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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2003;23:2002.)
© 2003 American Heart Association, Inc.

Vascular Biology

Vascular Endothelial Growth Factor–Regulated Gene Expression in Endothelial Cells

KDR-Mediated Induction of Egr3 and the Related Nuclear Receptors Nur77, Nurr1, and Nor1

Dan Liu; Haiyan Jia^*; David Ian Roderick Holmes^*; Anita Stannard; Ian Zachary

From BHF Laboratories, Department of Medicine, University College London, London, UK.

Correspondence to Ian Zachary, BHF Laboratories, Department of Medicine, Rayne Bldg, University College London, 5 University St, London WC1E 6JJ, UK. E-mail I.Zachary{at}ucl.ac.uk

	Abstract

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Objective— The program of gene expression regulated by vascular endothelial growth factor (VEGF) remains poorly understood. The aim of this study was to identify VEGF-regulated genes in human umbilical vein endothelial cells.

Methods and Results— VEGF-regulated gene expression was analyzed by screening Affymetrix oligonucleotide arrays and quantitative, real-time, reverse transcription–polymerase chain reaction. The most strongly induced genes were the NR4A nuclear receptor family members Nur77, Nurr1, and Nor1 and the zinc-finger transcription factor Egr3. VEGF also induced rapid expression of Down syndrome candidate region 1, cyclooxygenase-2, tissue factor, stanniocalcin-1, the serine/threonine kinase Cot, and eps15 homology domain-containing protein. VEGF-induced NR4A family and Egr3 expression was blocked by a KDR inhibitor, and placental growth factor and basic fibroblast growth factor weakly increased expression of these genes. Induction of NR4A genes was mediated via intracellular Ca²⁺, protein kinase C- and calcineurin-dependent pathways. VEGF increased protein expression of Nurr1 and Nur77 and decreased Nur77 phosphorylation at the negative regulatory site serine 351.

Conclusions— VEGF induces expression of NR4A nuclear receptors and Egr3 via KDR and KDR-mediated signaling mechanisms. The genes identified here are novel candidates as key early mediators of VEGF-induced endothelial functions.

Key Words: vascular endothelial growth factor • basic fibroblast growth factor • gene array

	Introduction

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Vascular endothelial growth factor (VEGF, VEGF-A) is essential for endothelial cell differentiation and angiogenesis during development and plays a major role in neovascularization in a variety of disease states.¹ Two tyrosine kinase receptors mediate the biologic effects of VEGF, KDR/Flk-1 and Flt-1, but signal transduction and biologic responses triggered by VEGF in endothelia are mediated primarily via KDR.¹ After binding to KDR, VEGF activates multiple early signaling cascades in endothelial cells and subsequently elicits an array of biologic activities in vivo and in vitro, including endothelial survival, proliferation, migration, and increased production of nitric oxide and prostaglandin I₂.^2–9

Upregulation of several genes by VEGF has been reported,^10–13 but the overall program of endothelial gene expression regulated by VEGF remains unclear. In particular, there is a lack of information regarding the early gene expression events that are likely to play a central role in signal transduction downstream of KDR and in mediating long-term biologic responses to VEGF. To provide novel insights into the molecular mechanisms through which VEGF exerts its biologic activities, we examined the pattern of gene expression regulated by VEGF by analysis of oligonucleotide arrays and real-time, quantitative reverse transcription–polymerase chain reaction (RT-PCR). A striking feature of these findings is the rapid upregulation of several immediate-early genes encoding transcription factors, including the NR4A family of orphan nuclear receptors and the transcription factor Egr3. These findings should advance our understanding of the mechanisms underlying the endothelial response to VEGF and generate new insights into the molecular basis for angiogenesis and other endothelial cellular functions.

	Methods

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Experimental Procedures
Cell Culture
Human umbilical vein endothelial cells (HUVECs; TCS CellWorks, Buckingham, UK) were cultured as described,³ and fully confluent HUVECs at passage 2 or 3 were preincubated with 0.3% fetal bovine serum without other supplements overnight. Cells were subsequently washed with medium and incubated with VEGF (R&D Systems Ltd). Cells were washed twice with phosphate-buffered saline before RNA or protein extraction.

Oligonucleotide Microarrays
Total RNA was extracted with TRIzol reagent (Invitrogen) and purified with use of a commercially available kit (RNeasy kit, Qiagen). Preparation of cRNA, hybridization, and scanning of Affymetrix GeneChips (U133A array) representing 18 000 human genes were performed according to the manufacturer’s instructions (Affymetrix). Preparation and hybridization of cRNA to arrays were performed in triplicate at each time point with RNA from 3 independent cell cultures and 3 arrays. Arrays at each time point were then compared with 3 control arrays hybridized with 1 of 3 RNAs from untreated cells (3x3 comparison).

Data from arrays were analyzed with Microarray Suite Software5.0 (Affymetrix) and Data Mining Tool Software (Affymetrix). Transcript abundance was determined from the average of the differences between perfect match and mismatch intensities for each probe family. For comparison of 2 chips, the software generated a change probability value, a difference call, and a signal log ratio. Nine pairwise comparisons (3 control compared with each of 3 VEGF RNAs) were performed for each time point. A gene was considered differentially expressed according to the following criteria: (1) a change in probability value <0.003 (increased expression) or >0.997 (decreased expression); (2) an increase or decrease in at least 8 of 9 cross comparisons; and (3) an average signal log ratio in 9 cross comparisons of >1 (>2-fold increase) or <-1 (>2-fold decrease).

RT and Real-Time PCR
Total RNA was extracted with an RNeasy kit (Qiagen), and after DNAse I treatment, single-stranded cDNA was synthesized from 2 µg total RNA with an oligo(dT)_12–18 primer and the SuperScript First-Strand Synthesis System (Invitrogen) and diluted for real-time PCR. Primers for real-time PCR (Genosys, Sigma) were designed by Primer3 software (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi) with the 3'-untranslated gene sequence to ensure the specificity of the fragment and that amplified fragments were 200 to 250 bp. Amplification of predicted fragments was verified by conventional RT-PCR. Real-time PCR was performed with the LightCycler-FastStart DNA Master SYBR Green I kit and Lightcycler system (Roche Diagnostics). For each primer pair, a melting curve was used to identify a temperature at which only the amplicon, and not the primer dimers, accounted for SYBR green–bound fluorescence. Real-time RT-PCR was conducted in duplicate for each sample with RNA preparations from at least 2 independent experiments. Data were normalized to the reference gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and presented as the mean fold change (±SD) compared with control.

Preparation of Nuclear and Cytosolic Fractions
Cells were collected by centrifugation (13 000 rpm for 1 minute), resuspended in 400 µL cold buffer A (10 mmol/L HEPES. pH 7.9; 10 mmol/L KCl; 0.1 mmol/L EDTA; 0.1 mmol/L EGTA; 1 mmol/L dithiothreitol; and a protease inhibitor mixture [Complete, Roche Diagnostics]), and incubated on ice for 15 minutes. Cells were solubilized with 25 µL of 10% Igepal CA630. The homogenates were centrifuged (13 000 rpm for 1 minute), supernatants (cytosolic fraction) were stored at -80°C, and nuclear pellets were solubilized with buffer B (20 mmol/L HEPES, pH 7.9; 0.4 mol/L NaCl; 1 mmol/L EDTA; 1 mmol/L EGTA; 1.5 mmol/L MgCl₂; 20% glycerol; 1 mmol/L dithiothreitol; and a protease inhibitor mixture [Complete]). After centrifugation (13 000 rpm for 5 minutes), supernatants (nuclear protein) were stored at -80°C. Protein concentration was determined by the bicinchoninic acid protein assay (Pierce).

Immunoblotting
Immunoblotting was performed as described.³ Membranes were incubated at 4°C overnight with 2 µg/mL phosphoserine 351–Nur77 antibody (Santa Cruz Biotechnology Inc); 0.25 µg/mL Nurr1 antibody (BD Biosciences); Nur77 antibody at 1:250 dilution (Active Motive); and antibody to extracellular signal–regulated kinases (ERKs) 1 and 2 at 1:500 dilution (Cell Signaling Technology Inc). Immunoreactive bands were detected by chemiluminescence with enhanced chemiluminescence plus reagents (Amersham Plc).

Materials
Growth factors were from R&D Systems Ltd. SU5614, cyclosporin A, GF109203X, U0126, rapamycin, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), and [1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) were from Calbiochem Inc. Antibodies to -tubulin and lamin-A were from Santa Cruz Inc.

	Results

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Oligonucleotide Array and Real-Time RT-PCR Analysis of VEGF-Regulated Gene Expression
The results of a 3x3 comparison between Affymetrix oligonucleotide arrays hybridized with RNA from VEGF-treated and control HUVECs showed a significant >2-fold VEGF-induced upregulation of 88 different genes and a >2-fold downregulation of 100 genes. The results for selected genes induced by >3-fold are shown in online Table I (please see http://atvb.ahajournals.org).

Transcriptional regulators made up the largest functional group of VEGF-regulated genes at 45 minutes and also comprised the most strongly induced set of genes (online Table I). In particular, at 45 and 90 minutes, VEGF markedly induced expression of genes encoding 3 related nuclear receptors, Nur77 (also known as NR4A1, NGFI-B, NAK-1, TR3), Nurr1 (NR4A2, MINOR), and Nor1 (NR4A3, NOT) and Egr3, a member of the Egr family of zinc-finger transcription factors.^14–17

Further analysis of VEGF-regulated expression of selected genes was performed by real-time, quantitative RT-PCR with gene-specific primer pairs (online Table II; please see http://atvb.ahajournals.org) and GAPDH as a reference gene, which was not significantly affected by VEGF (results not shown). The time courses of VEGF-induced expression of Nur77, Nurr1, Nor 1, and Egr3 were very similar, with detectable expression after 10 to 20 minutes and maximum expression at approximately 45 minutes, after which expression declined, remaining significantly increased after 90 minutes and decreasing to near control levels after 3 hours (Figure 1). In 4 independent experiments, the maximum VEGF-induced fold-increases in expression of Nur77, Nurr1, Nor1, and Egr3 above control levels were 40, 130, 60, and 220, respectively. The receptor specificity of VEGF-induced gene expression was examined with the specific KDR inhibitor SU5614. As shown in Figure 1, SU5614 markedly inhibited the induction of Nur77, Nurr1, Nor1, and Egr3. Treatment of HUVECs parallel to those used in Figure 1 with placental growth factor (P1GF) or basic fibroblast growth factor (bFGF) weakly increased expression of Nur77, Nurr1, Nor1, and Egr3 to maximum values of 2.5-, 3.5-, 3.0-, and 7.5-fold above controls, respectively, for P1GF and 2-, 7-, 8-, and 25-fold above controls for bFGF (online Figure I). Real-time RT-PCR also confirmed VEGF-induced expression of several other genes identified from arrays, including Down syndrome candidate region 1 (DSCR1), cyclooxyganase-2, tissue factor, stanniocalcin-1, eps 15 homology domain-containing protein, and Cot (online Figures II through IV; please see http://atvb.ahajournals.org).

View larger version (19K): [in this window] [in a new window]   Figure 1. VEGF-induced expression of NR4A nuclear receptors and Egr3. HUVECs were treated with 25 ng/mL VEGF for the times indicated, and real-time, quantitative RT-PCR was performed for Nur77, Nurr1, Nor1, and Egr3. Insert: Cells were pretreated with dimethyl sulfoxide (control [con], VEGF) or with 5 µmol/L SU5614 (V+SU) for 30 minutes and then treated for a further 45 minutes with either no addition (con) or with 25 ng/mL VEGF (VEGF and V+SU). Results are expressed as mean fold-increases ±SD above control level normalized to GAPDH expression or to (insert) the percentage of VEGF-induced expression at 45 minutes. Results are representative of 4 independent experiments, each performed on duplicate samples. The effects of SU5614 on NR4A and Egr3 genes were highly significant (P<0.0002).

Mechanism of VEGF-Induced Nuclear Receptor Expression
The intracellular mechanisms mediating VEGF-induced gene expression were investigated with the use of inhibitors of several signal transduction pathways. Nuclear receptor expression was not inhibited by rapamycin, whereas the phosphatidyl inositol 3-kinase inhibitor LY294002 reduced Nor 1 expression by 35% but had little effect on Nur77 and Nurr1 (Figure 2). The mitogen-activated protein kinase/ERK inhibitor U0126 caused a significant 59%, 47%, and 55% inhibition of Nur77, Nurr1, and Nor1, respectively. In contrast, the protein kinase C inhibitor GF109203X, the cell-permeable Ca²⁺ chelator BAPTA-AM, and the calcineurin inhibitor cyclosporin A all strikingly inhibited expression of NR4A genes. In addition, the Src inhibitor PP2 significantly enhanced VEGF-induced expression of Nur77, Nurr1, and Nor1 by 2.8-, 2.9-, and 1.6-fold, respectively. The protein synthesis inhibitor cycloheximide also augmented VEGF-induced nuclear receptor expression.

View larger version (20K): [in this window] [in a new window]   Figure 2. Signaling pathways mediating VEGF-induced nuclear receptor expression. HUVECs were pretreated for 30 minutes with dimethyl sulfoxide (con, V), 3 µmol/L GF109203X (GF), 20 µg/mL BAPTA-AM (BP), 10 µmol/L U0126 (U0), 500 ng/mL cyclosporin A (Cs), 10 µmol/L LY294002 (Ly), 20 nmol/L rapamycin (Rp), 3 µmol/L PP2 (PP), or 5 µmol/L cycloheximide (CX) and then treated for 45 minutes with nothing (con), 25 ng/mL VEGF alone (V), or 25 ng/mL VEGF in the presence of inhibitors. Some cells were treated with PP2 in the absence of VEGF (PP). Real-time RT-PCR was performed for Nur77, Nurr1, and Nor1. Results are representative of 2 to 4 independent experiments, each performed on duplicate samples. The dotted line indicates maximum gene expression stimulated by VEGF alone (100%). Probability values for VEGF vs VEGF+GF109203X, VEGF+BAPTA-AM, VEGF+U0126, and VEGF+cyclosporin A were all <0.00025 for Nur77, Nurr1, and Nor1 expression. For Nor1 only, P<0.0007 for VEGF vs VEGF+LY294002. P<0.05 for VEGF vs VEGF+PP2 for Nur77, Nurr1, and Nor1. Other effects were not significant

Egr3 expression was strongly decreased by GF109203X, BAPTA-AM, and U0126, whereas cyclosporin A had no significant effect (Figure 3). Rapamycin and LY294002 had little effect on Egr3 induction, but expression was significantly enhanced by PP2 and cycloheximide. VEGF-induced expression of DSCR1, encoding the calcineurin inhibitor, myocyte-enriched calcineurin inhibitory protein 1 (MCIP1),¹⁸ was strongly inhibited by cyclosporin A, BAPTA/AM, and GF109203X; partially inhibited by U0126; and also significantly reduced by PP2 (Figure 3).

View larger version (22K): [in this window] [in a new window]   Figure 3. Signaling pathways mediating VEGF-induced Egr3 and DSCR1 expression. HUVECs were pretreated with inhibitors and subsequently with 25 ng/mL VEGF as indicated in the legend to Figure 2. Real-time RT-PCR was performed for Egr3 and DSCR1. Results are representative of 2 to 4 independent experiments, each performed on duplicate samples. The dotted line indicates maximum gene expression stimulated by VEGF alone (100%). Probability values for Egr3 for VEGF vs VEGF+GF109203X, VEGF+BAPTA-AM, VEGF+U0126, and VEGF+PP2 are, respectively, <8.2x10-7, 9x10-9, 3.5x10-7, and 0.04. Cyclosporin A, LY294002, and rapamycin had no significant effect on Egr3 expression (P>0.09). For DSCR1, P<0.025 for VEGF vs VEGF+GF109203X, VEGF+BAPTA-AM, VEGF+U0126, VEGF+cyclosporin A, VEGF+PP2, and VEGF+SU5614 (result not shown). LY294002 and rapamycin had no significant effect on DSCR1 expression (P>0.79).

VEGF Regulation of Nur77 and Nurr1 Protein Expression and Phosphorylation
Western blotting showed that Nur77 protein increased after a 30-minute treatment with VEGF, reached a maximum after 3 hours, and remained above basal levels for up to 6 hours (Figure 4A). Nur77 is phosphorylated in T lymphocytes and PC12 cells, and phosphorylation at serine 351 within the DNA-binding domain has been shown to inhibit Nur77 DNA binding and transactivating activity.^19,20 Western blotting with antibodies to Nur77 phosphorylated at serine 351 showed that concomitantly with increased Nur77 expression, Nur77 serine 351 phosphorylation decreased in response to VEGF (Figure 4A). A reduction in serine 351 phosphorylation was evident after 1 hour and reached a maximum after 6 hours. VEGF-induced Nurr1 protein expression in total cell extracts was modestly increased after 3 and 6 hours (results not shown). To examine whether VEGF might preferentially increase Nurr1 expression in a nuclear pool, Nurr1 blots were performed in nuclear and cytosolic extracts. VEGF-induced Nurr1 protein expression was detectable in a crude nuclear fraction after 90 minutes, reached a maximum after 3 hours, and remained above control levels for 24 hours, whereas cytosolic Nurr1 showed little change (Figure 4B).

View larger version (52K): [in this window] [in a new window]   Figure 4. VEGF-induced Nur77 and Nurr1 protein expression and decreased Nur77 phosphorylation at serine 351. A, HUVECs were untreated (control, C) or treated with 25 ng/mL VEGF for the times indicated; total cell lysates were prepared; and equal amounts of protein were immunoblotted with antibodies to total Nur77, Nur77 phosphorylated at serine 351, or total ERKs 1 and 2. The results shown are representative of 2 independent experiments. Fold-changes relative to control are indicated below each lane. B, HUVECs were treated with 25 ng/mL VEGF for the times indicated, nuclear (Nuc) and cytosolic (Cyt) fractions were prepared, and equal amounts of protein were immunoblotted with antibody to total Nurr1. Cytosolic and nuclear extracts were also blotted with antibodies to -tubulin and lamin A, respectively. The results shown are representative of 2 independent experiments.

	Discussion

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Array analysis identified several known VEGF-induced genes, including cyclooxygenase-2, heparin-binding epidermal growth factor, tissue factor, and interleukin-8.^10–13 Five of the VEGF-regulated genes reported here (Cot, Nur77, DSCR1, dual specificity phosphatase 5, and Egr3) were increased by VEGF in HUVECs in a cDNA microarray representing 7267 human genes,²¹ and stanniocalcin-1 was upregulated by a 24-hour VEGF treatment in an oligonucleotide array representing 8900 genes.²² Our results showed limited overlap with other studies of VEGF-regulated gene expression.^23–25 Stanniocalcin-1, interleukin-8, phospholipase A₂-, CAT1, cyclooxygenase-2, and ₂-macroglobulin were also induced in models of capillary morphogenesis.^26–29

A major finding of this study is that VEGF strikingly induced mRNA expression of Nur77, Nurr1, and Nor1, 3 closely related members of the NR4A family of nuclear receptors. The conclusion that KDR mediates VEGF-induced nuclear receptor expression is supported by inhibition of gene expression by the specific KDR inhibitor SU5614 and the lack of effect of the Flt-1–specific ligand P1GF. Consistent with the contention that NR4A family members might play a functional role in the endothelial response to VEGF, VEGF induced expression of Nur77 and Nurr1 protein and concomitantly decreased Nur77 phosphorylation at serine 351, a negative regulatory site that inhibits Nur77 transcriptional activity.^19,20 The mechanism of VEGF-induced Nur77 dephosphorylation is unclear but might involve activation of a serine/threonine phosphatase. It is noteworthy that Akt has been shown to phosphorylate Nur77 at serine 351 and inhibit its transcriptional activity in T cells and Nur77-overexpressing cells.^30,31 Because induction of Nur77 mRNA was independent of the phosphatidyl inositol 3-kinaseK/Akt pathway and VEGF did not stimulate Nur77 phosphorylation, our results suggest that Akt-mediated Nur77 phosphorylation does not occur in response to VEGF.

Nur77 was originally identified as an immediate-early response gene induced by nerve growth factor, corticotropin, and membrane depolarization,^32,33 whereas Nurr1 expression is induced by PGE₂ and parathyroid hormone.^34,35 Differential regulation of members of this family through distinct signaling pathways is suggested by the finding that platelet-derived growth factor and bFGF had only a modest effect on Nurr1 expression, whereas PGE₂ selectively induced Nurr1 expression with little effect on Nur77 and Nor1.³⁴ VEGF might be unusual in causing strong induction of all 3 members of the NR4A nuclear receptor subfamily. Because bFGF had little effect on expression of NR4A genes, upregulation of these genes might play a specific role in VEGF-mediated endothelial biologic functions.

The marked inhibition of Nur77, Nurr1, and Nor1 expression by specific inhibitors of protein kinase C, increased intracellular [Ca²⁺], and calcineurin indicates that these signals play key roles in the pathway mediating their induction by VEGF. Previous findings have demonstrated central roles for protein kinase C and mobilization of intracellular Ca²⁺ in mediating VEGF stimulation of early signaling events, such as ERK activation and later cellular responses,^3,4,6 whereas calcineurin-dependent activation of nuclear factor of activated T cell transcription factors is implicated in VEGF-induced expression of cyclooxygenase-2.¹⁰ It is likely that rapid mobilization of intracellular Ca²⁺ is a key mediator of calcineurin activity in response to VEGF. The signaling mechanisms mediating VEGF regulation of NR4A genes partly diverged from those responsible for expression of Egr3 and DSCR1. VEGF-induced expression of Egr3 and DSCR1 was mediated by protein kinase C, intracellular Ca²⁺, and ERK but largely unaffected by cyclosporin A, whereas DSCR1 expression was strikingly cyclosporin A–sensitive and, unlike either Egr3 or NR4A genes, was partially inhibited by PP2. These results indicate that VEGF signaling leading to expression of NR4A genes and DSCR1 converges through a calcineurin-mediated pathway, whereas the mechanism underlying Egr3 expression diverges proximal to calcineurin, and DSCR1 expression might also be regulated via Src (Figure 5). Because DSCR1 encodes the calcineurin inhibitor MCIP1,¹⁸ VEGF-induced DSCR1 induction via calcineurin might participate in feedback inhibition of calcineurin-mediated endothelial gene expression.

View larger version (24K): [in this window] [in a new window]   Figure 5. Signaling mechanisms mediating VEGF-regulated endothelial gene expression. Signaling pathways mediating VEGF-induced expression of the NR4A nuclear receptors Nur77, Nurr1, and Nor1; Egr3; and DSCR1 are shown schematically. Activation of phospholipase C (PLC-) and subsequent mobilization of intracellular Ca2+ and activation of PKC and ERK play key roles in VEGF-induced gene expression. Ca2+ is essential for activation of calcineurin, which mediates expression of NR4A family members and DSCR1, whereas Egr3 expression is largely unaffected by cyclosporin A. ERK activation is important for Egr3 induction and contributes to NR4A receptor expression, and VEGF-induced DSCR1 expression is also partially mediated via an Src-dependent pathway. Calcineurin-mediated activation of NFAT (nuclear factor of activated T cells) transcription factors has also been reported to mediate VEGF-induced cyclooxygenase (COX)-2 expression.

Neither the biologic functions of NR4A family nuclear receptors nor their target genes are understood. Gene knockout and transgenic mice studies point to a role for Nurr1 in development of dopamine neurons and redundant roles for Nur77 and Nor1 in T-cell apoptosis.^15,36,37 Although NR4A genes have no clearly defined role in VEGF or vascular function, they might function in partially redundant and compensatory functions important for regulation of endothelial gene transcription. This possibility is supported by recent findings that Nur77 is implicated in both tumor necrosis factor-–induced expression of plasminogen activator inhibitor-1 and control of endothelial cell proliferation.^38,39

Although little is currently known regarding the early and later gene-expression events stimulated by VEGF, it is likely that induction of transcriptional regulators will play a pivotal role in triggering and coordinating its complex, long-term, biologic effects. The VEGF-regulated genes identified here will be important candidates for the key early mediators of VEGF-induced angiogenesis and might also have implications for the mechanisms underlying the roles of VEGF in endothelial progenitor cell differentiation and hematopoietic stem cell development.

	Acknowledgments

 
Acknowledgments

This work was supported by British Heart Foundation grant BS/94001 to I.Z.

	Footnotes

 
*These authors contributed equally to this work.

Received July 22, 2003; accepted August 26, 2003.

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