Title: An Autocrine Loop Mediates Expression of Vascular Endothelial Growth Factor in Human Dermal Microvascular Endothelial Cells
Abstract: The expression of vascular endothelial growth factor mRNA and protein is regulated by a number of agents including growth factors, cytokines, and phorbol esters. Here we report that vascular endothelial growth factor is able to increase its own level in cultured human dermal microvascular endothelial cells. Accumulation of vascular endothelial growth factor mRNA and polypeptide can be detected as early as 4 h after addition of vascular endothelial growth factor to the cell culture medium. The autocrine action of vascular endothelial growth factor appears to be mediated by the KDR receptor. The increase of its own message by vascular endothelial growth factor is blocked by the transcription inhibitor actinomycin D. Transient transfection experiments performed with human dermal microvascular endothelial cells and using a 3.2 kb human vascular endothelial growth factor promoter fragment showed that vascular endothelial growth factor auto-induction can be mimicked at the promoter level. This indicates that the observed vascular endothelial growth factor mRNA increase after vascular endothelial growth factor treatment is occurring at the level of transcription. Furthermore, vascular endothelial growth factor auto-induction is inhibited by PD 098059, showing that phosphorylation events, catalyzed by mitogen activated protein kinases, are a prerequisite for the vascular endothelial growth factor effect. Examination of extracellular signal-regulated kinase and c-Jun N-terminal protein kinase catalytic activities showed that both enzymes have to be activated to mediate the vascular endothelial growth factor signal. Our data demonstrate for the first time the existence of an autocrine loop for vascular endothelial growth factor in endothelial cells. Most probably this represents an amplification mechanism for the action of vascular endothelial growth factor in the microvascularization process. The expression of vascular endothelial growth factor mRNA and protein is regulated by a number of agents including growth factors, cytokines, and phorbol esters. Here we report that vascular endothelial growth factor is able to increase its own level in cultured human dermal microvascular endothelial cells. Accumulation of vascular endothelial growth factor mRNA and polypeptide can be detected as early as 4 h after addition of vascular endothelial growth factor to the cell culture medium. The autocrine action of vascular endothelial growth factor appears to be mediated by the KDR receptor. The increase of its own message by vascular endothelial growth factor is blocked by the transcription inhibitor actinomycin D. Transient transfection experiments performed with human dermal microvascular endothelial cells and using a 3.2 kb human vascular endothelial growth factor promoter fragment showed that vascular endothelial growth factor auto-induction can be mimicked at the promoter level. This indicates that the observed vascular endothelial growth factor mRNA increase after vascular endothelial growth factor treatment is occurring at the level of transcription. Furthermore, vascular endothelial growth factor auto-induction is inhibited by PD 098059, showing that phosphorylation events, catalyzed by mitogen activated protein kinases, are a prerequisite for the vascular endothelial growth factor effect. Examination of extracellular signal-regulated kinase and c-Jun N-terminal protein kinase catalytic activities showed that both enzymes have to be activated to mediate the vascular endothelial growth factor signal. Our data demonstrate for the first time the existence of an autocrine loop for vascular endothelial growth factor in endothelial cells. Most probably this represents an amplification mechanism for the action of vascular endothelial growth factor in the microvascularization process. human dermal microvascular cells vascular endothelial growth factor Vascular endothelial growth factor (VEGF) or vascular permeability factor, a glycosylated polypeptide of 40–45 kDa, is a specific mitogen of endothelial cells and is the major factor responsible for the induction of angiogenesis (Keck et al., 1989Keck P.J. Hauser S.D. Krivi G. Sanzo K. Warren T. Feder J. Connoly D.T. Vascular permeability factor, an endothelial cell mitogen related to PDGF.Science. 1989; 246: 1309-1312Crossref PubMed Scopus (1759) Google Scholar;Leung et al., 1989Leung D.W. Cachianes G. Kuang W.J. Goeddel D.V. Ferrara N. Vascular endothelial growth factor is a secreted angiogenic mitogen.Science. 1989; 246: 1306-1309Crossref PubMed Scopus (4300) Google Scholar;Ferrara, 1993Ferrara N. Vascular endothelial growth factor.Trends Cardiovasc Med. 1993; 3: 244-250Abstract Full Text PDF PubMed Scopus (168) Google Scholar). Several isoforms of VEGF resulting from the alternative splicing of its gene have been described (Tischer et al., 1991Tischer E. Mitchell R. Hartman T. Silva M. Gospodarowicz D. Fiddes J.C. Abraham J.A. The human gene for vascular endothelial growth factor: multiple protein forms are encoded through alternative exon splicing.J Biol Chem. 1991; 266: 11947-11954Abstract Full Text PDF PubMed Google Scholar); the larger forms, VEGF189 and VEGF206, remain cell-associated whereas the two smaller forms, VEGF121 and VEGF165, are secreted (Ferrara et al., 1992Ferrara N. Houck K. Jakeman L. Leung D.W. Molecular and biological properties of the vascular endothelial growth family of proteins.Endocr Rev. 1992; 13: 18-32Crossref PubMed Scopus (1515) Google Scholar). The level of VEGF is upregulated in a number of pathologic situations such as diabetic retinopathy, psoriasis (Detmar et al., 1994Detmar M. Brown L.F. Claffey K.P. et al.Overexpression of vascular permeability factor/vascular endothelial growth factor and its receptors in psoriasis.J Exp Med. 1994; 180: 1141-1146Crossref PubMed Scopus (627) Google Scholar), rheumatoid arthritis (Fava et al., 1994Fava R.A. Olsen N.J. Spencer-Green G. et al.Vascular permeability factor/endothelial growth factor (VPF/VEGF): accumulation and expression in human synovial fluids and rheumatoid synovial tissue.J Exp Med. 1994; 180: 341-346Crossref PubMed Scopus (486) Google Scholar), and cancers (Folkman, 1995Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease.J Nat Med. 1995; 1: 27-31Crossref PubMed Scopus (7013) Google Scholar). In psoriasis, for example, epidermal keratinocytes over-express VEGF, which in turn induces neo-vascularization through the proliferation and increased migration of dermal microvascular endothelial cells (Detmar, 1996Detmar M. Molecular regulation of angiogenesis in the skin.J Invest Dermatol. 1996; 106: 207-208Crossref PubMed Scopus (76) Google Scholar). VEGF has been shown to exert its effects in endothelial cells by interacting with two high affinity membrane receptors displaying tyrosine kinase activity, Flt-1 and KDR (Terman et al., 1992Terman B.I. Dougher-Vermazen M. Carrion M.E. Dimitrov D. Armellino D.C. Gospodarowicz D. Bohlen P. Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor.Biochem Biophys Res Commun. 1992; 187: 1579-1586Crossref PubMed Scopus (1371) Google Scholar;Quinn et al., 1993Quinn T.P. Peters K.G. De Vries C. Ferrara N. Williams L.T. Fetal liver kinase 1 is a receptor for vascular endothelial growth factor and is selectively expressed in vascular endothelium.Proc Natl Acad Sci USA. 1993; 90: 7533-7537Crossref PubMed Scopus (658) Google Scholar). A third class of receptor for VEGF, neuropiline-1 and -2, has been recently described (Soker et al., 1998Soker S. Takashima S. Miao H.Q. Neufeld G. Klagsbrun M. Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor.Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (1997) Google Scholar). Upregulation of VEGF mRNA and protein expression has been demonstrated in a number of in vitro and in vivo models. VEGF level is controlled by certain growth factors and cytokines such as interleukin-6 (Cohen et al., 1996Cohen T. Nahari D. Cerem L.W. Neufeld G. Levi B.Z. Interleukin 6 induces the expression of vascular endothelial growth factor.J Biol Chem. 1996; 271: 736-741Crossref PubMed Scopus (891) Google Scholar), epidermal growth factor, platelet derived growth factor, transforming growth factor β1 (TGF-β1), tumor necrosis factor α (TNFα), and keratinocyte growth factor (Frank et al., 1995Frank S. Hubner G. Breier G. Longaker M.T. Greenhalgh D.G. Werner S. Regulation of vascular endothelial growth factor expression in cultured keratinocytes. Implications for normal and impaired wound healing.J Biol Chem. 1995; 270: 12607-12613Abstract Full Text Full Text PDF PubMed Scopus (647) Google Scholar). Hypoxia occurring in certain pathologic situations also leads to an increase in VEGF expression (Ikeda et al., 1995Ikeda E. Achen M. Breier G. Risau W. Hypoxia-induced transcriptional activation and increased mRNA stability of vascular endothelial growth factor in C6 glioma cells.J Biol Chem. 1995; 270: 19761-19766Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar;Stein et al., 1995Stein I. Neeman M. Shweiki D. Itin A. Keshet E. Stabilization of vascular endothelial growth factor mRNA by hypoxia and hypoglycemia and coregulation with other ischemia-induced genes.J Biol Chem. 1995; 270: 19761-19766Crossref PubMed Scopus (522) Google Scholar;Shima et al., 1996Shima D.T. Deutsch U. D'Amore P.A. Hypoxic induction of vascular endothelial growth factor (VEGF) in human epithelial cells is mediated by increases in mRNA stability.J Biol Chem. 1996; 271: 2746-2753Crossref PubMed Scopus (547) Google Scholar). A 3.4 kb VEGF promoter fragment has been cloned (Tischer et al., 1991Tischer E. Mitchell R. Hartman T. Silva M. Gospodarowicz D. Fiddes J.C. Abraham J.A. The human gene for vascular endothelial growth factor: multiple protein forms are encoded through alternative exon splicing.J Biol Chem. 1991; 266: 11947-11954Abstract Full Text PDF PubMed Google Scholar) and shown to contain functional sites for the binding of transcription factors important for controlling VEGF expression. These factors include hypoxia induced factor (Forsythe et al., 1996Forsythe J. Jiang B. Iyer N. Agan F. Leung S. Koos R. Semenza G. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1.Mol Cell Biol. 1996; 16: 4604-4613Crossref PubMed Scopus (3019) Google Scholar), AP1 (Vega-Diaz et al., 2000Vega-Diaz B. Lenoir M.C. Ladoux A. Frelin C. Démarchez M. Michel S. Regulation of vascular endothelial growth factor in human keratinocytes by retinoids.J Biol Chem. 2000; 275: 642-650Crossref PubMed Scopus (108) Google Scholar), SP1 (Finkenzeller et al., 1997Finkenzeller G. Sparacio A. Technau A. Marme D. Siemeister G. SP1 recognition sites in the proximal promoter of the human vascular endothelial growth factor gene are essential for the platelet-derived growth factor-induced gene expression.Oncogene. 1997; 15: 669-676Crossref PubMed Scopus (180) Google Scholar), and AP2 (Gille et al., 1997Gille J. Swerlick R.A. Caughman S.W. Transforming growth factor-alpha-induced transcriptional activation of the vascular permeability factor (VPF/VEGF) gene requires AP2 dependent DNA binding and transactivation.EMBO J. 1997; 16: 750-759https://doi.org/10.1093/emboj/16.4.750Crossref PubMed Scopus (248) Google Scholar). In this report, we characterize a VEGF autocrine loop occurring in human dermal microvascular cells (HDMEC), one of the specific targets of VEGF action. This autocrine loop certainly provides an amplification mechanism for VEGF-driven angiogenesis. Recombinant VEGF was from Pepro Tech EC (Rocky Hill, NJ). Antibodies and protein substrates for the determination of MAP kinase activity were from Santa Cruz Biotechnology (Santa Cruz, CA) and Sigma. PD 098059 was purchased from TEBU (Paris). Actinomycin D was purchased from Sigma. Antibodies against KDR or Flt-1 VEGF receptors and VEGF were from R&D Systems (Abingdon, U.K.). HDMEC were either purchased from Clonetics or isolated from human skin obtained from plastic surgery. They were grown in endothelial basal medium (EBM-2; Clonetics) supplemented with 5% (vol/vol) fetal bovine serum and bovine pituitary extract, epidermal growth factor, hydrocortisone, gentamicin, amphotericin-B, VEGF, and fibroblast growth factor (FGF) at concentrations recommended by the manufacturer. For all experiments, third passage subconfluent HDMEC cultures grown in 60 mm dishes were first incubated for 24 h in EBM-2 devoid of FGF and VEGF but containing 2% (vol/vol) fetal bovine serum. Then they were treated for different periods of time with or without recombinant VEGF. In some experiments, 1 h before the addition of recombinant VEGF, a polyclonal antibody directed against VEGF, KDR VEGF receptor, or Flt-1 VEGF receptor was added to the culture medium, at concentrations recommended by the manufacturer. Total RNA was isolated from cultured HDMEC using the Trizol method (Gibco), according to the manufacturer's procedure, and stored at -80°C until use. The following primers for PCR were synthesized by Gibco (France): glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sense (5′-AATCCCATCACCATCTTCCA-3′) and antisense (5′-GTCATCATATTTGGCAGGTT-3′) oligonucleotides; VEGF sense (5′-CCATGAACTTTCTGCTGTCTT-3′) and antisense (5′-ATCGCATCAGGGGCACACAG-3′) oligonucleotides. The amplification products were predicted to be 558 bp for GAPDH and 249 bp for VEGF. The VEGF primers were chosen in exon 1 and exon 3 of the VEGF gene resulting in a PCR product of 294 bp irrespective of the splice form produced (Vega-Diaz et al., 2000Vega-Diaz B. Lenoir M.C. Ladoux A. Frelin C. Démarchez M. Michel S. Regulation of vascular endothelial growth factor in human keratinocytes by retinoids.J Biol Chem. 2000; 275: 642-650Crossref PubMed Scopus (108) Google Scholar). RT-PCR was carried out using 5 µg of total RNA extracted from cultured endothelial cells. After denaturation in diethylpyrocarbonate-treated water for 10 min at 70°C, RNA was reverse-transcribed into first-strand cDNA using SuperScriptII Rnase H reverse transcriptase (10 units per reaction, Gibco BRL) and 0.5 µg of oligo (dT) as primer, at 42°C for 50 min in a total volume of 20 µl in a buffer containing 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 1 mM dNTP, 10 mM dithiothreitol, and 20 units Rnasin. Reverse transcriptase was inactivated at 70°C for 15 min and the RNA template was digested by Rnase H at 37°C for 20 min. Each experiment included samples devoid of reverse transcriptase (negative controls) to exclude amplification from contaminating genomic DNA. Semiquantitative RT-PCR amplification was performed with a PTC 225 thermal cycler (MJ Research), following a 1 min period of denaturation at 94°C, under the following conditions: denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, for the total number of cycles indicated below. The assay mixture contained 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 0.1 µM of oligonucleotide primers, dNTPs (100 µM of dATP, dGTP, dTTP, 10 µM dCTP), 0.5 µCi of [32P]-dCTP, 0.5 units of Taq DNA polymerase, and 5 µl of a 100-fold diluted cDNA mixture. The final product was extended for 3 min at 72°C. In each experiment, reverse transcriptase positive controls (templates containing cDNA encoding for VEGF) and negative control (without DNA) were included. The PCR products were subjected to electrophoresis on 6% (wt/vol) acrylamide gels. Radioactivity in each band was quantified by the storage phosphorimaging technique. The screens were scanned using a Fuji BAS 2000. The signal was quantified in photo stimulating luminescence units using the Tina image analysis software. Results were expressed for each sample as band intensity relative to that of GAPDH. An optimum number of PCR cycles was determined in the region of exponential amplification. It was determined to be 28 cycles for VEGF and 22 cycles for GAPDH. Logarithmic dilutions of the cDNA mixture were used to verify the linear correlation between the intensity of the radioactive signal and the initial amount of cDNA. HDMEC cells were labeled overnight with 250 µCi [35S]-cysteine per ml of culture medium before being incubated with VEGF at 20 ng per ml. At different time points, the medium was collected. The attached cells were lyzed with 50 mM HEPES, pH 7.6, 250 mM NaCl, 3 mM ethylenediamine tetraacetic acid (EDTA), 3 mM ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 0.5% (vol/vol) Nonidet P-40, and 1% (vol/vol) Triton X-100 containing 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg per ml leupeptin, and 10 µg per ml aprotinin as protease inhibitors. The lysates were subjected to low speed centrifugation. The cell supernatant and the culture medium were added to an enzyme-linked immunosorbent assay (ELISA) plate (R&D Systems) and VEGF was bound by the immobilized antibody. VEGF ELISA was performed as described by the manufacturer except that addition of the conjugate was omitted and the plates were directly subjected to scintillation counting (Wallac Microbeta Trilux EG & G). HDMEC were labeled overnight with 250 µCi [35S]-cysteine per ml of culture medium before being incubated with VEGF at 20 ng per ml. At different time points, the medium was collected. The attached cells were lyzed with 50 mM HEPES, pH 7.6, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 0.5% (vol/vol) Nonidet P-40, and 1% (vol/vol) Triton X-100 containing 1 mM PMSF, 10 µg per ml leupeptin, and 10 µg per ml aprotinin as protease inhibitors. The lysates were subjected to low speed centrifugation and the supernatants were incubated with a polyclonal anti-VEGF antibody or with an unrelated polyclonal anti-PPAR-α antibody used as a control. The mixture was then incubated with Protein G-Sepharose. After centrifugation, the Sepharose beads were incubated with sodium dodecyl sulfate (SDS) sample buffer. The immunoprecipitated proteins were then separated by SDS polyacrylamide gel electrophoresis using non-reducing conditions. The gels were exposed to a phosphorimager screen. HDMEC were transiently transfected in 60 mm culture dishes using Superfect (Quiagen) according to the manufacturer's procedure. We used 5 µg of a construct that contained a 3.2 kb fragment of the human VEGF promoter cloned into the pGl2-basic vector luciferase reporter plasmid (Ikeda et al., 1995Ikeda E. Achen M. Breier G. Risau W. Hypoxia-induced transcriptional activation and increased mRNA stability of vascular endothelial growth factor in C6 glioma cells.J Biol Chem. 1995; 270: 19761-19766Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). The plasmid was kindly provided by Drs A. Damert and W. Risau (Max-Plank-Institut für physiologische und klinische Forschung, Bad Nauheim, Germany) with the permission of Dr. J. Abraham (Scios Nova, Sunnyvale, CA). Transfected cells were transferred into 10 wells of a 96-well plate for 48 h and treated either with or without 20 ng per ml VEGF or 100 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) (Sigma) for 4 h. Luciferase activity was determined using the LucLite kit (Packard) and the luminescence counter Wallac Microbeta Trilux. HMDEC were lyzed in cell extraction buffer containing 50 mM HEPES, pH 7.6, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 0.5% Nonidet P-40, 100 mM Na3VO4, 50 mM β-glycerophosphate, 2 mM sodiumpyrophosphate, 1% Triton X-100 and the protease inhibitors PMSF (1 mM), leupeptin (10 µg per ml), and aprotinin (10 µg per ml). The cell lysate was clarified by centrifugation (15,000 × g, 10 min, 4°C) and equal amounts of lysate were immunoprecipitated with antijun kinase JNK or anti-ERK antibodies and 30 µl of protein A-Sepharose for 3 h at 4°C. Immunoprecipitates were washed three times with cold lysis buffer followed by two washes with kinase assay buffer containing 50 mM HEPES, pH 7.6, 25 mM β-glycerophosphate, 0.1 mM Na3VO4, and 25 mM MgCl2. Then 5 µg of myelin basic protein (MBP) (for ERK) or 10 µg of GST-cJun (1–79) (for JNK) in kinase buffer containing 50 µM of cold ATP and 5 µCi of [γ-32P]-ATP (> 4000 µCi per mmol; ICN) were added. The mixture was incubated at 30°C for 20 min and the reaction was stopped by the addition of SDS sample buffer. The samples were subjected to SDS-polyacrylamide gel electrophoresis on a 12% (wt/vol) polyacrylamide gel. The gels were dried and the radioactivity contained in MBP or GST-cJun was quantified by the storage phosphorimaging technique. The screens were scanned and quantified as described before. The results are presented as means (± SEM) of the duplicates of three independent experiments. They were analyzed using the two-sided Student's t test (NS, not significant; *p < 0.1; **p < 0.05; ***p < 0.01. The expression of VEGF mRNA and protein is regulated in a paracrine fashion by a number of factors that include cytokines and several growth factors (Frank et al., 1995Frank S. Hubner G. Breier G. Longaker M.T. Greenhalgh D.G. Werner S. Regulation of vascular endothelial growth factor expression in cultured keratinocytes. Implications for normal and impaired wound healing.J Biol Chem. 1995; 270: 12607-12613Abstract Full Text Full Text PDF PubMed Scopus (647) Google Scholar). Cells of different origin are able to synthesize and secrete VEGF, which is over-expressed by malignant tumor cells (Levy et al., 1989Levy A. Tamargo R. Brem H. Nathans D. An endothelial cell growth factor from the mouse neuroblastoma cell line NB41.Growth Factors. 1989; 2: 9-19Crossref PubMed Scopus (53) Google Scholar;Conn et al., 1990Conn G. Soderman D. Schaeffer M. Wile M. Hatcher V. Thomas K. Amino acid and cDNA sequences of a vascular endothelial cell mitogen that is homologous to platelet-derived growth factor.Proc Natl Acad Sci USA. 1990; 87: 1323-1327Crossref PubMed Scopus (214) Google Scholar;Phillips et al., 1990Phillips H. Hains J. Leung D. Ferrara N. Vascular endothelial growth factor is expressed in rat corpus luteum.Endocrinology. 1990; 127: 965-967Crossref PubMed Scopus (246) Google Scholar;Garrido et al., 1993Garrido C. Saule S. Gospodarowicz D. Transcriptional regulation of vascular endothelial growth factor gene expression in ovarian bovine granulosa cells.Growth Factors. 1993; 8: 109-117Crossref PubMed Scopus (131) Google Scholar). VEGF is produced at the mRNA level by cultured cells from rat brain capillary endothelium (Ladoux and Frelin, 1993Ladoux A. Frelin C. Expression of vascular endothelial growth factor by cultured endothelial cells from brain microvessels.Biochem Biophys Res Commun. 1993; 194: 799-803https://doi.org/10.1006/bbrc.1993.1892Crossref PubMed Scopus (63) Google Scholar) or by cultures of bovine glomerula endothelial cells (Simorre-Pinatel et al., 1994Simorre-Pinatel V. Guerrin M. Chollet P. Penary M. Clamens S. Malecaze F. Plouet J. Vasculotropin-VEGF stimulates retinal capillary endothelial cells through an autocrine pathway.J Invest Ophthalmol Vis Sci. 1994; 35: 3393-3400PubMed Google Scholar;Uchida et al., 1994Uchida K. Uchida S. Nitta K. Yumura W. Marumo F. Nihei H. Glomerular endothelial cells in culture express and secrete vascular endothelial growth factor.Am J Physiol. 1994; 266: 81-88PubMed Google Scholar). A different situation occurs in human cells, where VEGF does not seem to be synthesized in resting cultured umbilical vein endothelial cells or HDMEC but can be produced as a consequence of TPA treatment or hypoxia (Namiki et al., 1995Namiki A. Brogi E. Kearney M. et al.Hypoxia induces vascular endothelial growth factor in cultured human endothelial cells.J Biol Chem. 1995; 270: 31189-31195Crossref PubMed Scopus (421) Google Scholar;Detmar et al., 1997Detmar M. Brown L.F. Berse B. Jackman R.W. Elicker B.M. Dvorak H.F. Claffey K.P. Hypoxia regulates the expression of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF); and its receptors in human skin.J Invest Dermatol. 1997; 108: 263-268Crossref PubMed Scopus (225) Google Scholar). The fact that human endothelial cells can, under certain circumstances, synthesize VEGF prompted us to verify whether or not VEGF could regulate its own synthesis in these cells. As model we used HDMEC in culture starved for growth factors over 24 h and treated for different periods of time with VEGF. Total RNA was prepared and the level of VEGF mRNA was quantitated by using RT-PCR. As shown in Figure 1(a), small amounts of VEGF mRNA were expressed at basal level. When VEGF at 10 ng per ml was added to the culture medium for 4 h, however, a substantial increase in VEGF mRNA could be observed. This upregulation by a factor of 2.5–3 was transient as the amount returned to normal after 16 h of incubation. The increase in VEGF message was paralleled by an augmentation of the level of matrix metalloproteinase 1 and 3 mRNAs (results not shown), two markers of endothelial cell activation, and comparable to that obtained after treatment of HDMEC with TNFα (Figure 1b). A similar induction level was also observed after treatement of HDMEC with TPA (result not shown). Figure 2 shows that the maximal increase in VEGF mRNA level was obtained at 10–20 ng per ml depending on the batch of recombinant VEGF used. No effect was observed at high concentration of VEGF (50 ng per ml), however, certainly indicating downregulation of the VEGF signal in the presence of an excess of VEGF. This may represent a protective mechanism of HDMEC against an excess of VEGF. Figure 3 clearly shows the specificity of the response as the action of VEGF was blocked by preincubation with a specific antibody recognizing the mature VEGF121 and VEGF165 isoforms. To determine whether the increase of VEGF transcript induced by VEGF was accompanied by a corresponding rise in the production of the polypeptide, a radioimmunoassay experiment was performed using a specific antibody recognizing the mature VEGF121 and VEGF165 isoforms. After labeling of the cells with [35S]-cysteine, the amount of VEGF was determined both in the cell culture supernatant and in the total cell lysate. VEGF was not detected, even after treatment with VEGF, in the cell culture medium (not shown). As shown in Figure 4(a), cell-associated VEGF polypeptides were detected in non-treated cells and were increased up to 24 h after VEGF treatment. In addition, cell-associated VEGF was immunoprecipitated from total cell lysate of HDMEC treated for 4 h with VEGF (Figure 4b). The fact that we were not able to detect significant amounts of VEGF in the cell culture supernatant is consistent with previous findings (Houck et al., 1992Houck K. Leung D. Rowland A. Winer J. Ferrara N. Dual regulation of vascular endothelial growth factor bioavailability by genetic and proteolytic mechanisms.J Biol Chem. 1992; 267: 26031-26037Abstract Full Text PDF PubMed Google Scholar) showing that the VEGF165 isoform can be sequestered by the extracellular matrix of endothelial cells. These results point to the existence of an autocrine loop for VEGF in HDMEC. An analogous autoregulation process has already been described for TGF-β1, which causes in various normal or transformed fibroblasts a rapid increase in the synthesis of its own message and in TGF-β1 secretion (Van-Obberghen-Schilling et al., 1988Van-Obberghen-Schilling E. Roche N.S. Flanders K.C. Sporn M.B. Roberts A.B. Transforming growth factor beta 1 positively regulates its own expression in normal and transformed cells.J Biol Chem. 1988; 263: 7741-7746PubMed Google Scholar;Kim et al., 1989aKim S.J. Denhez F. Kim K.Y. Holt J.T. Sporn M.B. Roberts A.B. Activation of the second promoter of the transforming growth factor-beta 1 gene by transforming growth factor-beta 1 and phorbol ester occurs through the same target sequences.J Biol Chem. 1989; 264: 19373-19378Abstract Full Text PDF PubMed Google Scholar, Kim et al., 1989bKim S.J. Jeang K.T. Glick A.B. Sporn M.B. Roberts A.B. Promoter sequences of the human transforming growth factor-beta 1 gene responsive to transforming growth factor-beta 1 autoinduction.J Biol Chem. 1989; 264: 7041-7045Abstract Full Text PDF PubMed Google Scholar,Kim et al., 1990Kim S.J. Angel P. Lafyatis R. et al.Autoinduction of transforming growth factor beta 1 is mediated by the AP-1 complex.Mol Cell Biol. 1990; 10: 1492-1497Crossref PubMed Google Scholar). It is thought that the TGF-β1 autocrine loop could amplify the physiologic process of wound healing. The stimulation by VEGF of its own RNA synthesis and polypeptide production certainly provides an amplification mechanism for the vascularization process. HDMEC express the two VEGF receptors KDR and Flt-1, and an interesting question was to know which of these two receptors was involved in the transmission of the VEGF signal. HDMEC were preincubated with anti-KDR or anti-Flt-1 antibodies before addition of VEGF. As shown in Figure 5, preincubation with anti-KDR antibody, but not with anti-Flt-1 antibody, completely blocked the increase of VEGF message. This shows that KDR might be involved in the VEGF autocrine loop. This is consistent with the fact that KDR is involved in the control of mitogenesis of endothelial cells (Waltenberger et al., 1994Waltenberger J. Claesson-Welsh L. Siegbahn A. Shibuya M. Heldin C.H. Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor.J Biol Chem. 1994; 269: 26988-26995Abstract Full Text PDF PubMed Google Scholar;Keyt et al., 1996Keyt B.A. Nguyen H.V. Berleau L.T. Duarte C.M. Park J. Chen H. Ferrara N. Identification of vascular endothelial growth factor determinants for binding KDR and FLT-1 receptors. Generation of receptor-selective VEGF variants by site-directed mutagenesis.J Biol Chem. 1996; 271: 5638-5646Crossref PubMed Scopus (413) Google Scholar) and other cell types (Dias et al., 2000Dias S. Hattori K. Zhu Z. et al.Autocrine stimulation of VEGFR-2 activates human leukemic cell growth and migration.J Clin Invest. 2000; 106: 511-521Crossref PubMed Scopus (403) Google Scholar).Figure 2Induction of VEGF mRNA level in cultured HDMEC treated with different concentrations of VEGF. Total RNA was isolated at the indicated time points from HDMEC following incubation in EBM-2 in the absence or presence of the indicated concentrations of VEGF. The expression level of VEGF mRNA was determined relative to that of GAPDH mRNA by semiquantitative RT-PCR analysis as described in Materials and Methods. Results were normalized to the values obtained with untreated cells.View Large Image Figure ViewerDownload (PPT)Figure 3Effect of anti-VEGF antibody on VEGF mRNA expression in cultured HDMEC treated with VEGF. HDMEC were preincubated for 1 h with a polyclonal antibody raised against VEGF (2 µg per ml) before the addition of VEGF (20 ng per ml). After 4 h, total RNA was isolated and the expression level of VEGF mRNA was determined relative to that of GAPDH mRNA by semiquantitative RT-PCR analysis as described in Materials and Methods. Results were normalized to the values obtained with untreated cells.View Large Image Figure ViewerDownload (PPT)Figure 4Induction of VEGF polypeptide production by cultured HDMEC treated with VEGF. (a) HDMEC were labeled with [35S]-cysteine in EBM-2 for 24 h and then treated until the indicated time points with or without VEGF at 10 ng per ml. The cell lysates were collected and VEGF was quantitated using a radioimmunoassay with a specific anti-VEGF antibody. (b) HDMEC were labeled with [35S]-cysteine in EBM-2 for 24 h and then treated for 4 h without (lanes 1 and 2) or with (lanes 3 and 4) VEGF at 10 ng per ml. The cell lysate was then immunoprecipitated with a control (lanes 1 and 3) or an anti-VEGF antibody (lanes 2 and 4). Immunoprecipitated VEGF was revealed by polyacrylamide gel electrophoresis and autoradiography.View Large Image Figure ViewerDownload (PPT)Figure 5Effect of anti-KDR or anti-Flt-1 antibody on VEGF mRNA expression in cultured HDMEC treated with VEGF. HDMEC were preincubated for 1 h with an antibody raised against KDR (1 µg per ml) or Flt-1 (100 µg per ml) before the addition of VEGF (20 ng per ml). After 4 h, total RNA was isolated and the expression level of VEGF mRNA was determined relative to that of GAPDH mRNA by semiquantitative RT-PCR analysis as described in Materials and Methods. Results were normalized to the values obtained with untreated cells.View Large Image Figure ViewerDownload (PPT) To prove that the induction of VEGF mRNA by VEGF in culture involves a transcriptional event, actinomycin D, an inhibitor of transcription, was used. As shown in Figure 6, actinomycin D reduces the basal level of VEGF mRNA and completely abolishes the VEGF-induced mRNA synthesis. Cycloheximide, an inhibitor of protein synthesis, could not be used to check if the increase of VEGF mRNA level required de novo protein synthesis, as treatment of HDMEC with this compound alone led to a large increase in VEGF mRNA expression. This effect of cycloheximide on VEGF level has been well documented in other cellular systems (Ladoux and Frelin, 1993Ladoux A. Frelin C. Expression of vascular endothelial growth factor by cultured endothelial cells from brain microvessels.Biochem Biophys Res Commun. 1993; 194: 799-803https://doi.org/10.1006/bbrc.1993.1892Crossref PubMed Scopus (63) Google Scholar). HDMEC in culture were then transfected with a 3.2 kb human VEGF promoter fragment linked to a luciferase reporter gene. As a control, cells were also treated with TPA, a well-known activator of VEGF promoter transactivation (Vega-Diaz et al., 2000Vega-Diaz B. Lenoir M.C. Ladoux A. Frelin C. Démarchez M. Michel S. Regulation of vascular endothelial growth factor in human keratinocytes by retinoids.J Biol Chem. 2000; 275: 642-650Crossref PubMed Scopus (108) Google Scholar). As shown in Figure 7, treatment of transfected HDMEC with VEGF led to an increase in reporter gene activity that was even greater than that obtained with TPA. This further confirms that VEGF auto-induction is occurring at the transcriptional level.Figure 7Effect of VEGF on the transactivation of the human VEGF promoter in HDMEC. HDMEC were transiently transfected with a plasmid containing a 3.2 kb human VEGF promoter fragment cloned upstream of a luciferase reporter gene. Transfected cells grown in EBM-2 were treated with or without VEGF 20 ng per ml or 100 nM TPA for 4 h. Luciferase activity was determined as described in Materials and Methods. Results were normalized to the values obtained with untreated cells.View Large Image Figure ViewerDownload (PPT) Previously it has been shown that distinct phosphorylation pathways control the transcription of the VEGF gene. For example, the protein kinase C zeta isoform has been implicated in the transcription of the VEGF gene induced by the SP1 transcription factor (Pal et al., 1998Pal S. Claffey K.P. Cohen H.T. Mukhopadhyay D. Activation of Sp1-mediated vascular permeability factor/vascular endothelial growth factor transcription requires specific interaction with protein kinase C zeta.J Biol Chem. 1998; 273: 26277-26280Crossref PubMed Scopus (157) Google Scholar). Phosphatidylinositol 3-kinase is activated in endothelial cells when VEGF expression is induced by platelet-derived growth factor (Wang et al., 1999Wang D. Huang H.J.S. Kazlaukas A. Cavence W.K. Induction of vascular endothelial growth factor expression in endothelial cells by platelet-derived growth factor through the activation of phosphatidylinositol 3-kinase.Cancer Res. 1999; 59: 1464-1472PubMed Google Scholar). In fibroblasts the p42/p44 mitogen activated protein (MAP) kinases play an important role in VEGF expression (Milanini et al., 1998Milanini J. Vinals F. Pouyssegur J. Pagès G. p42/p44 MAP kinase module plays a key role in the transcriptional regulation of the vascular endothelial growth factor gene in fibroblasts.J Biol Chem. 1998; 273: 18165-18172Crossref PubMed Scopus (287) Google Scholar). VEGF binding to the VEGF receptors activates the extracellular signal-regulated kinase (ERK) and the c-Jun N-terminal protein kinase (JNK) in human umbilical vein endothelial cells as well as the MAP kinase pathway in rat cardiac myocytes (Seko et al., 1998Seko Y. Takahashi N. Tobe K. Ueki K. Kadowaki T. Yazaki Y. Vascular endothelial growth factor (VEGF) activates Raf-1, mitogen-activated protein (MAP) kinases, and S6 kinase (p90rsk) in cultured rat cardiac myocytes.J Cell Physiol. 1998; 175: 239-246https://doi.org/10.1002/(sici)1097-4652(199806)175:3<239::aid-jcp1>3.0.co;2-pCrossref PubMed Scopus (0) Google Scholar). To gain some insights into the molecular events occurring between the interaction of VEGF with KDR and the increase of VEGF at the transcriptional level, HDMEC treated with VEGF were incubated with the MAP kinase inhibitor PD 098059. As shown in Figure 8, PD 098059 completely abolished the induction of VEGF mRNA by VEGF. This result led us to investigate the time course of activation of ERK and JNK in HDMEC following VEGF stimulation. As shown in Figure 9, activation of JNK occurred as early as 5 min after VEGF addition whereas ERK activation increased after 5 min and was maximum after 10 min. Our results are consistent with recent findings (Pedram et al., 1998Pedram A. Razandi M. Levin E.R. Extracellular signal-regulated protein kinase/Jun kinase cross-talk underlies vascular endothelial growth factor-induced endothelial cell proliferation.J Biol Chem. 1998; 273: 26722-26728Crossref PubMed Scopus (178) Google Scholar) showing that VEGF treatment of human umbilical vein endothelial cells increases both JNK and ERK activation. In this study ERK preceded JNK activation, however.Figure 9Effect of VEGF treatment of cultured HDMEC on JNK and ERK activation. HDMEC were incubated in EBM-2 for 24 h and then treated with or without VEGF at 10 ng per ml. Immunoprecipitated ERK and JNK were incubated in the presence of [32P]-ATP with their respective substrate MBP or GST-c-jun. 32P incorporation into the substrates was revealed by polyacrylamide gel electrophoresis and autoradiography.View Large Image Figure ViewerDownload (PPT) In conclusion, we have demonstrated the existence of an autocrine loop for VEGF in microvascular endothelial cells. This certainly provides an amplification mechanism for the physiologic and pathologic microvascularization processes. The fact that the expression by microvascular endothelial cells of the VEGF receptor KDR is increased after VEGF treatment implies that KDR may also contribute to the amplification of the VEGF signal (Shen et al., 1998Shen B.Q. Lee D.Y. Gerber H.P. Keyt B.A. Ferrara N. Zioncheck T.F. Homologous up-regulation of KDR/Flk-1 receptor expression by vascular endothelial growth factor in vitro.J Biol Chem. 1998; 273: 29979-29985Crossref PubMed Scopus (185) Google Scholar). While this work was in progress, another study reported that autocrine stimulation of endothelial cells by VEGF can lead to reorganization of the vascular network (Helmlinger et al., 2000Helmlinger G. Endo M. Ferrara N. Hlatky L. Jain K.J. Formation of endothelial cell networks.Nature. 2000; 405: 139-141https://doi.org/10.1038/35012129Crossref PubMed Scopus (63) Google Scholar). Future studies will be dedicated to the detailed pathways involved in VEGF auto-induction. Thanks are due to Prof. U. Reichert, Dr. M. Démarchez, and Prof. D. Dhouailly for helpful discussions.