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Vascular Permeability Factor (VPF)/Vascular Endothelial Growth Factor (VEGF) Receptor-1 Down-modulates VPF/VEGF Receptor-2-mediated Endothelial Cell Proliferation, but Not Migration, through Phosphatidylinositol 3-Kinase-dependent Pathways*

  • Huiyan Zeng
    Affiliations
    Departments of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215
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  • Harold F. Dvorak
    Affiliations
    Departments of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215
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  • Debabrata Mukhopadhyay
    Correspondence
    A Eugene P. Schonfeld National Kidney Cancer Association Medical Research Awardee. To whom correspondence should be addressed: Depts. of Pathology, RN270H, Beth Israel Deaconess Medical Center and Harvard Medical School, 330 Brookline Ave., Boston, MA 02215. Tel.: 617-667-7853; Fax: 617-667-3591;
    Affiliations
    Departments of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215
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  • Author Footnotes
    * This work was supported in part by National Institutes of Health Grants CA78383 (to D. M.) and CA50453 (to H. F. D.), by the Massachusetts Department of Public Health (D. M.), and under terms of a contract from the National Foundation for Cancer Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:July 20, 2001DOI:https://doi.org/10.1074/jbc.M103213200
      Vascular permeability factor (VPF)/vascular endothelial growth factor (VEGF) achieves its multiple functions by activating two receptor tyrosine kinases, Flt-1 (VEGF receptor-1) and KDR (VEGF receptor-2), both of which are selectively expressed on primary vascular endothelium. To dissect the respective signaling pathways and biological functions mediated by these receptors in primary endothelial cells with these two receptors intact, we developed a chimeric receptor system in which the N terminus of the epidermal growth factor receptor was fused to the transmembrane domain and intracellular domain of KDR (EGDR) and Flt-1 (EGLT). We observed that KDR, but not Flt-1, was responsible for VPF/VEGF-induced human umbilical vein endothelial cell (HUVEC) proliferation and migration. Moreover, Flt-1 showed an inhibitory effect on KDR-mediated proliferation, but not migration. We also demonstrated that the inhibitory function of Flt-1 was mediated through the phosphatidylinositol 3-kinase (PI-3K)-dependent pathway because inhibitors of PI-3K as well as a dominant negative mutant of p85 (PI-3K subunit) reversed the inhibition, whereas a constitutively activated mutant of p110 introduced the inhibition to HUVEC-EGDR. We also observed that, in VPF/VEGF-stimulated HUVECs, the Flt-1/EGLT-mediated down-modulation of KDR/EGDR signaling was at or before intracellular Ca2+mobilization, but after KDR/EGDR phosphorylation. By mutational analysis, we further identified that the tyrosine 794 residue of Flt-1 was essential for its antiproliferative effect. Taken together, these studies contribute significantly to our understanding of the signaling pathways and biological functions triggered by KDR and Flt-1 and describe a unique mechanism in which PI-3K acts as a mediator of antiproliferation in primary vascular endothelium.
      VPF
      vascular permeability factor
      VEGF
      vascular endothelial growth factor
      HUVEC
      human umbilical vein endothelial cell
      PI-3K
      phosphatidylinositol 3-kinase
      EC
      endothelial cell
      PLC
      phospholipase C PAE, porcine aortic endothelial
      EGF
      epidermal growth factor
      EGFR
      epidermal growth factor receptor
      PCR
      polymerase chain reaction
      FACS
      fluorescence-activated cell-sorting
      PBS
      phosphate-buffered saline
      EGDR
      the fusion receptor of EGFR N terminus with KDR C terminus
      EGLT
      the fusion receptor of EGFR N terminus with Flt-1 C-terminus
      To grow beyond minimal size, tumors must generate a new vascular supply for purposes of gas exchange, cell nutrition, and waste disposal (
      • Folkman J.
      ,
      • Folkman J.
      ,
      • Folkman J.
      • Klagsburn M.
      ,
      • Folkman J.
      • Watson K.
      • Ingber D.
      • Hanahan D.
      ). They do so by secreting angiogenic cytokines that induce the formation of new blood vessels (
      • Folkman J.
      • Klagsburn M.
      ,
      • Folkman J.
      • Watson K.
      • Ingber D.
      • Hanahan D.
      ,
      • Dvorak H.F.
      • Nagy J.A.
      • Feng D.
      • Brown L.F.
      • Dvorak A.M.
      ,
      • Risau W.
      ). Tumor-secreted angiogenic cytokines include fibroblast growth factor, platelet-derived growth factor B, and vascular permeability factor (VPF)1/vascular endothelial growth factor (VEGF) (
      • Risau W.
      ,
      • Benezra M.
      • Vlodasky I.
      • Ishai-Michaeli R.
      • Neufeld G.
      • Bar-Shavit R.
      ,
      • Senger D.R.
      • Perruzzi C.A.
      • Feder J.
      • Dvorak H.F.
      ,
      • Vlodavsky I.
      • Fuks Z.
      • Ishai-Michaeli R.
      • Bashkin P.
      • Levi E.
      • Korner G.
      • Bar-Shavit R.
      • Klagsbrun M.
      ). VPF/VEGF is likely the most important of these because it is expressed abundantly by a wide variety of human and animal tumors and because of its potency, selectivity for endothelial cells, and ability to regulate most and perhaps all of the steps in the angiogenic cascade (
      • Dvorak H.F.
      • Nagy J.A.
      • Feng D.
      • Brown L.F.
      • Dvorak A.M.
      ,
      • Risau W.
      ,
      • Dvorak H.F.
      ,
      • Dvorak H.F.
      • Orenstein N.S.
      • Carvalho A.C.
      • Churchill W.H.
      • Dvorak A.M.
      • Galli S.J.
      • Feder J.
      • Bitzer A.M.
      • Rypysc J.
      • Giovinco P.
      ,
      • Dvorak H.F.
      • Senger D.R.
      • Dvorak A.M.
      ,
      • Ferrara N.
      ). Moreover, a number of other angiogenic cytokines act, at least in part, by up-regulating VPF/VEGF expression (
      • Dvorak H.F.
      • Nagy J.A.
      • Feng D.
      • Brown L.F.
      • Dvorak A.M.
      ,
      • Seghezzi G.
      • Patel S.
      • Ren C.J.
      • Gualandris A.
      • Pintucci G.
      • Robbins E.S.
      • Shapiro R.L.
      • Galloway A.C.
      • Rifkin D.B.
      • Mignatti P.
      ). VPF/VEGF extensively reprograms endothelial cell expression of proteases, integrins, and glucose transporters, stimulates endothelial cell migration and division, protects endothelial cells from apoptosis and senescence, and induces angiogenesis in both in vitro and in vivo models (for review, see Refs.
      • Dvorak H.F.
      • Nagy J.A.
      • Feng D.
      • Brown L.F.
      • Dvorak A.M.
      ,
      • Risau W.
      ,
      • Ferrara N.
      , and
      • Leung D.W.
      • Cachianes G.
      • Kuang W.J.
      • Goeddel D.V.
      • Ferrara N.
      ). In addition, VPF/VEGF is the only angiogenic cytokine identified thus far that renders microvessels hyperpermeable to circulating macromolecules, a characteristic feature of angiogenic blood vessels (
      • Senger D.R.
      • Perruzzi C.A.
      • Feder J.
      • Dvorak H.F.
      ,
      • Dvorak H.F.
      ,
      • Dvorak H.F.
      • Orenstein N.S.
      • Carvalho A.C.
      • Churchill W.H.
      • Dvorak A.M.
      • Galli S.J.
      • Feder J.
      • Bitzer A.M.
      • Rypysc J.
      • Giovinco P.
      ,
      • Dvorak H.F.
      • Senger D.R.
      • Dvorak A.M.
      ,
      • Senger D.R.
      • Galli S.J.
      • Dvorak A.M.
      • Perruzzi C.A.
      • Harvey V.S.
      • Dvorak H.F.
      ).
      Most of the biological activities of VPF/VEGF are thought to be mediated by its interaction with two high-affinity receptor tyrosine kinases, Flt-1 (VEGF receptor-1) and KDR (VEGF receptor-2; flk-1 in mice) (
      • Fong G.H.
      • Rossant J.
      • Gertsenstein M.
      • Breitman M.L.
      ,
      • Millauer B.
      • Wizigmann-Voos S.
      • Schnurch H.
      • Martinez R.
      • Meller N.P.H.
      • Risau W.
      • Ullrich A.
      ,
      • Quinn T.P.
      • Peters K.G.
      • De Vries C.
      • Ferrara N.
      • Williams L.T.
      ,
      • Shalaby F.
      • Ho J.
      • Stanford W.L.
      • Fischer K.D.
      • Schuh A.C.
      • Schwartz L.
      • Bernstein A.
      • Rossant J.
      ,
      • Terman B.
      • Dougher-Vermazen M.
      • Carrion M.
      • Dimitrov D.
      • Armellino D.
      • Gospodarowicz D.
      • Bohlen P.
      ). A third receptor, neuropilin, has been recognized, but little is known about its capacity to initiate endothelial cell signaling (
      • Soker S.
      • Takashima S.
      • Miao H.Q.
      • Neufeld G.
      • Klagsbrun M.
      ,
      • Gagnon M.L.
      • Bielenberg D.R.
      • Gechtman Z.
      • Miao H.Q.
      • Takashima S.
      • Soker S.
      • Klagsbrun M.
      ). Both Flt-1 and KDR are selectively expressed on vascular endothelium but bind VPF/VEGF with different affinities; thus, Flt-1 binds VPF/VEGF with a K d of ∼10 pm, whereas the K d for KDR binding is 400–900 pm (
      • Joukov V.
      • Sorsa T.
      • Kumar V.
      • Jeltsch M.
      • Claesson-Welsh L.
      • Cao Y.
      • Saksela O.
      • Kalkkinen N.
      • Alitalo K.
      ,
      • Waltenberger J.
      • Claesson-Welsh L.
      • Siegbahn A.
      • Shibuya M.
      • Heldin C.H.
      ). Both receptors possess tyrosine kinase domains, potential ATP-binding sites, and long kinase insert regions that contain phosphorylation sites with binding capacity for different signaling molecules. Flt-1 and KDR also have different ligand specificities. Thus, Flt-1 interacts with VPF/VEGF (also known as VEGF-A) and with two other members of the VPF/VEGF family, PlGF and VEGF-B. KDR, on the other hand, interacts with VEGF-C and VEGF-D, in addition to VPF/VEGF (
      • Petrova T.V.
      • Makinen T.
      • Alitalo K.
      ). Both Flt-1 and KDR are essential for normal vascular development (
      • Fong G.H.
      • Rossant J.
      • Gertsenstein M.
      • Breitman M.L.
      ,
      • Shalaby F.
      • Ho J.
      • Stanford W.L.
      • Fischer K.D.
      • Schuh A.C.
      • Schwartz L.
      • Bernstein A.
      • Rossant J.
      ).
      At present, the signaling cascades following VPF/VEGF interaction with cultured endothelial cells (ECs) are only partially understood but are known to involve a series of protein phosphorylations, beginning with receptor phosphorylation and subsequently with tyrosine phosphorylation of phospholipase C-γ (PLC-γ) and phosphatidylinositol 3-kinase (PI-3K) (for review, see Refs.
      • Petrova T.V.
      • Makinen T.
      • Alitalo K.
      and
      • English J.
      • Pearson G.
      • Wilsbacher J.
      • Swantek J.
      • Karandikar M.
      • Xu S.
      • Cobb M.H.
      ). Like other endothelial cell agonists such as thrombin and histamine, VPF/VEGF activates protein kinase C, increases [Ca2+]i, and stimulates inositol-1,4,5-triphosphate accumulation (
      • Brock T.A.
      • Dvorak H.F.
      • Senger D.R.
      ).
      Because most cultured endothelial cells express both Flt-1 and KDR, it has been difficult to delineate the distinct signaling pathways and biological functions triggered individually by each, and much of our current information comes from studies with cell lines, particularly porcine aortic endothelial (PAE) cells, which do not normally express detectable levels of either KDR or Flt-1 and do not respond to VPF/VEGF. However, when PAE cells were engineered to express KDR, VPF/VEGF induced striking changes in cell morphology and behavior including actin reorganization, membrane ruffling, cell division, and chemotaxis (
      • Waltenberger J.
      • Claesson-Welsh L.
      • Siegbahn A.
      • Shibuya M.
      • Heldin C.H.
      ). Less is known about the consequences of VPF/VEGF interaction with Flt-1. In PAE cells engineered to express Flt-1 but not KDR, VPF/VEGF stimulated tissue factor expression but not cell migration or proliferation (
      • Waltenberger J.
      • Claesson-Welsh L.
      • Siegbahn A.
      • Shibuya M.
      • Heldin C.H.
      ). Most recently, in PAE cells overexpressing KDR and Flt-1, it was reported that Flt-1 repressed KDR-mediated proliferation (
      • Rahimi N.
      • Dayanir V.
      • Lashkari K.
      ). By inhibiting the expression of either receptor with antisense oligonucleotides, it was also found that KDR was required for HUVEC proliferation (
      • Bernatchez P.N.
      • Soker S.
      • Sirois M.G.
      ). A similar result was also obtained with KDR- or Flt-1-specific antibodies that blocked receptor interaction with VPF/VEGF (
      • Kanno S.
      • Oda N.
      • Abe M.
      • Terai Y.
      • Ito M.
      • Shitara K.
      • Tabayashi K.
      • Shibuya M.
      • Sato Y.
      ) and VPF/VEGF mutants that specifically bind to KDR (
      • Gille H.
      • Kowalski J.
      • Li B.
      • LeCouter J.
      • Moffat B.
      • Zioncheck T.F.
      • Pelletier N.
      • Ferrara N.
      ).
      Available data suggest that KDR and Flt-1 have different and perhaps complementary roles in vasculogenesis and angiogenesis. However, many of the data have been obtained from immortalized cell lines that may differ significantly in behavior from early-passage cells derived from primary endothelial cultures. Therefore, to elucidate the respective roles of KDR and Flt-1 in early-passage HUVECs expressing both KDR and Flt-1, we engineered chimeric constructs of both receptors, replacing the extracellular domain of each with the extracellular domain of epidermal growth factor receptor (EGFR). We used retroviral vectors to express these chimeric receptors in HUVECs that expressed both KDR and Flt-1 but not EGFR. Using this system, we demonstrated that HUVEC proliferation and migration were mediated exclusively through the KDR signaling pathway, a conclusion consistent with that of others (
      • Waltenberger J.
      • Claesson-Welsh L.
      • Siegbahn A.
      • Shibuya M.
      • Heldin C.H.
      ,
      • Bernatchez P.N.
      • Soker S.
      • Sirois M.G.
      ,
      • Kanno S.
      • Oda N.
      • Abe M.
      • Terai Y.
      • Ito M.
      • Shitara K.
      • Tabayashi K.
      • Shibuya M.
      • Sato Y.
      ,
      • Gille H.
      • Kowalski J.
      • Li B.
      • LeCouter J.
      • Moffat B.
      • Zioncheck T.F.
      • Pelletier N.
      • Ferrara N.
      ). Interestingly, however, Flt-1 activation mediated a distinctive inhibitory signaling pathway through PI-3K that down-regulated the cell proliferation pathway triggered by KDR.

      DISCUSSION

      VPF/VEGF is an important, multifunctional angiogenic cyokine that exerts a variety of biological activities on vascular endothelium. These include induction of microvascular hyperpermeability, stimulation of proliferation and migration, significant reprogramming of gene expression, endothelial cell survival, and prevention of senescence (for review, see Refs.
      • Dvorak H.F.
      • Nagy J.A.
      • Feng D.
      • Brown L.F.
      • Dvorak A.M.
      ,
      • Risau W.
      ,
      • Ferrara N.
      , and
      • Petrova T.V.
      • Makinen T.
      • Alitalo K.
      ). All of these functions are thought to be mediated by two receptor tyrosine kinases, KDR and Flt-1, that are selectively expressed on vascular endothelium and up-regulated at sites of VPF/VEGF overexpression as in tumors, healing wounds, chronic inflammation, etc. (for review, see Ref.
      • Risau W.
      ). Because both receptors are expressed on vascular endothelium, it has been difficult to define the respective role of each in mediating the various signaling events and biological activities induced in endothelium by VPF/VEGF. Therefore, current information has been gleaned largely from studies with a cell line, PAE, that does not express either receptor unless engineered to do so (
      • Joukov V.
      • Sorsa T.
      • Kumar V.
      • Jeltsch M.
      • Claesson-Welsh L.
      • Cao Y.
      • Saksela O.
      • Kalkkinen N.
      • Alitalo K.
      ,
      • Waltenberger J.
      • Claesson-Welsh L.
      • Siegbahn A.
      • Shibuya M.
      • Heldin C.H.
      ); from studies with PlGF, a ligand that binds Flt-1 but not KDR (
      • Risau W.
      ); from the use of a Flt-1-specific antibody (
      • Kanno S.
      • Oda N.
      • Abe M.
      • Terai Y.
      • Ito M.
      • Shitara K.
      • Tabayashi K.
      • Shibuya M.
      • Sato Y.
      ); from studies with antisense oligonucleotides that block Flt-1 expression (
      • Bernatchez P.N.
      • Soker S.
      • Sirois M.G.
      ); and from VPF/VEGF mutants that specifically bind to Flt-1 (
      • Gille H.
      • Kowalski J.
      • Li B.
      • LeCouter J.
      • Moffat B.
      • Zioncheck T.F.
      • Pelletier N.
      • Ferrara N.
      ).
      We sought to delineate the respective roles of KDR and Flt-1 in early-passage endothelial cells in which both receptors remained intact and functional. To that end, we engineered chimeric receptors, fusing the extracellular domain of the EGF receptor with the transmembrane and intracellular domains of either KDR or Flt-1. We transduced these constructs (or, as a control, LacZ) into HUVECs with a retroviral vector. This approach was feasible because, under the conditions of our experiments (≤80% confluence), HUVECs did not express the EGFR and did not respond to EGF. This strategy was also attractive because endogenous KDR and Flt-1 persisted in HUVECs transduced with either one or both of the chimeric receptors; therefore, the signaling and biological responses induced by VPF/VEGF or EGF could be compared in the same cells.
      Transducing our chimeric receptor into HUVECs, we found that KDR, but not Flt-1, mediated VPF/VEGF-induced EC proliferation and migration, extending earlier work that had reached the same conclusion using different approaches. Waltenberger et al. (
      • Waltenberger J.
      • Claesson-Welsh L.
      • Siegbahn A.
      • Shibuya M.
      • Heldin C.H.
      ) stably transfected KDR and Flt-1 into PAE, an endothelial cell line that uniquely fails to express either KDR or Flt-1. KDR-transfected PAE responded to VPF/VEGF stimulation with strong mitogenic and chemotactic responses, in contrast to Flt-1-transfected PAE, which failed to proliferate or migrate in response to VPF/VEGF. Bernatchezet al. (
      • Bernatchez P.N.
      • Soker S.
      • Sirois M.G.
      ) made use of antisense oligomers to demonstrate that repression of Flk-1/KDR, but not of Flt-1 expression, inhibited VPF/VEGF-mediated proliferation and migration in bovine aortic ECs. More recently, Gille et al. (
      • Gille H.
      • Kowalski J.
      • Li B.
      • LeCouter J.
      • Moffat B.
      • Zioncheck T.F.
      • Pelletier N.
      • Ferrara N.
      ) used a VPF/VEGF mutant that reacted selectively with KDR to determine that activated KDR can stimulate HUVEC proliferation and migration. Therefore, taken together, similar results have been obtained using different experimental approaches and three different types of endothelium, firmly establishing and generalizing the finding that KDR, but not Flt-1, mediates VPF/VEGF-induced EC proliferation and migration.
      The function of Flt-1 has been much less clear. One proposal has been that Flt-1 is a decoy receptor rather than a signal transducer because Flt-1 kinase domain null mice develop normally (
      • Hiratsuka S.
      • Minowa O.
      • Kuno J.
      • Noda T.
      • Shibuya M.
      ), in contrast to Flt-1 knockout mice, which are embryonic lethal (
      • Petrova T.V.
      • Makinen T.
      • Alitalo K.
      ). It was also reported that VPF/VEGF failed to stimulate Flt-1 tyrosine phosphorylation in PAE cells engineered to overexpress Flt-1 and did so poorly in HUVECs (
      • Waltenberger J.
      • Claesson-Welsh L.
      • Siegbahn A.
      • Shibuya M.
      • Heldin C.H.
      ,
      • de Vries C.
      • Escobedo J.A.
      • Ueno H.
      • Houck K.
      • Ferrara N.
      • Williams L.T.
      ). However, the expression level of Flt-1 in HUVECs is only about one-tenth that of KDR (
      • Joukov V.
      • Sorsa T.
      • Kumar V.
      • Jeltsch M.
      • Claesson-Welsh L.
      • Cao Y.
      • Saksela O.
      • Kalkkinen N.
      • Alitalo K.
      ,
      • Waltenberger J.
      • Claesson-Welsh L.
      • Siegbahn A.
      • Shibuya M.
      • Heldin C.H.
      ), and therefore measurement of Flt-1 phosphorylation is difficult and may have been underestimated. To take account of the large differences in receptor expression, we used three times as many HUVECs for measurement of phosphorylation of Flt-1 as for KDR and had no difficulty in demonstrating phosphorylation of both receptors (Fig.1 d, A).
      We then used our chimeric receptors to elucidate the distinct signaling pathways mediated by KDR and Flt-1. After transduction into HUVECs, both EGDR and EGLT were expressed at equivalent levels, and both underwent equivalent phosphorylation in response to EGF. This result stands in contrast to that reported by the study of Rahimi et al. (
      • Rahimi N.
      • Dayanir V.
      • Lashkari K.
      ), in which a chimeric receptor generated by fusing the N-terminal domain of colony-stimulating factor-1 receptor to Flt-1 showed no significant tyrosine phosphorylation when it was overexpressed in PAE cells and stimulated with colony-stimulating factor-1. It must be remembered, however, that PAE is a cell line with atypical properties (e.g., it doesn't normally express KDR or Flt-1) and therefore may be less representative of vascular endothelium than early-passage HUVECs.
      Of particular interest was the finding that EGDR/HUVEC cells responded to EGF with a proliferative response that was ∼50% greater than that induced by VPF/VEGF. Moreover, when HUVECs were co-transduced with EGLT and EGDR, the proliferation response to EGF was reduced to levels induced by VPF/VEGF in parental HUVECs. EGLT dose-response experiments further supported the inhibitory effect of Flt-1/EGLT on KDR/EGDR-mediated HUVEC proliferation. They also correlated well with the situation in parental HUVECs, where the ratio of KDR:Flt-1 expression is ∼ 10:1, but the binding affinity of VPF/VEGF to Flt-1 is 10 times higher than that to KDR. In our dose-response experiment, equivalent amounts of EGLT and EGDR were required to achieve the same inhibitory effect seen in parental HUVECs because the binding affinity of EGF to both EGDR and EGLT is similar.
      Several possible explanations have been proposed for the inhibitory effect of Flt-1 on KDR signaling. The possibility that Flt-1 sequestered VPF/VEGF, making it unavailable to KDR, as suggested by Hiratsuka et al. (
      • Hiratsuka S.
      • Minowa O.
      • Kuno J.
      • Noda T.
      • Shibuya M.
      ) with Flt-1 kinase domain null mice and by Rahimi et al. after activation of the colony-stimulating factor-1/Flt-1 chimera in PAE (
      • Rahimi N.
      • Dayanir V.
      • Lashkari K.
      ), was excluded by the finding that Flt-1/EGLT inhibited EC proliferation but had no inhibitory effect on migration. Moreover, pretreatment of EGLT/HUVEC cells with EGF before the addition of VPF/VEGF led to the same inhibitory effect on proliferation observed in parental HUVECs treated with VPF/VEGF alone. Also, co-transduction with EGLT, even in amounts equivalent to those of EGDR that greatly reduced proliferation, had no inhibitory effect on migration. Taken together, these results indicated that Flt-1/EGLT most likely acted by activating an inhibitory signaling pathway that down-modulated KDR/EGDR-mediated HUVEC proliferation.
      To extend these findings, we made use of inhibitors of specific metabolic pathways. The PLC inhibitor U73122 completely inhibited VPF/VEGF-induced HUVEC proliferation and migration, whereas the PI-3K inhibitor wortmannin increased VPF/VEGF-induced proliferation but did not affect migration (Fig. 4). This last finding seems to contradict the observation of Thakker et al. (
      • Thakker G.D.
      • Hajjar D.P.
      • Muller W.A.
      • Rosengart T.K.
      ) that PI-3K was required for VPF/VEGF stimulation of HUVEC proliferation; however, the author did not include certain controls (wortmannin-treated and VPF/VEGF-untreated cells) that turned out to be critical. HUVEC proliferation experiments with wortmannin involve two distinct variables, cell survival after serum starvation and VPF/VEGF stimulation, both of which must be controlled. Like Thakker et al. (
      • Thakker G.D.
      • Hajjar D.P.
      • Muller W.A.
      • Rosengart T.K.
      ), we found that wortmannin significantly reduced the survival of serum-starved HUVECs over a 24-h period when present at a concentration of 10 µm but had a much less deleterious effect under the conditions of our proliferation assay (≤100 nm in cells serum-starved for 24 h). Therefore, it was necessary to use wortmannin-treated HUVECs as a control to determine the effect of wortmannin on VPF/VEGF-induced activation.
      Because Flt-1/EGLT mediated an inhibitory effect on KDR/EGDR-mediated HUVEC proliferation (Figs. 2 a and 3), and wortmannin removed an inhibitory effect in VPF/VEGF-stimulated HUVECs that did not exist in EGF-stimulated EGDR/HUVEC proliferation (Fig. 4, a andb), we considered the possibility that PI-3K was involved in the Flt-1/EGLT-mediated inhibitory pathway. Our results with p85(DN) and the constitutively active PI-3K, p110CAAX, confirmed this hypothesis (Fig. 5, b and c). Treatment with VPF/VEGF induced more extensive proliferation (∼3-fold) in p85(DN)-overexpressing HUVECs than in LacZ-transduced HUVECs (∼2-fold). On the other hand, overexpression of p110CAAX did not inhibit proliferation further. Whereas p85(DN) had no effect on EGF-induced proliferation of EGDR/HUVEC, p110CAAX inhibited proliferation in these cells by 50% (Fig. 5 c), a finding consistent with the inhibitory effect of Flt-1/EGLT (Fig. 3). p85(DN) also removed the inhibitory effect of EGLT on EGDR-mediated proliferation induced with EGF (Fig. 5 c). However, neither p85(DN) nor p110CAAX had any effect on HUVEC migration induced by VPF/VEGF. Interestingly, Flt-1 was found to interact with the p85 subunit of PI-3K, whereas no association was observed between KDR and p85 (Fig. 5 a). These results corroborated the findings of Cunningham et al. (
      • Cunningham S.A.
      • Waxham M.N.
      • Arrate P.M.
      • Brock T.A.
      ), which suggested an association between Flt-1 and p85 using the yeast two-hybrid system. Furthermore, PI-3K (p110α) knockout mice (Pik3cadel/del) demonstrated extravasation blood, suggestive of defective angiogenesis. These embryos are developmentally retarded and die between embryonic day 9.5 and embryonic day 10.5 (
      • Bi L.
      • Okabe I.
      • Bernard D.J.
      • Wynshaw-Boris A.
      • Nussbaum R.L.
      ). The similarities of the Pik3cadel/del phenotype to that of Flt-1 knockout mice are consistent with our results that PI-3K is involved in Flt-1 signaling pathway.
      Intracellular Ca2+ mobilization is an important consequence of HUVEC stimulation by VPF/VEGF. We have now demonstrated that the Ca2+ response is mediated by KDR and not by Flt-1 signaling, a finding consistent with that obtained using an antisense approach (
      • Bernatchez P.N.
      • Soker S.
      • Sirois M.G.
      ). However, our results also indicated that cells transduced with EGLT or p110CAAX exerted an inhibitory effect on EGDR or KDR-mediated intracellular Ca2+ mobilization (Fig. 6,b and e), an effect also observed in parental HUVECs. However, inhibition could be relieved by the PI-3K inhibitors wortmannin and LY294002, both of which increased both the slope of Ca2+ mobilization and the overall magnitude (Fig. 6,c and d). We did not detect any difference in EGDR phosphorylation in cells that were or were not transduced with EGLT, and we did not detect any difference in KDR phosphorylation in parental HUVECs that were or were not pretreated with wortmannin. Therefore, we conclude that the Flt-1/EGLT pathway intersects KDR signaling and inhibits KDR-mediated HUVEC proliferation at a step after receptor phosphorylation and at or before the step at which KDR mediates intracellular Ca2+ mobilization.
      Finally, to define the domain(s) of Flt-1 responsible for its antiproliferative activity, we engineered several EGLT mutants, EGLT(793stop), EGLT(824stop), and EGLT(Y794F). EGLT(824stop) had an inhibitory effect on KDR-mediated cell proliferation similar to that of full-length EGLT, whereas EGLT(793stop) and EGLT(Y794F) completely lost this inhibitory activity. Tyrosine 794 may reside in the SH2 domain of the juxta-membrane portion of Flt-1 and possibly interacts with a downstream signaling molecule to inhibit cell proliferation. In fltkinase−/− knockout mice, Flt-1 was truncated at amino acid 785 but also introduced a six-amino acid sequence from the following intron. In this six-amino acid sequence, there is a Gln residue, which, like tyrosine, often has an important role in protein function. On the other hand, tyrosine 794 is also six amino acids away from the three serine “repressor” residues that have been described by others (
      • Gille H.
      • Kowalski J., Yu, L.
      • Chen H.
      • Pisabarro M.T.
      • Davis-Smyth T.
      • Ferrara N.
      ). One explanation of the difference between our results and those with fltkinase−/− mice is that this 6-amino acid sequence may introduce an unexpected function. Because Flt-1 does not inhibit KDR-mediated migratory signaling, our data fail to support the idea that Flt-1 behaves as a decoy receptor but do support the hypothesis that its inhibitory function reflects a negative signaling event.
      In summary, we have engineered chimeric receptors to distinguish the signaling events triggered by KDR and Flt-1 in early-passage HUVECs. These tools have allowed us to identify a number of downstream signaling pathways that are stimulated by activation of KDR and Flt-1. KDR-mediated proliferation and migration involve activation of PLC, whereas Flt-1 was found to exert an inhibitory effect on HUVEC proliferation, but not on migration, through the PI-3K pathway. The Flt-1-mediated antiproliferative pathway acts after KDR phosphorylation but at or before KDR-mediated intracellular Ca2+mobilization. This study thus represents the first direct analysis of Flt-1 and KDR function in early-passage ECs and has demonstrated cross-talk between the pathways mediated by these two receptors. However, neither the KDR nor Flt-1 pathways are fully defined, and further work is needed to demonstrate the complete complement of signaling steps and pathway regulators that govern such other VPF/VEGF-mediated effects on ECs as increased microvascular permeability, gene expression reprogramming, survival, and prevention of senescence.

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