The clinical toxicity profile of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor (VEGFR) targeting angiogenesis inhibitors; A review
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Aim: To determine vascular endothelial growth factor C (VEGF-C) expression in retinal endothelial cells, its antiapoptotic potential and its putative role in diabetic retinopathy. Method: Cultured retinal endothelial cells and pericytes were exposed to tumour necrosis factor (TNF)α and VEGF-C expression determined by reverse transcriptase-polymerase chain reaction. Secreted VEGF-C protein levels in conditioned media from endothelial cells were examined by western blotting analysis. The ability of VEGF-C to prevent apoptosis induced by TNFα or hyperglycaemia in endothelial cells was assessed by flow cytometry. The expression of VEGF-C in diabetic retinopathy was studied by immunohistochemistry of retinal tissue. Result: VEGF-C was expressed by both vascular endothelial cells and pericytes. TNFα up regulated both VEGF-C and vascular endothelial growth factor receptor-2 (VEGFR)-2 expression in endothelial cells in a dose-dependent manner, but had no effect on VEGFR-3. Flow cytometry results showed that VEGF-C prevented endothelial cell apoptosis induced by TNFα and hyperglycaemia and that the antiapoptotic effect was mainly via VEGFR-2. In pericytes, the expression of VEGF-C mRNA remained stable on exogenous TNFα treatment. VEGF-C immunostaining was increased in retinal vessels in specimens with diabetes compared with retinal specimens from controls without diabetes. Conclusion: In retinal endothelial cells, TNFα stimulates the expression of VEGF-C, which in turn protects endothelial cells from apoptosis induced by TNFα or hyperglycaemia via VEGFR-2 and thus helps sustain retinal neovascularisation.
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Objective To determine the effect of PTK787 on the expression of vascular endothelial growth factor and vascular endothelial growth factor receptor-2 mRNA,and further discuss the role of PTK787 on anti-acute myeloid leukemia.Methods The acute myeloid leukemia model was established on 40 severe combined immunodeficiency mice by HL-60 cells transplantation.The mice were divided into five group randomly,the normal group,the sicken group,the treated group with 50mg/kg PTK787,the treated group with 100mg/kg PTK787,the treated group with 200mg/kg PTK787.Logarithmic phase cells were implanted into the sicken group and the treated group by celiac injection.The expression of vascular endothelial growth factor was detected by enzyme linked immunosorbent assay.The expression of vascular endothelial growth factor receptor-2 mRNA was detected by reverse transcription-polymerase chain reaction.Results(1)Expression of vascular endothelial growth factors and vascular endothelial growth factor receptor-2 mRNA were determined on all mice.(2)Compared with the normal group,the mRNA level of vascular endothelial growth factor and vascular endothelial growth factor receptor-2 in the sicken group was significantly and gradually increased with the course of disease.(3)Compared with the sicken group,the expression of vascular endothelial growth factor and vascular endothelial growth factor receptor-2 mRNA of treated group decreased obviously.Conclusion The anti-effect on acute myeloid leukemia of PTK787 is related with the decrease expression of vascular endothelial growth factor and vascular endothelial growth factor receptor-2.
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Systemic vascular endothelial growth factor inhibition, in combination with chemotherapy, improves the outcome of patients with metastatic cancer. Peripheral sensory neuropathies occurring in patients receiving both drugs are attributed to the chemotherapy. Here, we provide unprecedented evidence that vascular endothelial growth factor receptor inhibitors trigger a painful neuropathy and aggravate paclitaxel-induced neuropathies in mice. By using transgenic mice with altered neuronal vascular endothelial growth factor receptor expression, systemic inhibition of vascular endothelial growth factor receptors was shown to interfere with the endogenous neuroprotective activities of vascular endothelial growth factor on sensory neurons. In vitro, vascular endothelial growth factor prevented primary dorsal root ganglion cultures from paclitaxel-induced neuronal stress and cell death by counteracting mitochondrial membrane potential decreases and normalizing hyperacetylation of α-tubulin. In contrast, vascular endothelial growth factor receptor inhibitors exerted opposite effects. Intriguingly, vascular endothelial growth factor or vascular endothelial growth factor receptor inhibitors exerted their effects through a mechanism whereby Hdac6, through Hsp90, controls vascular endothelial growth factor receptor-2-mediated expression of the anti-apoptotic Bcl2. Our observations that systemic anti-vascular endothelial growth factor therapies interfere with the neuroprotective activities of vascular endothelial growth factor may have important implications for the application of anti-vascular endothelial growth factor therapies in cancer patients.
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Although the importance of the vascular endothelial growth factor (VEGF)/VEGF tyrosine kinase receptor (VEGFR) system in angiogenesis is well established, very little is known about the regulation of VEGFR expression in vascular endothelial cells. We have cloned partial cDNAs encoding bovine VEGFR-1 (flt) and -2 (flk-1) and used them to study VEGFR expression by bovine microvascular- and large vessel-derived endothelial cells. Both cell lines express flk-1, but not flt. Transforming growth factor β1 (TGF-β1) reduced the high affinity 125I-VEGF binding capacity of both cell types in a dose-dependent manner, with a 2.0-2.7-fold decrease at 1-10 ng/ml. Cross-linking experiments revealed a decrease in 125I-VEGF binding to a cell surface monomeric protein corresponding to Flk-1 on the basis of its affinity for VEGF, molecular mass (185-190 kDa), and apparent internalization after VEGF binding. Immunoprecipitation and Western blot experiments demonstrated a decrease in Flk-1 protein expression, and TGF-β1 reduced flk-1 mRNA levels in a dose-dependent manner. These results imply that TGF-β1 is a major regulator of the VEGF/Flk-1 signal transduction pathway in endothelial cells. Although the importance of the vascular endothelial growth factor (VEGF)/VEGF tyrosine kinase receptor (VEGFR) system in angiogenesis is well established, very little is known about the regulation of VEGFR expression in vascular endothelial cells. We have cloned partial cDNAs encoding bovine VEGFR-1 (flt) and -2 (flk-1) and used them to study VEGFR expression by bovine microvascular- and large vessel-derived endothelial cells. Both cell lines express flk-1, but not flt. Transforming growth factor β1 (TGF-β1) reduced the high affinity 125I-VEGF binding capacity of both cell types in a dose-dependent manner, with a 2.0-2.7-fold decrease at 1-10 ng/ml. Cross-linking experiments revealed a decrease in 125I-VEGF binding to a cell surface monomeric protein corresponding to Flk-1 on the basis of its affinity for VEGF, molecular mass (185-190 kDa), and apparent internalization after VEGF binding. Immunoprecipitation and Western blot experiments demonstrated a decrease in Flk-1 protein expression, and TGF-β1 reduced flk-1 mRNA levels in a dose-dependent manner. These results imply that TGF-β1 is a major regulator of the VEGF/Flk-1 signal transduction pathway in endothelial cells.
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Vascular endothelial growth factor (VEGF) plays a central role in tumor angiogenesis. It stimulates endothelial cell proliferation and vessel hyperpermeability, promotes cell migration, and inhibits apoptosis. All these actions of VEGF are mediated by receptor tyrosine kinase, vascular endothelial growth factor receptor (VEGFR). Selective targeting VEGFR signal transduction pathway may be proved to be useful in developing tumor angiogensis inhibitors.
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Decay-accelerating factor (DAF), a membrane-bound complement regulatory protein, is up-regulated on endothelial cells (ECs) following treatment with vascular endothelial growth factor (VEGF), providing enhanced protection from complement-mediated injury. We explored the signaling pathways involved in this response. Incubation of human umbilical vein ECs with VEGF induced a 3-fold increase in DAF expression. Inhibition by flk-1 kinase inhibitor SU1498 and failure of placental growth factor (PlGF) to up-regulate DAF confirmed the role of VEGF-R2. The response was also blocked by pretreatment with phospholipase C-γ (PLCγ) inhibitor U71322 and protein kinase C (PKC) antagonist GF109203X. In contrast, no effect was seen with nitric oxide synthase inhibitor NG-monomethyl-l-arginine (l-NMMA). Use of PKC agonists and isozyme-specific pseudosubstrate peptide antagonists suggested a role for PKCα and -ϵ in VEGF-mediated DAF up-regulation. This was confirmed by transfection of ECs with PKCα and -ϵ dominant-negative constructs, which in combination completely abrogated induction of DAF by VEGF. In contrast, LY290042, a phosphoinositide 3-kinase (PI3K) inhibitor, significantly augmented DAF expression, suggesting a negative regulatory role for phosphoinositide 3-kinase. The widely used immunosuppressive drug cyclosporin A (CsA) inhibited DAF induction by VEGF in a dose-dependent manner. The VEGF-induced DAF expression was functionally effective, significantly reducing complement-mediated EC lysis, and this cytoprotective effect was reversed by CsA. These data provide evidence for a VEGF-R2-, phospholipase C-γ-, and PKCα/ϵ-mediated cytoprotective pathway in ECs. This may represent an important mechanism for the maintenance of vascular integrity during chronic inflammation involving complement activation. Moreover, inhibition of this pathway by CsA may play a role in CsA-mediated vascular injury. Decay-accelerating factor (DAF), a membrane-bound complement regulatory protein, is up-regulated on endothelial cells (ECs) following treatment with vascular endothelial growth factor (VEGF), providing enhanced protection from complement-mediated injury. We explored the signaling pathways involved in this response. Incubation of human umbilical vein ECs with VEGF induced a 3-fold increase in DAF expression. Inhibition by flk-1 kinase inhibitor SU1498 and failure of placental growth factor (PlGF) to up-regulate DAF confirmed the role of VEGF-R2. The response was also blocked by pretreatment with phospholipase C-γ (PLCγ) inhibitor U71322 and protein kinase C (PKC) antagonist GF109203X. In contrast, no effect was seen with nitric oxide synthase inhibitor NG-monomethyl-l-arginine (l-NMMA). Use of PKC agonists and isozyme-specific pseudosubstrate peptide antagonists suggested a role for PKCα and -ϵ in VEGF-mediated DAF up-regulation. This was confirmed by transfection of ECs with PKCα and -ϵ dominant-negative constructs, which in combination completely abrogated induction of DAF by VEGF. In contrast, LY290042, a phosphoinositide 3-kinase (PI3K) inhibitor, significantly augmented DAF expression, suggesting a negative regulatory role for phosphoinositide 3-kinase. The widely used immunosuppressive drug cyclosporin A (CsA) inhibited DAF induction by VEGF in a dose-dependent manner. The VEGF-induced DAF expression was functionally effective, significantly reducing complement-mediated EC lysis, and this cytoprotective effect was reversed by CsA. These data provide evidence for a VEGF-R2-, phospholipase C-γ-, and PKCα/ϵ-mediated cytoprotective pathway in ECs. This may represent an important mechanism for the maintenance of vascular integrity during chronic inflammation involving complement activation. Moreover, inhibition of this pathway by CsA may play a role in CsA-mediated vascular injury. Vascular endothelial growth factor (VEGF) 1The abbreviations used are: VEGF, vascular endothelial growth factor; VEGF-R, VEGF receptor; NO, nitric oxide; eNOS, endothelial nitric oxide synthase; EC, endothelial cell; PLC, phospholipase C; PKC, protein kinase C; MAPK, mitogen-activated protein kinase; PI3K, phosphoinositide 3-kinase; CsA, cyclosporin A; l-NMMA, NG-monomethyl-l-arginine; myr-, myristoylated; HUVEC, human umbilical vein endothelial cells; PlGF, placental growth factor; DN, dominant-negative; MOI, multiplicity of infection; RFI, relative fluorescent intensity; DAF, decay-accelerating factor; Akt, antiapoptotic kinase; ERK, extracellular signal-regulated kinase; MFI, mean fluorescent intensity; mAb, monoclonal antibody; PI, propidium iodide; PBu, phorbol 12,13-dibutyrate; c-, classical; n-, novel; DETA, diethylenetriamine.1The abbreviations used are: VEGF, vascular endothelial growth factor; VEGF-R, VEGF receptor; NO, nitric oxide; eNOS, endothelial nitric oxide synthase; EC, endothelial cell; PLC, phospholipase C; PKC, protein kinase C; MAPK, mitogen-activated protein kinase; PI3K, phosphoinositide 3-kinase; CsA, cyclosporin A; l-NMMA, NG-monomethyl-l-arginine; myr-, myristoylated; HUVEC, human umbilical vein endothelial cells; PlGF, placental growth factor; DN, dominant-negative; MOI, multiplicity of infection; RFI, relative fluorescent intensity; DAF, decay-accelerating factor; Akt, antiapoptotic kinase; ERK, extracellular signal-regulated kinase; MFI, mean fluorescent intensity; mAb, monoclonal antibody; PI, propidium iodide; PBu, phorbol 12,13-dibutyrate; c-, classical; n-, novel; DETA, diethylenetriamine. represents a family of multifunctional glycoproteins centrally involved in vasculogenesis, angiogenesis, regulation of vascular permeability, and cytoprotection (1Ferrara N. Gerber H.-P. LeCouter J. Nat. Med. 2003; 9: 669-676Crossref PubMed Scopus (7777) Google Scholar). In addition, VEGF may act as a proinflammatory cytokine regulating cellular adhesion, molecule expression, and T lymphocyte trafficking (2Kim I. Moon S.-O. Hoon Kim S. Jin Kim H. Soon Koh Y. Young Koh G. J. Biol. Chem. 2001; 276: 7614-7620Abstract Full Text Full Text PDF PubMed Scopus (640) Google Scholar, 3Reinders M.E. Sho M. Izawa A. Wang P. Mukhopadhyay D. Koss K.E. Geehan C.S. Luster A.D. Sayegh M.H. Briscoe D.M. J. Clin. Investig. 2003; 112: 1655-1665Crossref PubMed Scopus (199) Google Scholar). The importance of VEGF in angiogenesis is exemplified by the embryonic lethality observed in VEGF and VEGF receptor-1 and -2 (VEGF-R1 and -2) gene-targeted mice (4Fong G.-H. Rossant J. Gertsenstein M. Breitman M.L. Nature. 1995; 376: 66-70Crossref PubMed Scopus (2204) Google Scholar, 5Shalaby F. Rossant J. Yamaguchi T.P. Gertsenstein M. Wu X.F. Breitman M.L. Schuh A.C. 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These mechanisms may contribute to the capacity of VEGF to facilitate vascular repair during nephritis and thrombotic microangiopathy (18Ostendorf T. Kunter U. Eitner F. Loos A. Regele H. Kerjaschki D. Henninger D.D. Janjic N. Floege J. J. Clin. Investig. 1999; 104: 913-923Crossref PubMed Scopus (277) Google Scholar). The actions of VEGF on vascular ECs are mediated via tyrosine-kinase signaling receptors VEGF-R1 (flt-1) and VEGF-R2 (flk-2/KDR), which are capable of interacting with a variety of downstream signaling pathways (1Ferrara N. Gerber H.-P. LeCouter J. Nat. Med. 2003; 9: 669-676Crossref PubMed Scopus (7777) Google Scholar, 19Zachary I. Am. J. Physiol. Cell Physiol. 2001; 280: C1375-C1386Crossref PubMed Google Scholar). The formation of a VEGF-R2·c-Src complex results in activation of phospholipase C-γ (PLCγ), leading to the generation of diacylglycerol and inositol 1,4,5-triphosphate (13He H. Venema V.J. Guo X.L. Venema R.C. Marrero M.B. Caldwell R.B. J. Biol. 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In addition, PI3K activity has been implicated in VEGF-induced up-regulation of eNOS and generation of NO, which may in turn exert antiapoptotic effects by inhibiting the cysteine protease activities of caspases (30Dimmeler S. Haendeler J. Nehls M. Zeiher A.M. J. Exp. Med. 1997; 185: 601-608Crossref PubMed Scopus (785) Google Scholar, 31Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2214) Google Scholar). Evidence is now emerging that endothelial injury, leading to widespread persistent global EC dysfunction, is common in systemic inflammatory diseases and predisposes a patient to premature atherosclerosis and cardiovascular mortality. Endothelial injury has also been linked to the obliterative vasculopathy and accelerated atherosclerosis seen following organ transplantation (32Hruban R.H. Beschorner W.E. Baumgartner W.A. Augustine S.M. Ren H. Reitz B.A. Hutchins G.M. Am. J. Pathol. 1990; 137: 871-882PubMed Google Scholar). Complement activation has an established role in the pathogenesis of inflammatory cardiovascular diseases including atherosclerosis, myocardial infarction, and accelerated arteriosclerosis following cardiac transplantation (33Torzewski J. Bowyer D.E. Waltenberger J. Fitzsimmons C. Atherosclerosis. 1997; 132: 131-138Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 34Qian Z. Hu W. Liu J. Sanfilippo F. Hruban R.H. Baldwin III, W.M. Transplantation. 2001; 72: 900-906Crossref PubMed Scopus (36) Google Scholar). Innate mechanisms for the control of complement activation on the cell surface include the membrane-bound regulatory proteins DAF (CD55), membrane cofactor protein (MCP, CD46), and CD59 (35Liszewski M.K. Farries T.C. Lublin D.M. Rooney I.A. Atkinson J.P. Adv. Immunol. 1996; 61: 201-283Crossref PubMed Google Scholar). DAF can be induced on the EC surface by treatment with C-reactive protein, proinflammatory cytokines, thrombin, VEGF, and basic fibroblast growth factor via distinct signaling pathways (16Mason J.C. Lidington E.A. Yarwood H. Lublin D.M. Haskard D.O. Arthritis Rheum. 2001; 44: 138-150Crossref PubMed Scopus (52) Google Scholar, 36Mason J.C. Yarwood H. Sugars K. Morgan B.P. Davies K.A. Haskard D.O. Blood. 1999; 94: 1673-1682Crossref PubMed Google Scholar, 37Lidington E.A. Haskard D.O. Mason J.C. Blood. 2000; 96: 2784-2792Crossref PubMed Google Scholar, 38Mason J.C. Lidington E.A. Ahmad S.R. Haskard D.O. Am. J. Physiol. Cell Physiol. 2002; 282: C578-C587Crossref PubMed Scopus (45) Google Scholar, 39Li S.-H. Szmitko P.E. Weisel R.D. Wang C.-H. Fedak P.W.M. Li R.-K. Mickle D.A.G. Verma S. Circulation. 2004; 109: 833-836Crossref PubMed Scopus (79) Google Scholar). DAF is a glycosylphosphatidylinositol-anchored glycoprotein that acts to prevent the formation and accelerate the decay of C3 and C5 convertases (40Lublin D.M. Atkinson J.P. Annu. Rev. Immunol. 1989; 7: 35-58Crossref PubMed Scopus (396) Google Scholar). The cytoprotective importance of DAF is revealed by the increased susceptibility of DAF-deficient mice to glomerular injury in models of glomerulonephritis (41Sogabe H. Nangaku M. Ishibashi Y. Wada T. Fujita T. Sun X. Miwa T. Madaio M.P. Song W.-C. J. Immunol. 2001; 167: 2791-2797Crossref PubMed Scopus (76) Google Scholar, 42Lin F. Emancipator S.N. Salant D.J. Medof M.E. Lab. Investig. 2002; 82: 563-569Crossref PubMed Scopus (56) Google Scholar). The demonstration that VEGF is fundamental to the maintenance of vascular homeostasis highlights the importance of precisely defining the mechanisms underlying these functions. Herein we describe in detail the VEGF-R2- and PKC-dependent signaling pathway by which VEGF induces DAF expression on the EC surface, so enhancing cytoprotection against complement-mediated injury. We demonstrate dependence upon activation of PKCα and PKCϵ, PI3K-mediated constraint, and inhibition of VEGF-mediated cytoprotection against complement by the immunosuppressive drug cyclosporin A (CsA). Monoclonal Antibodies and Other Reagents—Monoclonal antibodies 1H4 (anti-DAF) and RMAC8 (antiendoglin) were kind gifts from Dr. D. Lublin (St. Louis, MO) and Dr. A. d'Apice (Victoria, Australia), respectively. DETA NONOate was from Cayman Chemicals (Ann Arbor, MI), and CsA, thymeleatoxin, ingenol dibenzoate, Gö6976, GF109203X, and SU1498 were from Merck Biosciences Ltd. (Nottingham, UK). The PKCβ inhibitor LY379196 was a kind gift from Dr. K. Ways (Eli Lilly, Indianapolis, IN). NG-Monomethyl-l-arginine (l-NMMA), U-73122, and LY294002 were from BIOMOL (Plymouth Meeting, PA). Myristoylated (myr) PKC peptide inhibitors (myr-ψPKC) (myr-Arg-Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln-Lys-Asn-Val) and PKCϵ V1–2 (myr-Glu-Ala-Val-Ser-Leu-Lys-Pro-Thr) were from Promega (Southampton, UK) and BIOMOL, respectively. myr-PKCθ (myr-Leu-His-Gln-Arg-Arg-Gly-Ala-Ile-Lys-Gln-Ala-Lys-Val-His-His-Val-Lys-Cys) and myr-PKCζ (myr-Ser-Ile-Tyr-Arg-Arg-Gly-Ala-Arg-Arg-Trp-Arg-Lys-Leu) were from Merck Biosciences. Recombinant human VEGF 165 was purchased from PeproTech EC Ltd. (London, UK). Placental growth factor (PlGF) was purchased from R&D Systems (Abingdon, UK). Other products were from Sigma. Cell Isolation and Culture—Human umbilical vein ECs (HUVEC), isolated from umbilical cords as described previously (36Mason J.C. Yarwood H. Sugars K. Morgan B.P. Davies K.A. Haskard D.O. Blood. 1999; 94: 1673-1682Crossref PubMed Google Scholar), were cultured in 1% gelatin-coated tissue culture flasks (Costar, Cambridge, MA) in medium 199 (M199) (ICN Biomedicals Inc., Costa Mesa, CA) supplemented with 20% fetal bovine serum (Hyclone Laboratories Inc., Logan, UT), 100 IU/ml penicillin, 0.1 mg/ml streptomycin, 2 mm L-glutamine (all from Invitrogen), 10 units/ml heparin (Leo Laboratories, Prince Risborough, UK), and 30 μg/ml EC growth factor (Sigma). Adenoviral Infection—Generation of adenovirus expression vectors for dominant-negative (DN) PKC isozymes has been described previously (43Ohba M. Ishino K. Kashiwagi M. Kawabe S. Chida K. Huh N.H. Kuroki T. Mol. Cell. Biol. 1998; 18: 5199-5207Crossref PubMed Google Scholar). DN-PKCα, DN-PKCϵ, and β-galactosidase adenoviruses were amplified in HEK-293A cells and purified using the BD Adeno-X™ purification kit (BD Biosciences). Viral titers were estimated by using the BD Adeno-X™ rapid titer kit. HUVEC were infected by incubation with the relevant adenovirus in serum-free M199 for 2 h at 37 °C. The medium was then changed to M199, 10% fetal bovine serum, and HUVEC were incubated overnight prior to addition of VEGF or carrier control for up to 48 h. Infection of HUVEC with a β-galactosidase control adenovirus demonstrated a transfection efficiency of 95%. Optimal multiplicity of infection (MOI) for the adenoviruses was determined by Western blotting. Flow Cytometry—Flow cytometry was performed as described previously (36Mason J.C. Yarwood H. Sugars K. Morgan B.P. Davies K.A. Haskard D.O. Blood. 1999; 94: 1673-1682Crossref PubMed Google Scholar). Pharmacological antagonists were added 60 min prior to the addition of VEGF. In some experiments the results are expressed as the relative fluorescent intensity (RFI), representing mean fluorescent intensity (MFI) with test mAb divided by the MFI using an isotype-matched irrelevant mAb. Cell viability was assessed by examination of EC monolayers using phase contrast microscopy, cell counting, and estimation of trypan blue exclusion. In all experiments EC monolayers were treated with the appropriate vehicle controls. Complement-mediated Cell Lysis—To estimate complement-mediated cell lysis, ECs in M199, 10% fetal calf serum were pretreated with VEGF, VEGF and CsA, or carrier controls for 48 h, then harvested with trypsin/EDTA, washed, opsonized with antiendoglin mAb RMAC8, and incubated with 5–20% rabbit serum (Serotec, Oxford, UK) in Veronal-buffered saline, 1% gelatin (VBS) for 75 min at 37 °C. Following further washing, ECs were resuspended in VBS, and propidium iodide (PI) (Sigma) was added to a final concentration of 50 μg/ml. ECs were analyzed by flow cytometry using the FL2 channel. Lysis was calculated in triplicate samples as the number of PI-positive cells expressed as a percentage of the total number of cells. Western Blotting—HUVEC were lysed (4 mm EDTA, 50 mm Tris/HCl, pH 7.4, in 150 mm NaCl with 25 mm sodium deoxycholic acid, 200 μm sodium orthovanadate, 10 mm sodium pyrophosphate, 100 mm sodium fluoride, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, and 5% protease inhibitor mixture), and the protein content was determined using the Bio-Rad Dc protein assay. Cell lysates were subjected to SDS-PAGE on 12.5% gels, and separated proteins were transferred to Immobilon™-P transfer membranes (Millipore Corporation, Bedford, MA). The membranes were probed with rabbit polyclonal antibodies against PKC isozymes (Santa Cruz Biotechnology Inc., Santa Cruz, CA) and against the phosphorylated and non-phosphorylated forms of p38 MAPK (New England Biolabs Ltd., Hitchin, Herts, UK). The blots were developed with an enhanced chemiluminescence substrate (Amersham Biosciences). Integrated density values for the test and control bands were obtained with an Alpha Innotech ChemiImager 5500 (Alpha Innotech, San Leandro, CA). Statistical Analysis—Differences between the results of experimental treatments were evaluated by analysis of variance with the Bonferroni multiple comparison test using GraphPad 4.0 software (GraphPad Software, San Diego, CA). Differences were considered significant at p values of <0.05. VEGF-induced Up-regulation of DAF Is Mediated via VEGF-R2 and PLCγ—DAF is expressed at a low level on resting ECs in culture. We have reported previously that VEGF induces a significant, unimodal increase in EC surface expression of DAF as measured by flow cytometry. This response is indirect, requiring the synthesis of one or more intermediate proteins, and is dependent upon an increase in steady-state DAF mRNA (16Mason J.C. Lidington E.A. Yarwood H. Lublin D.M. Haskard D.O. Arthritis Rheum. 2001; 44: 138-150Crossref PubMed Scopus (52) Google Scholar). DAF protein expression was significantly increased following 24 h of exposure to VEGF with maximal expression at 48–72 h (Fig. 1A). A dual approach was used to explore which VEGF receptor was involved in this response. First, preincubation of EC with SU1498, a selective inhibitor of VEGF-R2 tyrosine kinase, had no effect on basal DAF expression while abrogating VEGF-induced DAF up-regulation (Fig. 1B). Furthermore PlGF, which preferentially binds VEGF-R1, failed to induce DAF expression at concentrations up to 50 ng/ml (Fig. 1B). These data suggest that VEGF-R2 is the dominant receptor in the induction of DAF expression by VEGF. Ligation of VEGF-R2 by VEGF results in the phosphorylation of PLCγ, a response that has been implicated in VEGF-dependent signaling pathways in EC (13He H. Venema V.J. Guo X.L. Venema R.C. Marrero M.B. Caldwell R.B. J. Biol. Chem. 1999; 274: 25130-25135Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 44Takahashi T. Ueno H. Shibuya M. Oncogene. 1999; 18: 2221-2230Crossref PubMed Scopus (479) Google Scholar). To investigate the role of PLCγ, HUVEC were treated with the PLCγ antagonist U73122. This resulted in a dose-dependent inhibition of VEGF-induced DAF up-regulation (not shown), which was maximal following pretreatment with 10 μm U73122 (Fig. 1C). VEGF-induced DAF Up-regulation Is Regulated by PKCα and PKCϵ —To explore the role of PKC in VEGF-induced DAF up-regulation, we studied the expression, activation, and inhibition of specific PKC isozymes. Initial immunoblotting experiments demonstrated that HUVEC expressed PKCα, -βII, -δ, -ϵ, -θ, and -ζ (data not shown). Activation of PKC with phorbol 12,13-dibutyrate (PBu), which activates both classical (cPKCα, -β, and -γ) and novel (nPKCδ, -ϵ, and -θ,) PKC isozymes, resulted in a significant increase in DAF expression (Fig. 2A). Furthermore, as seen in Fig. 2, A and B, both ingenol dibenzoate, which preferentially activates nPKC (45Asada A. Zhao Y. Kondo S. Iwata M. J. Biol. Chem. 1998; 273: 28392-28398Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), and thymeleatoxin, which preferentially activates cPKC (46Kazanietz M.G. Areces L.B. Bahador A. Mischak H. Goodnight J. Mushinski J.F. Blumberg P.M. Mol. Pharmacol. 1993; 44: 298-307PubMed Google Scholar), were capable of inducing a significant dose-dependent increase in cell surface DAF expression. Previous studies have demonstrated activation and translocation of PKCα, PKCβ, PKCδ, PKCϵ, and PKCζ in ECs exposed to VEGF (20Xia 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. J. Clin. Investig. 1996; 98: 2018-2026Crossref PubMed Scopus (524) Google Scholar, 21Wellner M. Maasch C. Kupprion C. Lindschau C. Luft F.C. Haller H. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 178-185Crossref PubMed Scopus (71) Google Scholar, 23Gliki G. Abu-Ghazaleh R. Jezequl S. Wheeler-Jones C. Zachary I. Biochem. J. 2001; 353: 503-512Crossref PubMed Scopus (86) Google Scholar, 47Shen B.Q. Lee D.Y. Zioncheck T.F. J. Biol. Chem. 1999; 274: 33057-33063Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). Inhibition of cPKC and nPKC with GF109203X completely inhibited DAF induction by both PBu and VEGF (Fig. 2, C and D). However, Gö6976, a cPKC-specific inhibitor, only reduced DAF up-regulation by 30–50%, supporting additional involvement of nPKC isozymes. Subsequent experiments sought to establish which one of the cPKC isozymes, PKCα or -β, was involved in the response. As seen in Fig. 3A, both Gö6976 and a specific cell-permeable peptide antagonist of PKCα/β, myr-ψPKC, significantly inhibited VEGF-induced DAF expression. In contrast, LY379196, a PKCβ-specific antagonist, had no effect, thereby implicating PKCα in VEGF-induced DAF up-regulation. There was no significant effect of the antagonists alone on basal DAF expression (not shown). Pretreatment of ECs with a specific peptide antagonist of PKCϵ (myr-PKCϵ) resulted in a minimal reduction in VEGF-induced DAF expression. However, a combination of myr-ψPKC and myr-PKCϵ completely abrogated the response (Fig. 3A). In contrast, similar myristoylated peptides specific for PKCθ and PKCζ and rottlerin, an antagonist of PKCδ, had no significant inhibitory effect (not shown). The role of PKCα and -ϵ was explored further using adenoviruses expressing PKCα and -ϵ dominant-negative constructs. Infection with each DN adenovirus led to a dose-dependent increase in the specific PKC isozyme immunoreactivity (Fig. 3B), and an MOI of 50 and 200 was used for further experiments with DN-PKCα and DN-PKCϵ, respectively. Infection with the DN-PKC and β-galactosidase adenoviruses had no effect on basal DAF expression (not shown). However, infection with DN-PKCα or DN-PKCϵ alone resulted in 25–30% inhibition of VEGF-induced DAF, whereas use of the dominant-negative adenoviruses in combination completely abrogated the response, thereby confirming the requirement for activation of both PKCα and -ϵ by VEGF for a maximal response (Fig. 3C). VEGF-induced DAF Up-regulation Is Independent of Nitric Oxide Generation and Thrombin Release—Treatment of ECs with VEGF increases expression and phosphorylation of eNOS and local NO synthesis (13He H. Venema V.J. Guo X.L. Venema R.C. Marrero M.B. Caldwell R.B. J. Biol. Chem. 1999; 274: 25130-25135Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 47Shen B.Q. Lee D.Y. Zioncheck T.F. J. Biol. Chem. 1999; 274: 33057-33063Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 48Papapetropoulos A. Garcia-Cardena G. Madri J.A. Sessa W.C. J. Clin. Investig. 1997; 100: 3131-3139Crossref PubMed Scopus (1015) Google Scholar). To investigate the potential role of NO in VEGF-induced DAF expression, HUVEC were preincubated with the NO synthase inhibitor l
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Abstract Vascular endothelial growth factor (VEGF) is a newly identified growth and permeability factor with a unique specificity for endothelial cells. Recently the flt‐encoded tyrosine kinase was characterized as a receptor for VEGF. A novel tyrosine kinase receptor encoded by the KDR gene was also found to bind VEGF with high affinity when expressed in CMT‐3 cells. Screening for flt and KDR expression in a variety of species and tissue‐derived endothelial cells demonstrates that flt is predominantly expressed in human placenta and human vascular endothelial cells. Placenta growth factor (PIGF), a growth factor significantly related to VEGF, is coexpressed with flt in placenta and human vascular endothelial cells. KDR is more widely distributed and expressed in all vessel‐derived endothelial cells. These data demonstrate that cultured human endothelial cells isolated from different tissues express both VEGF receptors in relative high levels and, additionally, that all investigated nonhuman endothelial cells in culture are also positive for KDR gene expression.
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