), VEGF-E, which binds selectively to VEGFR-2 but not to VEGFR-1 (
      • Meyer M.
      • Clauss M.
      • Lepple-Wienhues A.
      • Waltenberger J.
      • Augustin H.G.
      • Ziche M.
      • Lanz C.
      • Buttner M.
      • Rziha H.J.
      • Dehio C.
      ), or receptor-specific mutants (
      • Gille H.
      • Kowalski J.
      • Li B.
      • LeCouter J.
      • Moffat B.
      • Zioncheck T.F.
      • Pelletier N.
      • Ferrara N.
      ). In terms of tissue factor induction in endothelial cells, VEGF-E was found to have a similar bioactivity as VEGF (also designated as VEGF-A) itself (
      • Meyer M.
      • Clauss M.
      • Lepple-Wienhues A.
      • Waltenberger J.
      • Augustin H.G.
      • Ziche M.
      • Lanz C.
      • Buttner M.
      • Rziha H.J.
      • Dehio C.
      ). In contrast, tissue factor was only slightly induced by PlGF (maximally at 2–3-fold) in comparison with similar concentrations of VEGF (maximally 25–80-fold) (
      • Clauss M.
      • Weich H.
      • Breier G.
      • Knies U.
      • Rockl W.
      • Waltenberger J.
      • Risau W.
      ). On top of these findings is our present observation that VEGFR-2 signaling is essential for the observed synergy between VEGF and PI3-kinase inhibitors in tissue factor induction. This is in line with the observation that VEGFR-2-mediated signaling is the major inducer of the PI3-kinase-Akt pathway (
      • Gille H.
      • Kowalski J.
      • Li B.
      • LeCouter J.
      • Moffat B.
      • Zioncheck T.F.
      • Pelletier N.
      • Ferrara N.
      ), a finding that can be also explained by the presence of a repressor sequence in the juxtamembrane domain of the VEGFR-1, which inhibits VEGF-dependent PI3-kinase activation (
      • Gille H.
      • Kowalski J., Yu, L.
      • Chen H.
      • Pisabarro M.T.
      • Davis-Smyth T.
      • Ferrara N.
      ). Furthermore, in the same study only a marginal activation of P38 and Erk-1/2 was observed with VEGFR-1-specific VEGF mutants. In conclusion, a VEGFR-2-dependent PI3-kinase and Akt-activation pathway, which can suppress MAP kinase activities and subsequently also tissue factor production, fits best with the data provided in this study.
      In conclusion, our finding that a PI3-kinase-dependent pathway is a negative regulator for the MAP kinase-mediated tissue factor production defines a novel regulatory principle in endothelial cells. We propose that in vascular endothelial cells under physiological conditions, continuous PI3-kinase activation by plasma components such as insulin and insulin-like growth factors occurs, leading to the suppression of tissue factor production. In tumors, however, blood flow and perfusion are impaired, and the supply with growth factors is suboptimal (
      • Sevick E.M.
      • Jain R.K.
      ,
      • Dewhirst M.W.
      • Kimura H.
      • Rehmus S.W.
      • Braun R.D.
      • Papahadjopoulos D.
      • Hong K.
      • Secomb T.W.
      ). In consequence, levels of PI3-kinase activation may be reduced in tumors. In addition, tumor-produced cytokines such as VEGF can elicit specific signaling events that are not induced by serum components, leading to tissue factor production. Thus, the striking observation that tumor endothelium is one rare exception of tissue factor expression on the endothelium may be explained by diminished PI3-kinase activity together with increased P38 and Erk-1/2 MAP kinase activity in comparison to the physiological condition.
      Our hypothesis that PI3-kinase activity is a suppressor for endothelial activation and tissue factor production is supported by preliminary experiments, in which wortmannin was applied to animals bearing tumors producing high amounts of VEGF. In these animals hemorrhagic necroses occurred that were restricted to the tumors
      S. Kröger, S. Blum, R. de Waal, A. Hilbig, C. Ebenebe, and M. Clauss, manuscript in preparation.
      and similar to that observed with systemic TNF application or targeting of tissue factor to the tumor endothelium (
      • Huang X.
      • Molema K.
      • King S.
      • Watkins L.
      • Edgington T.S.
      • Thorpe P.E.
      ). This is in line with a recent in vitro observation that wortmannin can also enhance the production of endothelial tissue factor protein when co-applied together with TNF and VEGF (
      • Shen B.Q.
      • Lee D.Y.
      • Cortopassi K.M.
      • Damico L.A.
      • Zioncheck T.F.
      ). Collectively, these observations give novel insights in the regulatory mechanism controlling endothelial activation and tissue factor production under physiological and pathological conditions. Further studies that address PI3-kinase induction in activatedversus quiescent endothelial cells are currently under investigation.

      ACKNOWLEDGEMENTS

      We are grateful to R. Müller (Institute of Molecular Biology and Tumor Research, University of Marburg) for the opportunity to use the S2 facilities and W. Ruf (Scripps Institute, La Jolla, CA) for supplying tissue factor monoclonal antibodies. The excellent technical assistance of C. Kremser is gratefully acknowledged.

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