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Vascular Endothelial Growth Factor Induces Expression of Connective Tissue Growth Factor via KDR, Flt1, and Phosphatidylinositol 3-Kinase-Akt-dependent Pathways in Retinal Vascular Cells*

Open AccessPublished:December 29, 2000DOI:https://doi.org/10.1074/jbc.M006509200
      Fibroblastic proliferation accompanies many angiogenesis-related retinal and systemic diseases. Since connective tissue growth factor (CTGF) is a potent mitogen for fibrosis, extracellular matrix production, and angiogenesis, we have studied the effects and mechanism by which vascular endothelial growth factor (VEGF) regulates CTGF gene expression in retinal capillary cells. In our study, VEGF increased CTGF mRNA levels in a time- and concentration-dependent manner in bovine retinal endothelial cells and pericytes, without the need of new protein synthesis and without altering mRNA stability. VEGF activated the tyrosine receptor phosphorylation of KDR and Flt1 and increased the binding of phosphatidylinositol 3-kinase (PI3-kinase) p85 subunit to KDR and Flt1, both of which could mediate CTGF gene induction. VEGF-induced CTGF expression was mediated primarily by PI3-kinase activation, whereas PKC and ERK pathways made only minimal contributions. Furthermore, overexpression of constitutive active Akt was sufficient to induce CTGF gene expression, and inhibition of Akt activation by overexpressing dominant negative mutant of Akt abolished the VEGF-induced CTGF expression. These data suggest that VEGF can increase CTGF gene expression in bovine retinal capillary cells via KDR or Flt receptors and the activation of PI3-kinase-Akt pathway independently of PKC or Ras-ERK pathway, possibly inducing the fibrosis observed in retinal neovascular diseases.
      VEGF
      vascular endothelial growth factor
      CTGF
      connective tissue growth factor
      Flt
      fms-like tyrosine kinase
      KDR/Flk1
      fetal liver kinase/kinase domain-containing receptor
      VEGFR
      vascular endothelial growth factor receptor
      PKB
      protein kinase B
      MAPK
      mitogen-activated protein kinase
      ERK
      extracellular signal-regulated kinase
      kb
      kilobase pair
      PKC
      protein kinase C
      PI
      phosphatidylinositol
      TGF-β
      transforming growth factor-β
      BREC
      bovine retinal endothelial cells
      PlGF
      placenta growth factor
      BRPC
      bovine retinal pericytes
      PCR
      polymerase chain reaction
      CAAkt
      constitutive active Akt
      DNAkt
      dominant negative Akt
      DNRas
      dominant negative K-Ras
      DNERK
      dominant negative extracellular signal-regulated kinase
      DNPKCζ
      dominant negative PKCζ
      Angiogenesis and fibrosis are key components in development, growth, wound healing, and regeneration (
      • Klagsbrun M.
      • D'Amore P.A.
      ). In addition, these processes commonly occur together in many disease states where neovascularization is believed to initiate the pathological cascade. Some of these diseases are proliferative diabetic retinopathy (
      • Aiello L.P.
      • Gardner T.W.
      • King G.L.
      • Blankenship G.
      • Cavallerano J.D.
      • Ferris III, F.L.
      • Klein R.
      ), rheumatoid arthritis (
      • Firestein G.S.
      ), and age-related macular degeneration (
      • Lopez P.F.
      • Sippy B.D.
      • Lambert H.M.
      • Thach A.B.
      • Hinton D.R.
      ). Thus, it is possible that the factors that regulate angiogenesis may also induce factors that stimulate extracellular matrix production and fibrosis. Accordingly, we have studied the ability of vascular endothelial growth factor (VEGF),1 an established angiogenic factor, to regulate the expression of connective tissue growth factor (CTGF), a growth factor with known actions on fibroblast proliferation, matrix production, and associated with fibrotic disorders.
      VEGF is expressed as a family of peptides of 121, 145, 165, 189, and 206 amino acid residues (
      • Neufeld G.
      • Cohen T.
      • Gengrinovitch S.
      • Poltorak Z.
      ). Its expression is induced by hypoxia (
      • Shweiki D.
      • Itin A.
      • Soffer D.
      • Keshet E.
      ) and is essential in the vasculogenesis process during development (
      • Ferrara N.
      • Carver-Moore K.
      • Chen H.
      • Dowd M.
      • Lu L.
      • O'Shea K.S.
      • Powell-Braxton L.
      • Hillan K.J.
      • Moore M.W.
      ). Several receptors have been shown to mediate the action of VEGF, and most of them belong to the tyrosine kinase receptor family (
      • Petrova T.V.
      • Makinen T.
      • Alitalo K.
      ). Upon the binding of VEGF to its receptors, multiple signaling cascades are activated, including the tyrosine phosphorylation of phospholipase Cγ, elevation of intracellular calcium and diacylglycerol, activation of protein kinase C (PKC), and extracellular signal-regulated kinase (MAPK/ERK) for endothelial cell proliferation (
      • Wu L.W.
      • Mayo L.D.
      • Dunbar J.D.
      • Kessler K.M.
      • Baerwald M.R.
      • Jaffe E.A.
      • Wang D.
      • 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.
      ,
      • Takahashi T.
      • Ueno H.
      • Shibuya M.
      ,
      • He H.
      • Venema V.J.
      • Gu X.
      • Venema R.C.
      • Marrero M.B.
      • Caldwell R.B.
      ). In addition, VEGF also stimulates activation of phosphatidylinositol (PI) 3-kinase leading to Akt/PKB activation and possibly enhancing endothelial cell survival (
      • Fujio Y.
      • Walsh K.
      ,
      • Gerber H.P.
      • McMurtrey A.
      • Kowalski J.
      • Yan M.
      • Keyt B.A.
      • Dixit V.
      • Ferrara N.
      ,
      • Thakker G.D.
      • Hajjar D.P.
      • Muller W.A.
      • Rosengart T.K.
      ). However, in non-endothelial cells such as capillary pericytes that predominantly express Flt1 receptor, the action of VEGF is poorly understood.
      Connective tissue growth factor (CTGF), a member of CCN family (CYR61, CTGF, and NOV) (
      • Bork P.
      ,
      • Lau L.F.
      • Lam S.C.
      ), is a potent and ubiquitously expressed growth factor that has been shown to play a unique role in fibroblast proliferation, cell adhesion, and the stimulation of extracellular matrix production (
      • Frazier K.
      • Williams S.
      • Kothapalli D.
      • Klapper H.
      • Grotendorst G.R.
      ,
      • Kireeva M.L.
      • Latinkic B.V.
      • Kolesnikova T.V.
      • Chen C.C.
      • Yang G.P.
      • Abler A.S.
      • Lau L.F.
      ). The 38-kDa protein was originally identified in conditioned medium from human umbilical vein endothelial cells (
      • Bradham D.M.
      • Igarashi A.
      • Potter R.L.
      • Grotendorst G.R.
      ), and the expression was shown to be selectively stimulated by transforming growth factor-β (TGF-β) in cultured fibroblasts (
      • Igarashi A.
      • Okochi H.
      • Bradham D.M.
      • Grotendorst G.R.
      ). Due to its mitogenic action on fibroblasts and its ability to induce the expression of the extracellular matrix molecules, collagen type I, fibronectin, and integrin α5 (
      • Frazier K.
      • Williams S.
      • Kothapalli D.
      • Klapper H.
      • Grotendorst G.R.
      ), CTGF is supposed to play an important role in connective tissue cell proliferation and extracellular matrix deposition as one of the mediators of TGF-β (
      • Kothapalli D.
      • Frazier K.S.
      • Welply A.
      • Segarini P.R.
      • Grotendorst G.R.
      ). CTGF also seems to be an important player in the pathogenesis of various fibrotic disorders, since it was shown to be overexpressed in scleroderma, keloids, and other fibrotic skin disorders (
      • Igarashi A.
      • Nashiro K.
      • Kikuchi K.
      • Sato S.
      • Ihn H.
      • Fujimoto M.
      • Grotendorst G.R.
      • Takehara K.
      ), as well as in stromal rich mammary tumors (
      • Frazier K.S.
      • Grotendorst G.R.
      ), and in advanced atherosclerotic lesions (
      • Oemar B.S.
      • Werner A.
      • Garnier J.M.
      • Do D.D.
      • Godoy N.
      • Nauck M.
      • Marz W.
      • Rupp J.
      • Pech M.
      • Luscher T.F.
      ). Recently, the integrin αvβ3 has been reported to serve as a receptor on endothelial cells for CTGF-mediated endothelial cell adhesion, migration, and angiogenesis (
      • Babic A.M.
      • Chen C.C.
      • Lau L.F.
      ,
      • Shimo T.
      • Nakanishi T.
      • Nishida T.
      • Asano M.
      • Kanyama M.
      • Kuboki T.
      • Tamatani T.
      • Tezuka K.
      • Takemura M.
      • Matsumura T.
      • Takigawa M.
      ).
      Besides TGF-β, the expression of CTGF is reported to be regulated by dexamethasone in BALB/c 3T3 cells (
      • Dammeier J.
      • Beer H.D.
      • Brauchle M.
      • Werner S.
      ), high glucose in human mesangial cells (
      • Murphy M.
      • Godson C.
      • Cannon S.
      • Kato S.
      • Mackenzie H.S.
      • Martin F.
      • Brady H.R.
      ), kinin in human embryonic fibroblasts (
      • Ricupero D.A.
      • Romero J.R.
      • Rishikof D.C.
      • Goldstein R.H.
      ), factor VIIa, and thrombin in WI-38 fibroblasts (
      • Pendurthi U.R.
      • Allen K.E.
      • Ezban M.
      • Rao L.V.
      ), tumor necrosis factor α in human skin fibroblast (
      • Abraham D.J.
      • Shiwen X.
      • Black C.M.
      • Sa S.
      • Xu Y.
      • Leask A.
      ), and cAMP in bovine endothelial cells (
      • Boes M.
      • Dake B.L.
      • Booth B.A.
      • Erondu N.E.
      • Oh Y.
      • Hwa V.
      • Rosenfeld R.
      • Bar R.S.
      ). Since many of these cytokines are known to induce VEGF, it is possible that increased VEGF expression can regulate the expression of CTGF. In the present study, we have investigated the regulation of CTGF by VEGF in retinal endothelial cells and pericytes via the PI3-kinase and several other signaling pathways.

      DISCUSSION

      In this study, we have shown that VEGF can increase the mRNA expression of CTGF in a time- and concentration-dependent manner in both microvascular endothelial cells and contractile cells (capillary pericytes) possibly indicating that the effects of VEGF on CTGF expression may occur in all cells with VEGF receptors. This possibility is supported by the results showing that both Flt1 (VEGFR1) and KDR/Flk1 (VEGFR2) can mediate the increases in CTGF mRNA expression. The ability of Flt1 to induce increases in CTGF mRNA levels is demonstrated in the pericytes that have predominantly Flt1 receptors and where the expression of KDR/Flk1 receptors were not significantly high enough to be determined by Northern blot analysis as reported in a previous publication (
      • Takagi H.
      • King G.L.
      • Aiello L.P.
      ). In addition, PlGF, a Flt1 receptor-specific ligand (
      • Park J.E.
      • Chen H.H.
      • Winer J.
      • Houck K.A.
      • Ferrara N.
      ), was able to induce CTGF mRNA levels in BRPC but not in BREC, again supporting the postulate that VEGF can induce CTGF mRNA by activating through Flt1 in pericytes. The KDR/Flk1 receptors in the endothelial cells can also induce CTGF gene expression since KDR/Flk1 receptors are the predominant VEGF receptors in endothelial cells (
      • Millauer B.
      • Wizigmann-Voos S.
      • Schnurch H.
      • Martinez R.
      • Moller N.P.
      • Risau W.
      • Ullrich A.
      ), and PlGF was not effective in inducing CTGF mRNA expression in endothelial cells. Further studies will be necessary to determine whether other types of VEGF receptors, such as Flt4 (
      • Joukov V.
      • Pajusola K.
      • Kaipainen A.
      • Chilov D.
      • Lahtinen I.
      • Kukk E.
      • Saksela O.
      • Kalkkinen N.
      • Alitalo K.
      ) and neuropilin-1 (
      • Soker S.
      • Takashima S.
      • Miao H.Q.
      • Neufeld G.
      • Klagsbrun M.
      ), which are present in endothelial cells, can also induce CTGF expression.
      The VEGF dose-response curves for CTGF in both BRPC and BREC are similar and suggest that VEGF binds to high affinity receptors, consistent with the known Kd values of Flt1 and KDR/Flk1 at 10–100 pm (
      • Millauer B.
      • Wizigmann-Voos S.
      • Schnurch H.
      • Martinez R.
      • Moller N.P.
      • Risau W.
      • Ullrich A.
      ,
      • de-Vries C.
      • Escobedo J.A.
      • Ueno H.
      • Houck K.
      • Ferrara N.
      • Williams L.T.
      ). VEGF-induced CTGF mRNA is most likely due to an induction of transcription rather than altering the half-life of CTGF mRNA since the addition of VEGF failed to change the degradation rates of CTGF mRNA. The time course of the action of VEGF on CTGF (which required 6–9 h) suggests this is potentially a chronic action of VEGF. In addition, the time needed to achieve maximum effect is also consistent with the calculated mRNA half-life of CTGF mRNA of 2–4 h.
      From a biological perspective, the effects of VEGF on CTGF mRNA could potentially have important physiological impact for several reasons. First is that the increase in CTGF mRNA results in increased protein levels. Second, the VEGF concentration that was minimally active (0.25 ng/ml) can easily bind and activate a significant percentage of the VEGFR-1, -2 receptors (
      • Millauer B.
      • Wizigmann-Voos S.
      • Schnurch H.
      • Martinez R.
      • Moller N.P.
      • Risau W.
      • Ullrich A.
      ,
      • de-Vries C.
      • Escobedo J.A.
      • Ueno H.
      • Houck K.
      • Ferrara N.
      • Williams L.T.
      ). Third, this low level of VEGF may exist even in non-pathological states, suggesting that low levels of VEGF may have physiological actions on maintaining extracellular matrix production via the induction of CTGF. At 2.5–25 ng/ml VEGF which are encountered in hypoxic and angiogenic states (
      • Aiello L.P.
      • Avery R.L.
      • Arrigg P.G.
      • Keyt B.A.
      • Jampel H.D.
      • Shah S.T.
      • Pasquale L.R.
      • Thieme H.
      • Iwamoto M.A.
      • Park J.E.
      • Nguyen H.V.
      • Aiello L.M.
      • Ferrara N.
      • King G.L.
      ), the induction of CTGF expression by VEGF could potentially induce the fibrosis that frequently accompanies neovascularization. This possibility is supported further by the demonstration that the protein levels of CTGF expression were increased 10 h after the addition of VEGF that was consistent with the maximum increase in the mRNA levels at 6–9 h. In addition, the potency of VEGF on CTGF expression appeared to be similar to TGF-β1, suggesting that both of them could induce fibrosis associated with neovascularization.
      The activation of the endogenous tyrosine kinases of KDR/Flk1s can stimulate multiple signaling pathways, including Ras-ERK (
      • Guo D.
      • Jia Q.
      • Song H.Y.
      • Warren R.S.
      • Donner D.B.
      ), PI3-kinase-Akt (
      • Fujio Y.
      • Walsh K.
      ,
      • Gerber H.P.
      • McMurtrey A.
      • Kowalski J.
      • Yan M.
      • Keyt B.A.
      • Dixit V.
      • Ferrara N.
      ,
      • Thakker G.D.
      • Hajjar D.P.
      • Muller W.A.
      • Rosengart T.K.
      ), and phospholipase Cγ-PKC (
      • Wu L.W.
      • Mayo L.D.
      • Dunbar J.D.
      • Kessler K.M.
      • Baerwald M.R.
      • Jaffe E.A.
      • Wang D.
      • 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.
      ,
      • Takahashi T.
      • Ueno H.
      • Shibuya M.
      ) cascades. Much less is known regarding the regulation of Flt1 receptors. The results in BREC confirmed previous publications that VEGF can increase the tyrosine phosphorylation of KDR/Flk1 and its interaction with p85 subunit of PI3-kinase. In addition, VEGF also activates the ERK1/2 pathway confirming earlier reports from many laboratories, including ours. In contrast, VEGF was unable to activate ERK1/2 but stimulated the activation of PI3-kinase and phosphorylation of Akt in BRPC. These results have provided further direct evidence that the signaling pathways for Flt1 in vascular cells are different from those for KDR/Flk1. The lack of effect on ERK1/2 activation also supports the hypothesis that Flt1, unlike KDR/Flk1, is not involved in mitogenic actions (
      • Kanno S.
      • Oda N.
      • Abe M.
      • Terai Y.
      • Ito M.
      • Shitara K.
      • Tabayashi K.
      • Shibuya M.
      • Sato Y.
      ). Further studies will be needed to determine the structural differences responsible for the inability of VEGFR1 to engage the Ras-ERK pathway.
      The present report does provide the strong evidence that VEGF is inducing CTGF gene expression in both endothelial cells and pericytes via VEGFR1 or -R2 by the activation of PI3-kinase and Akt. This evidence includes the ability of wortmannin, a PI3-kinase inhibitor, to inhibit the effects of VEGFs in both cell types, whereas PD98059, a MAPK/ERK kinase inhibitor, and GF109203X, a general classical PKC and novel PKC inhibitor (1 μm) (
      • Park J.Y.
      • Takahara N.
      • Gabriele A.
      • Chou E.
      • Naruse K.
      • Suzuma K.
      • Yamauchi T.
      • Ha S.W.
      • Meier M.
      • Rhodes C.J.
      • King G.L.
      ,
      • Toullec D.
      • Pianetti P.
      • Coste H.
      • Bellevergue P.
      • Grand-Perret T.
      • Ajakane M.
      • Baudet V.
      • Boissin P.
      • Boursier E.
      • Loriolle F.
      • Dunhanel L.
      • Charon D.
      • Kirilovsky J.
      ), did not have significant actions. Adenovirus containing dominant negative mutants of p85 subunit of PI3-kinase or Akt inhibited the action of VEGFs, whereas overexpression of dominant negative mutants of Ras and ERK1 by adenovirus vectors did not inhibit CTGF mRNA expression. Conversely, the overexpression of constitutive active Akt increased CTGF mRNA expression by 2.5-fold. The molecular steps between Akt activation and the enhancement of CTGF gene expression in the nucleus remain unclear, although the PKCζ isoform is most likely not involved since the overexpression of either the wild type or dominant negative of PKCζ isoform did not alter the effects of VEGF on CTGF mRNA levels.
      The molecular processes between Akt phosphorylation and CTGF gene expression in the nucleus have not been studied. However, Pendurthiet al. (
      • Pendurthi U.R.
      • Allen K.E.
      • Ezban M.
      • Rao L.V.
      ) reported that factor VII and thrombin induced CTGF gene expression through a PI3-kinase-dependent pathway. TGF-β has been reported to increase the transcription rates of CTGF. A promoter element of CTGF, which is responsive to TGF-β stimulation, has been reported to be present between −162 and −128 nucleotides in the 5′ region (
      • Grotendorst G.R.
      • Okochi H.
      • Hayashi N.
      ). However, it is unlikely that the effects of VEGF on CTGF mRNA levels are mediated via the expression of TGF-β since the addition of cycloheximide did not change these effects.
      In summary, these results have provided the first evidence that VEGF can induce the expression of CTGF via both Flt1 and KDR/Flk1 by the selectively activated PI3-kinase-Akt pathway but independent of the Ras-ERK pathway. In addition, the spectrum of signaling pathways may be different between Flt1 and KDR/Flk1, possibly reflecting their physiological roles. Biologically, these results support the conclusion that VEGF, through its effects on CTGF expression, may have physiological roles such as the maintenance of capillary strength and wound healing via the extracellular matrix production. In disease states, VEGF-induced CTGF may cause the proliferation of fibrocellular components in retinal neovascular diseases such as proliferative diabetic retinopathy and age-related macular degeneration.

      ACKNOWLEDGEMENT

      We thank Dr. Edward P. Feener for suggestions during the preparation of this manuscript.

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