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Vascular Endothelial Growth Factor Regulates Endothelial Cell Survival through the Phosphatidylinositol 3′-Kinase/Akt Signal Transduction Pathway

REQUIREMENT FOR Flk-1/KDR ACTIVATION*
Open AccessPublished:November 13, 1998DOI:https://doi.org/10.1074/jbc.273.46.30336
      Vascular endothelial growth factor (VEGF) has been found to have various functions on endothelial cells, the most prominent of which is the induction of proliferation and differentiation. In this report we demonstrate that VEGF or a mutant, selectively binding to the Flk-1/KDR receptor, displayed high levels of survival activity, whereas Flt-1-specific ligands failed to promote survival of serum-starved primary human endothelial cells. This activity was blocked by the phosphatidylinositol 3′-kinase (PI3-kinase)-specific inhibitors wortmannin and LY294002. Endothelial cells cultured in the presence of VEGF and the Flk-1/KDR-selective VEGF mutant induced phosphorylation of the serine-threonine kinase Akt in a PI3-kinase-dependent manner. Akt activation was not detected in response to stimulation with placenta growth factor or an Flt-1-selective VEGF mutant. Furthermore, a constitutively active Akt was sufficient to promote survival of serum-starved endothelial cells in transient transfection experiments. In contrast, overexpression of a dominant-negative form of Akt blocked the survival effect of VEGF. These findings identify the Flk-1/KDR receptor and the PI3-kinase/Akt signal transduction pathway as crucial elements in the processes leading to endothelial cell survival induced by VEGF. Inhibition of apoptosis may represent a major aspect of the regulatory activity of VEGF on the vascular endothelium.
      HUVE
      human umbilical vein endothelial
      PI3-kinase
      phosphatidylinositol 3′-kinase
      VEGF
      vascular endothelial growth factor
      rh
      recombinant human
      PlGF
      placenta growth factor
      PBS
      phosphate-buffered saline
      PI
      propidium iodide
      bFGF
      basic fibroblast growth factor
      sel
      selective
      KDR
      kinase domain region.
      Angiogenesis and vascular remodeling occur throughout growth and development and involve both proliferation and regression of vascular endothelial cells. Although extensive research has been dedicated to the elucidation of the factors that induce endothelial cell proliferation and differentiation (for review, see Refs.
      • Folkman J.
      ,
      • Ferrara N.
      • Davis-Smyth T.
      ), surprisingly little is known about the mechanisms that regulate regression of blood vessels. Rapid degeneration of vascular structures occurs under various physiological or pathological circumstances. Apoptosis of endothelial cells probably contributes to the maturation of vascular granulation tissue into avascular scar tissue (
      • Robbins S.L.
      • Angel M.
      • Kumar V.
      ) and to the pruning of retinal vessels during development (
      • Alon T.
      • Hemo I.
      • Itin A.
      • Pe'er J.
      • Stone J.
      • Keshet E.
      ). Apoptosis-like changes in endothelial cells have been also observed in the involuting corpus luteum, breast (
      • Walker N.I.
      • Bennett R.E.
      • Kerr J.F.
      ), parotid glands undergoing pressure atrophy (
      • Walker N.I.
      • Gobe G.C.
      ), and fibrotic lung lesions (
      • Polunovsky V.A.
      • Chen B.
      • Henke C.
      • Snover D.
      • Wendt C.
      • Ingbar D.H.
      • Bitterman P.B.
      ). However, experiments designed to study blood vessel regression in vivo have not been very successful so far at detecting apoptotic endothelial cells lining the blood vessels. This may be attributable, at least in part, to the fact that endothelial cells undergoing apoptosis tend to lose their attachment to the basement membrane. In addition, apoptotic endothelial cell may become the target of the immune system and thus are eliminated by phagocytes as soon as they manifest early signs of apoptosis (
      • Augustin H.G.
      • Braun K.
      • Telemenakis I.
      • Modlich U.
      • Kuhn W.
      ).In vitro, apoptosis often follows withdrawal of a critical trophic factor. Accordingly, human umbilical vein endothelial (HUVE)1 cells undergo apoptosis after loss of adhesion or serum deprivation (
      • Araki S.
      • Shimada Y.
      • Kaji K.
      • Hayashi H.
      ,
      • Meredeth J.E.
      • Fazeli B.
      • Schwartz M.A.
      ). Fibroblast growth factor and phorbol esters reduce the apoptosis of serum-deprived HUVE cells (
      • Araki S.
      • Simada Y.
      • Kaji K.
      • Hayashi H.
      ), and heat shock and endotoxin act together to increase apoptosis in porcine endothelial cells (
      • Buchman T.G.
      • Abello P.A.
      • Smith E.H.
      • Bulkley G.B.
      ).
      It has recently been shown that the survival signal mediated by various growth factors and cytokines may be dependent on the phosphatidylinositol 3′-kinase (PI3-kinase)/Akt signal transduction pathway (
      • Minshall C.
      • Arkins S.
      • Freund G.G.
      • Kelley K.W.
      ,
      • Yao R.
      • Cooper G.M.
      ,
      • Yao R.
      • Cooper G.M.
      ,
      • Dudek H.
      • Datta S.R.
      • Franke T.F.
      • Birnbaum M.J.
      • Yao R.
      • Cooper G.M.
      • Segal R.A.
      • Kaplan D.R.
      • Greenberg M.E.
      ,
      • Songyang Z.
      • Baltimore D.
      • Cantley L.C.
      • Kaplan D.R.
      • Franke T.F.
      ,
      • Kennedy S.G.
      • Wagner A.J.
      • Conzen S.D.
      • Jordan J.
      • Bellacosa A.
      • Tsichlis P.N.
      • Hay N.
      ,
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      • Toker A.
      ). However, not all survival signals require PI3-kinase activity, and Akt (also referred as protein kinase Bα or Racα)-independent survival pathways exist (
      • Borasio G.D.
      ,
      • Nobes C.D.
      • Reppas J.B.
      • Markus A.
      • Tolkovsky A.M.
      ,
      • Xia Z.
      • Dickens J.
      • Raingeaud R.J.
      • Greenberg M.E.
      ). It was shown previously that vascular endothelial growth factor (VEGF) can induce PI3-kinase activity in a variety of endothelial cell (
      • Guo D.
      • Jia Q.
      • Song H.Y.
      • Warren R.S.
      • Donner D.B.
      ,
      • 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.
      ,
      • Kevil C.G.
      • Payne D.K.
      • Mire E.
      • Alexander J.S.
      ), but so far a specific biological function of PI3-kinase in endothelial cells has not been demonstrated.
      The endothelial cell-specific mitogen VEGF has been shown to be a key positive regulator of normal and abnormal angiogenesis (
      • Ferrara N.
      • Davis-Smyth T.
      ,
      • Mustonen T.
      • Alitalo K.
      ). The critical role of VEGF in the development of the vascular system is emphasized by embryonic lethality after loss of a single VEGF allele. A growing body of evidence indicates that VEGF may also act as a survival factor for newly formed blood vessels. In the developing retina, vascular regression in response to hyperoxia has been correlated with inhibition of VEGF release by glial cells (
      • Alon T.
      • Hemo I.
      • Itin A.
      • Pe'er J.
      • Stone J.
      • Keshet E.
      ). Furthermore, administration of anti-VEGF monoclonal antibodies results in regression of already established tumor-associated vasculature in xenograft models (
      • Yuan F.
      • Chen Y.
      • Dellian M.
      • Safabakhsh N.
      • Ferrara N.
      • Jain R.K.
      ). More recently, using a tetracycline-regulated VEGF expression system in xenografted C6 glioma cells, it has been shown that decreased levels of VEGF production lead to detachment of endothelial cells from the walls of preformed vessels in the tumor, and detached apoptotic endothelial cells were identified by means of double staining for Van Willebrand factor and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling staining (
      • Benjamin L.E.
      • Keshet E.
      ).
      VEGF exerts its biological effects by binding to its respective transmembrane receptors VEGF receptor 1 (Flt-1) and VEGF receptor 2 (Flk-1/KDR), both of which are expressed on endothelial cells specifically and contain a cytoplasmic tyrosine kinase domain. Knockout studies in mice revealed that both receptors are essential for the development of the embryonic vasculature, and mouse embryos null for either receptor died in utero between days 8.5 and 9.5 (
      • Shalaby F.
      • Rossant J.
      • Yamaguchi T.P.
      • Gertsenstein M.
      • Wu X.F.
      • Breitman M.L.
      • Schuh A.C.
      ,
      • Fong G.H.
      • Rossant J.
      • Gertsenstein M.
      • Breitman M.L.
      ). Stimulation of endothelial cells with VEGF demonstrated that VEGF induces phosphorylation of a variety of proteins, including phosphatidylinositol 3-kinase, Ras GTPase-activating protein, p190-rhoGAP, p62, phospholipase C-γ, the oncogenic adaptor protein NcK, p125 focal adhesion kinase, paxilin, and several others (23, 31–37; for review, see Ref.
      • Merenmies J.
      • Parada L.F.
      • Henkemeyer M.
      ). Strong experimental evidence links Flk-1/KDR activation to VEGF-induced mitogenesis and angiogenesis (
      • Ferrara N.
      • Davis-Smyth T.
      ). In contrast, the maintenance and survival function of VEGF has been associated with the Flt-1 receptor, because the Flt-1 mRNA is highly expressed in quiescent endothelium (
      • Peters K.G.
      • De Vries C.
      • Williams L.T.
      ), whereas Flk-1/KDR is primarily expressed in proliferating vessels (
      • Quinn T.P.
      • Peters K.G.
      • De Vries C.
      • Ferrara N.
      • Williams L.T.
      ).
      In the present study, we investigated the survival role of VEGF in HUVE cells cultured in serum-free conditions. We found that VEGF potently prevents apoptosis and promotes survival in this system. Using several approaches, we demonstrate the critical role of the PI3-kinase-Akt pathway in such effects. We also show that Flk-1/KDR, not Flt-1, is the primary mediator of such a signal.

      DISCUSSION

      The elucidation of the mechanisms responsible for blood vessel regression is critical to address several fundamental biological questions. This process is prominent not only in the course of organogenesis and organ remodeling during embryonic life but also in a variety of physiological situations in the adult, such as corpus luteum regression (
      • Modlich U.
      • Kaup F.J.
      • Augustin H.G.
      ) and uterine or breast involution after delivery (
      • Walker N.I.
      • Bennett R.E.
      • Kerr J.F.
      ). Over the last few years, several inhibitors of angiogenesis have been identified, including angiostatin (
      • O'Reilly M.S.
      • Holmgren L.
      • Shing Y.
      • Chen C.
      • Rosenthal R.A.
      • Moses M.
      • Lane W.S.
      • Cao Y.
      • Sage E.H.
      • Folkman J.
      ) and endostatin (
      • O'Reilly M.O.
      • Boehm T.
      • Shing Y.
      • Fukai N.
      • Vasios G.
      • Lane W.S.
      • Flynn E.
      • Birkhead J.R.
      • Olsen B.R.
      • Folkman J.
      ). These agents are able to inhibit tumor growth by suppressing angiogenesis in xenograft models. However, it is presently unknown whether these molecules participate in the physiological regulation of blood vessel regression. Recently, members of the family of Tie-2 ligands such as angiopoietin-2 have been shown to be involved in blood vessel remodeling and pruning, at least during embryonic development (
      • Maisonpierre P.C.
      • Suri C.
      • Jones P.F.
      • Bartunkova S.
      • Wiegand S.J.
      • Radziejewski C.
      • Compton D.
      • McClain J.
      • Aldrich T.H.
      • Papadopoulos N.
      • Daly T.J.
      • Davis S.
      • Sato T.N.
      • Yancopoulos G.D.
      ).
      Increasing evidence supports the concept that the withdrawal of a positive effector such as VEGF is sufficient to result in blood vessel regression in various in vivo systems, including tumors (
      • Borgstrom P.
      • Bourdon M.A.
      • Hillan K.J.
      • Sriramarao P.
      • Ferrara N.
      ) and developing organs (
      • Alon T.
      • Hemo I.
      • Itin A.
      • Pe'er J.
      • Stone J.
      • Keshet E.
      ,
      • Benjamin L.E.
      • Keshet E.
      ,
      • Modlich U.
      • Kaup F.J.
      • Augustin H.G.
      ). These studies demonstrated the presence of an inverse correlation between VEGF expression and the levels of vessel regression, implicating VEGF as an important survival factor for endothelial cells present in newly formed vasculature.
      Using an in vitro model system of serum-starved HUVE cells, we attempted to elucidate the signal transduction pathways resulting in VEGF-mediated survival. The HUVE cells used in our experiments consisted of pooled primary isolates from 300 individual donor umbilical veins and thus are likely to represent an average population of endothelial cells. Both VEGF receptors, Flk-1/KDR and Flt-1, were previously found to be expressed on HUVE cells by cell binding and cross-linking studies with VEGF or the Flt-1-specific ligand PlGF (
      • Waltenberger J.
      • Claesson Welsh L.
      • Siegbahn A.
      • Shibuya M.
      • Heldin C.H.
      ,
      • Park J.E.
      • Chen H.H.
      • Winer J.
      • Houck K.A.
      • Ferrara N.
      ) or by real-time reverse transcription-polymerase chain reaction analysis (
      • Gerber H.P.
      • Condorelli F.
      • Park J.
      • Ferrara N.
      ). We first verified that serum removal indeed resulted in apoptotic cell death. This was confirmed by a series of morphological changes consistent with apoptosis, as evidenced by time lapse video recording. Furthermore, the finding that the caspase inhibitor ZVAD-fmk completely prevented apoptosis and mimicked VEGF activity demonstrates that this process is mediated by the universal cell death machinery.
      In our survival assay, we found decreased levels of VEGF-dependent survival when cells were exposed to wortmannin or LY 294002, two potent inhibitors of PI3-kinase. Activation of PI3-kinase increases the intracellular amounts of phosphatidylinositol-3,4,5-bisphosphate and phosphatidylinositol-3,4,5-triphosphate, which positively regulates Akt by binding to the pleckstrin homology domain of Akt. Akt activation by growth factors requires PI3-kinase activity (
      • Downward J.
      ). In our experiments we found that Akt becomes phosphorylated after VEGF stimulation, and such activation could be inhibited by the PI3-kinase-specific inhibitor wortmannin. In a previous report, PI3-kinase was found to be tyrosine phosphorylated after exposure of endothelial cells to VEGF (
      • Guo D.
      • Jia Q.
      • Song H.Y.
      • Warren R.S.
      • Donner D.B.
      ). However, another report failed to detect induction of PI3-kinase after stimulation of HUVE cells by VEGF (
      • Abedi H.
      • Zachary I.
      ). These latter findings do not necessarily conflict with our data, because those experiments were conducted in presence of 2% fetal bovine serum, which may induce the PI3-kinase/Akt pathway to high levels, which cannot be further increased by VEGF. Interestingly, Xia et al. (
      • 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.
      ) recently reported that PI3-kinase is activated 2.1-fold by VEGF in cultured endothelial cells but is not required for VEGF-dependent proliferation.
      The serine-threonine protein kinase Akt (
      • Bellacosa A.
      • Testa J.R.
      • Staal S.P.
      • Tsichlis P.N.
      ) is one of the major targets of PI3-kinase-generated signals. The ability of activated Akt to promote survival was found in fibroblasts (
      • Kennedy S.G.
      • Wagner A.J.
      • Conzen S.D.
      • Jordan J.
      • Bellacosa A.
      • Tsichlis P.N.
      • Hay N.
      ) and PC12 pheochromocytoma cells (
      • Parrizas M.
      • Saltiel A.R.
      • LeRoith D.
      ) and neuronal cells (
      • Dudek H.
      • Datta S.R.
      • Franke T.F.
      • Birnbaum M.J.
      • Yao R.
      • Cooper G.M.
      • Segal R.A.
      • Kaplan D.R.
      • Greenberg M.E.
      ). A number of different growth factors have been shown to rapidly activate Akt via PI3-kinase activation, such as platelet-derived growth factor, epidermal growth factor, bFGF, insulin, and insulin-like growth factor 1 (
      • Kohn A.D.
      • Kovacina K.S.
      • Roth R.A.
      ,
      • Franke T.F.
      • Yang S.I.
      • Chan T.O.
      • Datta K.
      • Kazlauskas A.
      • Morrison D.K.
      • Kaplan D.R.
      • Tsichlis P.N.
      ,
      • Burgering B.M.
      • Coffer P.J.
      ,
      • Cross D.A.
      • Alessi D.R.
      • Cohen P.
      • Andjelkovich M.
      • Hemmings B.A.
      ,
      • Andjelkovich M.
      • Jakubowicz T.
      • Cron P.
      • Ming X.-F.
      • Han J.-W.
      • Hemmings B.A.
      ,
      • Harrington E.A.
      • Bennett M.R.
      • Fanidi A.
      • Evan G.I.
      ). In our experiments, VEGF and a mutant VEGF form, which binds to the Flk-1/KDR receptor specifically, could strongly induce Akt phosphorylation, and this induction was PI3-kinase-dependent. Moreover, in transient transfection experiments, the kinase-inactive Akt mutant, which has been suggested to act in a dominant-negative manner (
      • Kohn A.D.
      • Kovacina K.S.
      • Roth R.A.
      ), was found to block VEGF survival activity on endothelial cells. These findings constitute, to the best of our knowledge, the first direct demonstration of a role played by the PI3-kinase/Akt pathway in mediating a VEGF biological activity.
      Although the significance of VEGF and its endothelial cell-specific receptors in angiogenesis is well established, the signal transduction cascades initiated by the two known VEGF receptors resulting either in proliferation or in survival of endothelial cells are incompletely characterized.
      Based on gene knockout experiments in mice and several other studies, it can be deduced that Flk-1/KDR is critical for VEGF-mediated angiogenesis, both in the developing and in the adult animal (
      • Shalaby F.
      • Rossant J.
      • Yamaguchi T.P.
      • Gertsenstein M.
      • Wu X.F.
      • Breitman M.L.
      • Schuh A.C.
      ,
      • Shalaby F.
      • Ho J.
      • Stanford W.
      • Fischer K.D.
      • Schuh A.C.
      • Schwartz L.
      • Bernstein A.
      • Rossant J.
      ). In contrast, Flt-1 appears to be primarily involved in endothelial cell morphogenesis, at least during embryonic development (
      • Fong G.H.
      • Rossant J.
      • Gertsenstein M.
      • Breitman M.L.
      ). Although the role of Flt-1 in the adult animal is less clearly defined, there is increasing evidence that Flt-1 is able to transduce a signal and mediate a biological response. Flt-1, but not Flk-1/KDR, has been implicated in monocyte migration in response to VEGF or PlGF via a GTP-dependent signaling pathway (
      • Barleon B.
      • Sozzani S.
      • Zhou D.
      • Weich H.A.
      • Mantovani A.
      • Marme D.
      ,
      • Clauss M.
      • Weich H.
      • Breier G.
      • Knies U.
      • Rockl W.
      • Waltenberger J.
      • Risau W.
      ). Furthermore, the finding that Flt-1 mRNA is expressed in both proliferating and quiescent endothelial cells suggests a role for this receptor in the maintenance of endothelial cells (
      • Peters K.G.
      • De Vries C.
      • Williams L.T.
      ).
      The motif YXXM required for binding of the SH2-domain of the p85 regulatory subunit of PI3-kinase is absent from the noncatalytic regions of the intracellular domain of Flt-1 as well as from Flk-1/KDR. Using the yeast two-hybrid system, two groups reported independently an association between tyrosine 1213 of the Flt-1 carboxyl terminus, present within the tyrosine kinase domain and the p85 subunit of phosphatidylinositol 3-kinase. (
      • Igarashi K.
      • Isohara T.
      • Kato T.
      • Shigeta K.
      • Yamano T.
      • Uno I.
      ,
      • Cunningham S.A.
      • Waxham M.N.
      • Arrate P.M.
      • Brock T.A.
      ). However, when we tested PlGF or an Flt-1-specific VEGF mutant form in different assay systems, we could not detect any significant survival effect. The same mutants did not induce Akt phosphorylation, as assessed by Western blotting using an antibody that specifically recognizes the phosphorylated Akt. Likewise, we were unable to detect any change in the percentage of annexin V-positive, apoptotic endothelial cells in response to the Flt-1-selective ligands. These findings lead us to the conclusion that the Flt-1 receptor, at least when present in the homodimeric form, is not sufficient to mediate survival activity. In contrast, a KDR-selective VEGF mutant completely mimicked the antiapoptotic and survival-promoting effects of VEGF and led to strong activation of Akt via PI3-kinase. Given the absence of a consensus binding site on the cytoplasmic domain of Flk-1/KDR, we speculate on the existence of an adaptor molecule mediating the activation of p85 by Flk-1/KDR. We consider the possibility that only heterodimeric VEGF receptors consisting of Flt-1 and Flk-1 subunits would allow for p85 binding and activation to be very unlikely, because the KDR-selective mutant was sufficient to induce survival and Akt activation.
      In conclusion, our findings provide evidence that VEGF survival signals in endothelial cells are mediated by the Flk-1/KDR receptor through the PI3-kinase/Akt signal transduction pathway. Further delineation of this signaling pathway may yield attractive targets for cancer therapy aimed to induce endothelial cell apoptosis and blood vessel regression.

      ACKNOWLEDGEMENTS

      We thank the fluorescence-activated cell sorting lab at Genentech for their excellent support, H. Nguyen for cell culture, M. Vasser, P. Ng, and P. Jhurani for oligonucleotide synthesis, and M. Winkler and T. Yee for purification of VEGF mutants. We also thank Hendrik Gille for valuable discussion and David Wood and Charles Hoffman for excellent graphic art work.

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