CCL2 Regulates Angiogenesis via Activation of Ets-1 Transcription Factor Svetlana M. Stamatovic, Richard F. Keep, Marija Mostarica-Stojkovic and Anuska V. Andjelkovic This information is current as of October 2, 2021. J Immunol 2006; 177:2651-2661; ; doi: 10.4049/jimmunol.177.4.2651 http://www.jimmunol.org/content/177/4/2651 Downloaded from References This article cites 54 articles, 16 of which you can access for free at: http://www.jimmunol.org/content/177/4/2651.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

CCL2 Regulates Angiogenesis via Activation of Ets-1 Transcription Factor1

Svetlana M. Stamatovic,* Richard F. Keep,*† Marija Mostarica-Stojkovic,§ and Anuska V. Andjelkovic2*‡

Although recent studies have suggested that CC chemokine CCL2 may directly affect the angiogenesis, the signaling events involved in such regulation remain to be determined. This study investigated a potential signal mechanism involved in CCL2- induced angiogenesis. Our in vitro and in vivo (hemangioma model of angiogenesis) experiments confirmed earlier findings that CCL2 can induce angiogenesis directly. Using a gene array analysis, CCL2 was found to induce expression of several angiogenic factors in brain endothelial cells. Among the most prominent was an up-regulation in Ets-1 transcription factor. CCL2 induced a significant increase in Ets-1 mRNA and protein expression as well as Ets-1 DNA-binding activity. Importantly, Ets-1 antisense oligonucleotide markedly abrogated in vitro CCL2-induced angiogenesis, suggesting that Ets-1 is critically involved in this process. ␤ Downloaded from Activation of Ets-1 by CCL2 further regulated some of Ets-1 target molecules including 3 integrins. CCL2 induced significant ␤ up-regulation of 3 mRNA and protein expression, and this effect of CCL2 was prevented by the Ets-1 antisense oligonucleotide. The functional regulation of Ets-1 activity by CCL2 was dependent on ERK-1/2 cascade. Inhibition of ERK1/2 activity by PD98509 prevented CCL2-induced increases in Ets-1 DNA-binding activity and Ets-1 mRNA expression. Based on these findings, we suggest that Ets-1 transcription factor plays a critical role in CCL2 actions on brain endothelial cells and CCL2-induced angiogenesis. The Journal of Immunology, 2006, 177: 2651–2661. http://www.jimmunol.org/

ngiogenesis, the formation of new blood vessels from be fully elucidated. Until very recently, it was generally accepted preexisting blood vessels, takes place in many physio- that CCL2 indirectly stimulates angiogenesis via its chemoattrac- A logical and pathological conditions such as embryo de- tant effects on monocytes/macrophages, which in turn may release velopment, ovulation, wound healing, rheumatoid arthritis, dia- direct-acting angiogenic factors (15, 16, 18). However, a few re- betic proliferative retinopathy, and tumorigenesis (1–5). cent studies have suggested that CCL2 may also exert direct an- Angiogenesis consists of series of highly ordered and tightly reg- giogenic effects (17). Support for this hypothesis comes from the ulated events including degradation of the existing basement mem- fact that endothelial cells express the CCL2 receptor CCR2

brane, chemotaxis, proliferation, and capillary tube formation (6). (19–21). by guest on October 2, 2021 Many factors have been found to influence angiogenesis. Some The current study was aimed at identifying critical intracellular have a stimulatory effect on angiogenesis, including vascular en- signaling molecules that might be involved in CCL2-induced an- dothelial growth factor (VEGF),3 basic fibroblast growth factor giogenesis. Attention was focused on Ets-1 because this transcrip- (bFGF), angiopoietin-1, ELRϩ CXC chemokines IL-8, NAP-2, tional factor is believed to be involved in regulating angiogenesis. ENA-78 and GRO (7–14). Others, such as endostatin, angiostatin, and ELRϪ chemokines IFN-␥-inducible protein and monokine in- Materials and Methods duced by IFN-␥, inhibit angiogenesis (13–14). All procedures were performed in strict accordance with the National In- Recently, the CC chemokine MCP-1 (CCL2/MCP-1/JE) has stitute of Health’s Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of been added to the growing list of angiogenic modulators (15–18). University of Michigan. Best known for its role in modulating inflammatory responses by inducing monocyte/macrophage recruitment to sites of inflamma- Cells tion, CCL2 also has a distinct role in angiogenesis. The molecular Brain endothelial cell line (bEnd.3) was purchased from American Type mechanisms by which CCL2 regulates angiogenesis have still to Culture Collection. Cells were grown in medium containing DMEM, 10% heat-inactivated FBS, 1ϫ antibiotic/antimycotic, and 2 mM glutamine (all purchased from Invitrogen Life Technologies). Cells from 22 to 25 pas-

† ‡ sages were used for all experiments. Primary cell cultures of brain micro- *Department of Neurosurgery, Molecular and Integrative Physiology and Pathol- vascular endothelial cells were prepared from CCR2 knockout mice or ogy, University of Michigan, Medical School, Ann Arbor, MI 48109; and §Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Bel- wild-type mice by a method already established in our laboratory and de- grade, Serbia and Montenegro scribed in detail previously (21). Received for publication December 29, 2005. Accepted for publication May 23, 2006. In vitro angiogenesis assay The costs of publication of this article were defrayed in part by the payment of page This assay was performed using previously described methods (22, 23). charges. This article must therefore be hereby marked advertisement in accordance Bovine fibrinogen (at final concentration of 2.5 mg/ml; Sigma-Aldrich) with 18 U.S.C. Section 1734 solely to indicate this fact. was dissolved in DMEM supplemented with aprotinin (200 ␮g/ml; Sigma- 1 This work was supported by Grant NS 044907 (to A.V.A.) from the National In- Aldrich) and dispensed into 12-well tissue culture plates. Polymerization stitutes of Health. was induced by addition of thrombin (25 U/ml; Sigma-Aldrich) for1hat 2 Address correspondence and reprint requests to Dr. Anuska V. Andjelkovic, De- 37°C. After that, bEnd.3 cells were seeded onto the fibrin gel at 5 ϫ partment of Neurosurgery and Pathology, University of Michigan, MI 48109. E-mail 104/well and allowed to attach for up to 2 h. The medium was then care- address: [email protected] fully removed, and fibrin solution, mixed with test agent(s), was added to 3 Abbreviations used in this paper: VEGF, vascular endothelial growth factor; bFGF, the cells. Fresh medium with matched test agent(s) was added on top of the basic fibroblast growth factor; uPA, urokinase. generated fibrin gel overlay, and tube formation was assessed 24 h later.

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 2652 MECHANISMS OF CHEMOKINE-INDUCED ANGIOGENESIS

The total length of tubular structures was calculated in five different areas nucleotide probe containing the DNA-binding motif for Ets-1 (provided by using Image J software (National Institutes of Health) (23). Panomics). For competition experiments and supershift assays, excess of For blocking experiments, bEnd.3 cells were first incubated in the se- the corresponding cold (unlabelled) probe or ant-Ets-1 Ab (2 ␮g/ml; Santa rum-free medium containing PD 98059 (20 ␮M; Calbiochem) for1hor Cruz Biotechnology) was added to the binding reactions and incubated for treated with antisense or sense oligonucleotides as described below. They 5 min before addition of the labeled probes. Reactions were subjected to were then trypsinized and plated onto the fibrin gels. electrophoresis on 6% polyacrylamide gel in 0.5ϫ Tris-borate-ethylene diamine tetraacetic acid (TBE) buffer followed by electroblotting onto ny- Hemangioma formation in nu/nu mice lon membranes (Biodyne B membrane) and UV cross-linking. Biotinylated Male nude mice (nu/nu; provided by The Jackson Laboratory), 10–12 wk oligonucleotides were then detected by probing with streptavidin conju- of age, were used. Mice were anesthetized with i.p. ketamine/xylazine (100 gated to HRP and visualized by ECL and autoradiography. mg/kg, 5 mg/kg). They were then inoculated with 0.5 ml of bEnd.3 cell Transfection suspension at 5 ϫ 106 cells/ml s.c. in the right femoral flank. Over 3 wk, hemangioma size was measured with caliper three times per week, and Ets-1 antisense and sense phosphorothioate oligonucleotides were pur- hemangioma weight (in grams) was estimated by the following formula: chased from Oligos Etc. The sequences used were as follows: sense, 5Ј- 2 length ϫ width /2. Experiments were terminated when hemangioma ATGAAGGCGGCCGTCGATCT-3Ј; and antisense, 5Ј-AGATCGACG weight reached ϳ2 g (3 wk postinoculation). The hemangiomas were ex- GCCGCCTTCAT-3Ј. Briefly, when bEnd.3 cell culture reached ϳ90% cised, weighed, photographed, frozen in OCT-embedding medium, and confluence, the cells were washed once with serum-reduced OptiMEM I processed for H&E staining and immunohistochemistry. Hemangioma tis- medium. The transfection of oligonucleotides was then performed with sue was also taken for RT-PCR analysis. OptiMEM I medium containing 500 nM oligonucleotides and 12 ␮g/ml To establish the contribution of CCL2 to hemangioma growth and an- lipofectamine (Invitrogen Life Technologies). Cells were incubated with giogenesis, inhibition studies were performed where (nu/nu) mice bearing o the transfection medium for4hat37C, 5% CO2, before changing back to bEnd.3 cells received either CCL2 antisense (5Ј-AAGCGTGACAGAG normal medium. After an additional incubation for 20 h, cells were used in Ј ␮ ACCTGCATAGTCGTGG-3 ) phosphorothioate oligonucleotide (5 M blocking experiments. Efficiency of transfection was established by West- Downloaded from each; Oligos Etc.) into the peritumor (hemangioma) area or a monoclonal ern blot analysis and EMSA. anti-CCL2-neutralizing Ab (25 ␮g/ml/mouse; R&D Systems) i.p. at days 1, 6, 9, 12, 21 after inoculation. Controls received either sense phosphoro- Western blotting thioate oligonucleotide (5Ј-CCACCACTATGCAGGTCTCTGTCACG TTT-3Ј,5␮M; Oligos Etc.) or vehicle (PBS). Cells were lysed in radioimmunoprecipitation assay buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, Proliferation assay 0.1% SDS, and 2 mM sodium orthovanadate) containing protease inhibi-

tors (10 ␮g/ml leupeptin, 10 ␮g/ml aprotinin, 1 mM EDTA, and 1 mM http://www.jimmunol.org/ Briefly, bEnd.3 cells were cultured in complete medium at a density of 1 ϫ 5 PMSF). The protein content was determined using Pierce protein assay kit. 10 cells/ml on 96-well microplates. After 12 h, the medium was removed Equal amounts of protein were electrophoretically separated by SDS- and replaced with DMEM containing 0.5% FBS plus murine recombinant PAGE on 7.5 or 10% gels and transferred to Trans-Blot nitrocellulose CCL2 or VEGF (PreproTech) at different concentrations (12.5–400 ng/ml) membrane (Bio-Rad). Immunoblotting was performed using rabbit anti- for 16 h. Medium alone was added as a negative control. bEnd.3 cell Ets-1 Ab (Santa Cruz Biotechnology), anti- Ets-1 phospho-(T38) Ab proliferation was measured using a colorimetric BrdU incorporation assay (Novus Biological), anti-phospho-ERK1/2 Ab (Cell Signaling Technol- (Cell Proliferation ELISA; Roche) according to the manufacturer’s ␤ ogy), or hamster anti-mouse CD61 (integrin 3 chain) Ab (BD Bio- instructions. sciences). Immunoreactive proteins were visualized using an ECL detec- Chemotaxis assay tion kit (Pierce). Autoradiographic images underwent semiquantitative densitometry analysis using National Institutes of Health image software Chemotaxis assays were performed in BD BioCoat Fibronectin Cell Cul- package (version 1.63). After probing for pERK1/2 or phospho-Ets-1 to by guest on October 2, 2021 ture Inserts (BD Biosciences). Briefly, bEnd.3 cells or CCR2Ϫ/Ϫ mouse examine ERK activation status or level of phosphorylated Ets-1, immuno- brain microvascular endothelial cells (5 ϫ 104 cells in 250 ␮l of DMEM blots were stripped using Restore Western Blot Stripping Buffer (Pierce containing 0.5% FBS) were added to the 3.0-␮m insert of the chemotaxis Biotechnology) and reprobed with rabbit anti-ERK1/2 Ab (Cell Signaling plates. Serial 2-fold dilutions of recombinant mouse CCL2 (range 0.8 to Technology) or anti-Ets-1 Ab. 400 ng/ml) in DMEM containing 0.5% FBS was added to the wells. VEGF (10 ng/ml) or medium alone was used as positive and negative controls. MAPK assays The cells were labeled with Calcein-AM (Molecular Probes) and allowed to migrate across a membrane insert for 22 h. The intensity of fluorescence In vitro MAPK assay was performed using a MAPK assay kit (Upstate was measured with a fluorescence plate reader (Applied Biosystems; Cyto- Biotechnology) according to the manufacturer’s recommendations. Flour 4000 plate reader) configured to read at excitation/emission wave- Immunofluorescence microscopy and histopathology lengths of 485/530 nm. The number of migrated cells was estimated from standard curve. Immunofluorescence staining was performed on 6-␮m-thick cryosections For blocking experiments, in addition to CCL2, medium was supple- of bEnd.3 cell-induced hemangiomas. The sections were preincubated in mented with 10 ␮g/ml neutralizing goat IgG anti-mouse CCL2 Ab (or the blocking solution (PBS containing 2% BSA and 0.5% Tween 20) for 1 h nonspecific isotype-matched control Ab; both obtained from R&D and then incubated with goat anti-mouse CCL2 Ab (R&D Systems), rat Systems). anti-mouse CD31 Ab (BD Biosciences), or rat anti-mouse F4/80 Ab (Se- cDNA array rotec). After overnight incubation at 4°C, the sections were washed and incubated with secondary Ab conjugated to FITC or Texas Red for1hat At different times after CCL2 treatment (0–16 h), bEnd.3 cells were har- room temperature. Sections were then analyzed by confocal microscopy vested, and total RNA was prepared using TRIzol reagents (Invitrogen Life (Zeiss; LSM 510). Some sections from each hemangioma were stained Technologies). The procedure for biotinylated cDNA probe synthesis was directly with H&E for standard histological examination. done using the AmpoLabeling-LPR kit (SuperArray Bioscience) according to the manufacturer’s instructions. The resulting cDNA probe was hybrid- RT-PCR ized to GEArray Q Series mouse angiogenesis gene array (SuperArray Total RNA was isolated from bEnd.3 cells or hemangioma using the Bioscience) according to the manufacturer’s instructions. The relative ex- TRIzol reagent (Invitrogen Life Technologies). Briefly, 2 ␮g from each pression level of each gene was analyzed using a software package pro- sample was reverse-transcribed into cDNA using an Invitrogen Life Tech- vided by SuperArray (SuperArray Bioscience). nologies cDNA Synthesis Kit, following the manufacturer’s protocol. Ets-1 EMSA cDNA was amplified as a 510-bp fragment by PCR with the forward primer 5Ј -TACCCTTCCGTCATTCTCC-3Ј and reverse primer 5Ј- TTTT Ј ␤ Nuclear extracts were prepared from bEnd.3 cells using a Nuclear Extract TCCTCTTTCCCCATC-3 . 3-integrin cDNA was amplified as 430 bp Kit (Active Motif) according to the manufacturer’s instructions. The pro- with the forward primer 5Ј-GGGGACTGCCTGTGTGACTC-3Ј and re- tein concentration was determined using the Bio-Rad protein assay. EMSA verse primer 5Ј-CTTTTCGGTCGTGGATGGTG-3. Samples were stan- was performed with the Panomics EMSA kit (Panomics) according to man- dardized using primers specific to cDNA encoding mouse ␤-actin. A total ␤ ufacturer’s protocol. In brief, Ets-1-binding reactions were conducted with of 40 cycles for Ets-1 and 30 cycles for 3 integrin were applied. The PCR 5 ␮g of nuclear proteins and 10 ng of biotinylated double-stranded oligo- cycles included 1-min denaturation at 94°C, 1-min annealing at 55°C, and The Journal of Immunology 2653

1-min extension at 72°C, except for the first cycle, which had 2-min de- tube formation was comparable to that observed after stimulation naturation, and the last cycle with 10-min elongation. The PCR products with VEGF, a known potent angiogenic factor (Fig. 2). ϫ were resolved using electrophoresis on a 2% agarose gel in 1 TBE buffer If CCL2 acts as a direct angiogenic factor, then it is expected (Tris-HCl/EDTA/boric acid; pH 8). The gel was stained with ethidium bromide and photographed. that endothelial cells possess CCR2, the receptor for CCL2. As already stated, CCL2 exerts its biological effects on endothelial ELISA cells through CCR2 (19–21). Fig. 3A shows that there is a low, Supernatants collected from in vitro angiogenic assays in fibrin gel were constitutive expression of the CCL2 and CCR2 transcripts in assayed for VEGF level by ELISA, using the mouse VEGF Quantikine kit bEnd.3 cells. However, CCL2 treatment of bEnd.3 cells for 4 h (R&D Systems). induced a significant increase in CCR2 expression. This increase in Statistics CCR2 expression was comparable to that observed after stimula- tion of bEnd 3 cells with TNF-␣ (10 ng/ml). Statistical analyses were performed using commercially available software Analyzing the hemangioma tissue, we also found a significant (Stat-View; SAS Institute). One-way ANOVA was used to compare the mean responses among the experimental groups. The Dunnett’s test was amount of CCL2 and CCR2 mRNA expression, the level of which used to determine significance between groups. was diminished by applying CCL2 antisense phosphorothioate oli- gonucleotide (Fig. 3A). Results Conditioned media were collected from bEnd 3 cells seeded on CCL2 as potent angiogenic factor the fibrin gel after 0–24 h of treatment with CCL2 and analyzed To examine the potential angiogenic effect of CCL2, the expres- for VEGF by ELISA. We found that the amount of VEGF was not significantly different between control (nontreated bEnd3 cells) sion and effect of CCL2 on hemangioma development in vivo was Downloaded from examined. Hemangiomas offer a unique model to study angiogen- and CCL-2-treated samples (data not shown). esis. They are a primary tumor of microvasculature in which an- CCL2 also induced proliferation of bEnd.3 cell in a concentra- giogenesis is initially excessive followed by inhibition and regres- tion-dependent manner (Fig. 3B). Quantification of incorporated sion of the newly formed blood vessels. Injection of 5 ϫ 106 BrdU in bEnd.3 cells indicated peak proliferation at CCL2 con- Polyoma middle T-transformed endothelial cells (bEnd.3 cells) s.c. centration of 50 ng/ml ( p Ͻ 0.01 vs control). This proliferative response was comparable to that observed with VEGF (50 ng/ml). in the right femoral flank of nu/nu mice was sufficient to induce http://www.jimmunol.org/ hemangioma formation over 21 days. As shown in Fig. 1A, the This strongly suggests that CCL2 has a potent mitogenic effect on hemangiomas were cystic vascular structures with high levels of bEnd.3 cells. The proliferative effect of CCL2 was blocked in the CCL2 in infiltrated macrophages (F4/80ϩ cells) and endothelial presence of CCL2-neutralizing Ab, and it was also CCR2 depen- cells (CD31ϩ cells). To establish that CCL2 plays a significant role dent. In experiments where brain endothelial cells were prepared Ϫ Ϫ in hemangioma formation, on days 1, 6, 9, 12, and 21 after bEnd.3 from mice genotype CCR2 / , CCL2 was not able to induce a injection mice received neutralizing anti-CCL2 Ab or CCL2 anti- proliferative response (Fig. 3B). sense phosphorothioate oligonucleotide. Inhibition of CCL2 activ- Stimulation of endothelial cell motility is a common feature of ity/synthesis markedly reduced hemangioma size (Fig. 1B). Thus, angiogenic factors. As shown in Fig. 3C, CCL2 also displays che-

CCL2 can exert a potent effect on hemangioma formation and motactic activity for endothelial cells. CCL2 induced directional by guest on October 2, 2021 angiogenesis. bEnd.3 cell migration (chemotaxis) in a concentration-dependent The effect of CCL2 on angiogenesis was further examined in manner (0.78–400 ng/ml). Maximal migration was obtained with vitro using a fibrin gel angiogenesis assay and bEnd.3 cells. CCL2 100 ng/ml CCL2. CCL2-induced bEnd.3 cell chemotaxis was sim- significantly enhanced the formation of tube-like structures com- ilar to that found with VEGF (Fig. 3C). Inhibiting CCL2 with an pared with controls (vehicle-treated cells; Fig. 2). The angiogenic anti-CCL2-neutralizing Ab completely blocked CCL2-enhanced response of bEnd 3 cells to CCL2 was dose dependent. After 24-h cell migration. In addition, brain endothelial cell migration toward stimulation with CCL2, the greatest increase in tube-like formation the CCL2 was CCR2 dependent. In the range of concentrations was observed at the concentration of 100 ng/ml. This increase in tested (0.78–400 ng/ml), CCL2 did not elicit migration of brain

FIGURE 1. CCL2 contributes to angiogenesis in vivo and induces angiogenesis in vitro. A, Hemangio- mas formed after inoculation of bEnd.3 cells into the right flank of a nu/nu mouse (see Material and Methods for details) were stained with H&E or used for fluores- cence immunohistochemistry using anti-CCL2, anti- F4/80 and anti-CD31 Abs. CCL2 was highly expressed in cystic walls of hemangioma, mostly in F4/80 positive macrophages and CD31 positive endothelial cells. Scale bars, 10 ␮m. B, Effect of inhibiting CCL2 activity using neutralizing Ab to CCL2 (25 ␮g/ml, i.p.) or CCL2 an- tisense phosphorothioate oligonucleotide (5 ␮M) on hemangioma growth. Photographs show hemangiomas at day 21. The graph shows the time course of heman- p Ͻ ,ء ;gioma weight. Values are mean Ϯ SD; n ϭ 6 .p Ͻ 0.001 vs nontreated bEnd.3 cells ,ءء ;0.01 2654 MECHANISMS OF CHEMOKINE-INDUCED ANGIOGENESIS

FIGURE 2. Effect of CCL2 on tube formation using an in vitro angiogenesis assay in fibrin gels. Media were supplemented with different CCL2 Downloaded from concentrations or VEGF or no growth factor (control). Tube formation was observed after 24 h and total tubular length was calculated (␮m). The graph shows average tubular length from five independent experiments, with each well being measured in five randomly selected areas. Values are mean Ϯ SD; p Ͻ 0.001 vs control. Scale bar, 100 ␮m ,ءء

Ϫ Ϫ endothelial cells prepared from CCR2 / mice. Thus, the chemo- Tie1, Tie2. Due to this, further studies focused on the activity of http://www.jimmunol.org/ tactic response of brain endothelial cells to CCL2 stimulation is Ets-1 during CCL2-induced angiogenesis. linked to the expression of CCR2 in these cells. Taken together, these results indicate that CCL2 may act as a CCL2-induced angiogenesis through activation of Ets-1 potent direct angiogenic factor. In subsequent experiments to in- transcription factor vestigate the mechanisms underlying such angiogenic activity, 100 To begin to investigate whether Ets-1 plays a critical role in CCL2- ng/ml CCL2 was used because this induced the peak angiogenic induced angiogenesis, Ets-1 mRNA and protein expression as well response. as Ets-1 activation (phosphorylation) and Ets-1 DNA-binding ac- tivity were analyzed during treatment of bEnd.3 cells with CCL2. by guest on October 2, 2021 Mechanisms of CCL2-induced angiogenesis Cells were exposed to 100 ng/ml CCL2 for the varying time pe- To identify potential mechanisms of CCL2-induced angiogenesis, riods (1–16 h). CCL2 induced significant up-regulation of Ets-1 a series of gene array experiments were performed to examine mRNA within 2 h. This up-regulation peaked at 4 h and persisted transcript abundance in endothelial cells exposed to CCL2. Table up to 16 h (Fig. 4A). Up-regulation of Ets-1 mRNA was followed I shows changes in angiogenic factor RNA expression after treat- by increased Ets-1 protein expression (peak at 8 h; Fig. 4A). Be- ment with CCL2 for 4 and 16 h. Values in the columns for every sides up-regulation of Ets-1 mRNA and proteins, CCL2 also in- listed gene represent expression as a percentage of an internal con- duced brief phosphorylation Ets-1 (on the threonine 38 residue) trol (␤-actin expression) present on every membrane. In addition, observed here in time period 0–4 h (Fig. 4B). This change in the expression of certain genes in cells treated with CCL2 for 4 and phosphorylation status is considered as a trigger for transfer of 16 h is compared with control (untreated) cells in the column la- Ets-1 to nucleus and binding to specific DNA sequences (24). beled percentage of fold increase. Only genes where expression Therefore, we analyzed nuclear extracts for Ets-1-binding activity. was increased 1.5-fold or greater were taken as up-regulated genes. As shown Fig. 4C, treatment of bEnd.3 cells with CCL2 induced From the table, it is evident that CCL2 induced expression of the an increased Ets-1-binding activity over time period 0–4 h. Spec- following: 1) transcription factors closely associated with angio- ificity of the Ets-1-binding activity was tested by 1) competition genesis (Ets-1, SMAD); 2) growth factors and their receptors with homologous DNA and 2) by supershift assay, and those (TGF␣, TGF␤, ephrin B2, ephrin B4, as well as receptors for the studies verified that CCL2 induced specific Ets-1-binding activity VEGF-KDR, neuropilin, angiopoietin Tie1, Tie2, TGF␣R1, (Fig. 4C). TGF␣R2; and 3) adhesion molecules, particularly integrins sub- To confirm that Ets-1 transcription factor plays a critical role in ␣ ␣ ␤ units 5, v, and 3 (CD61), then PECAM-1 and VCAM-1; and 4) CCL2-induced angiogenesis, bEnd.3 cells were transfected with matrix proteins, proteases, and protease inhibitors (collagen 18a1 Ets-1 sense, mismatched, or antisense phosphorothioate oligonu- and fibronectin, Adamts1, urokinase, MMP9, thrombospondin 1, cleotides. To determine transfection efficiencies, phosphorothioate plasminogen inhibitor 1) and other factors (NOS3, cox1, and oligonucleotides were end-labeled with FITC and transfected into cox2). bEnd.3 cells. Homogenous staining of the nuclei with pronounced To elucidate possible mechanisms that might be involved in accumulation in the nucleoli was noted after6hin50%ofbEnd.3 CCL2-induced angiogenesis, we correlated up-regulated genes cells, and this was used as the transfection time (data not shown). with specific transcription factor expression. The results showed In addition, to test whether antisense oligonucleotide effectively that the Ets-1 transcription factor was highly expressed after CCL2 blocked the DNA-binding activity of Ets-1, gel mobility shift as- treatment as were some of the genes that are Ets-1 dependent, such says were performed, which showed that binding activity of Ets-1 ␤ ␣ Ͼ as integrins 3 subunits (CD61), v subunits, urokinase (uPA), was reduced ( 50%) in bEnd.3 cells transfected with antisense MMP9, the VEGF receptor KDR, and the angiopoietin receptors oligonucleotides (data not shown). The Journal of Immunology 2655 Downloaded from http://www.jimmunol.org/

FIGURE 3. A, Expression of CCL2 and CCR2 mRNA in control bEnd.3 cells and hemangioma tissue with and without CCL2 antisense or sense phosphorothioate oligonucleotide treatment. bEnd.3 cells possess mRNA for CCL2 and its receptor, CCR2, under resting conditions. Increased expression of CCR2 was found in bEnd.3 cells during a treatment with CCL2 (100 ng/ml) or in the presence of TNF-␣ (10 ng/ml). High expression of CCL2 and CCR2 mRNA was found in hemangiomas and this was reduced by treatment with CCL2 antisense but not CCL2 sense treatment. Values from semiquantitative densitometric analysis represent means Ϯ SD from five independent experiments. B, CCL2 exerted a mitogenic effect on bEnd.3 cells in vitro. Cells were stimulated with CCL2 or VEGF at the indicated concentrations and allowed to proliferate for 16 h. BrdU incorporation was measured for the last 4 h. To test specificity of the CCL2 effect, CCL2-induced proliferation was also examined in the presence of a neutralizing CCL2 Ab (CCL2Ab) or a specific isotype by guest on October 2, 2021 Ab (goat IgG). The receptor dependence of CCL2-induced proliferation was examined by performing the proliferation assay on brain endothelial cells p Ͻ 0.001. C, CCL2 increases ,ءء ;p Ͼ 0.05 ,ء .prepared from mice phenotype CCR2Ϫ/Ϫ. Data represent mean Ϯ SD from five independent experiments bEnd.3 cell migration through Transwell filters. Calcein-AM-labeled bEnd.3 cells were layered on the top of Transwell filters. A chemotactic gradient was established by adding varying concentrations of CCL2 or VEGF (25 ng/ml) to the lower chamber and cell migration measured after 22 h. In controls, no growth factor was added. To test specificity, CCL2-induced migration was measured in the presence of a neutralizing CCL2 Ab (CCL2Ab) or a specific isotype Ab (goat IgG). The receptor dependence of CCL2-induced migration was examined by performing the assay on brain endothelial cells prepared .p Ͻ 0.001 vs nontreated controls ,ءء .from mice phenotype CCR2Ϫ/Ϫ. Values represent means Ϯ SD from five independent experiments

Reducing Ets-1 activity with antisense oligonucleotide signifi- Blocking Ets-1 activity with Ets-1 antisense oligonucleotide sig- ␤ cantly blocked CCL2-induced angiogenesis in the fibrin gel assay nificantly reduced integrin 3 mRNA and protein expression after compared with cells where Ets-1 was not blocked or bEnd.3 cells CCL2 treatment. In contrast, transfection of bEnd.3 cells with ␤ transfected with sense Ets-1 oligonucleotide (Fig. 4C). These sets sense Ets-1 oligonucleotide did not diminish 3 mRNA and pro- ␤ of experiments indicate that Ets-1 could be a critical transcription tein expression. Thus, increased expression of 3 integrin with factor for CCL2-induced angiogenesis. CCL2 treatment results from Ets-1 activation, and CCL2 probably exerts its angiogenic activity through ␤ -integrin function. ␤ 3 CCL2-regulated Ets-1 target molecule 3 integrin Ets-1 transcription factors are denoted as important angiogenic switch molecules that change quiescent endothelial cells to an an- CCL2 activates Ets-1 transcription factors through ERK1/2 giogenic phenotype (24–26). One of the factors regulated by Ets-1 activation ␤ is 3 integrin, a critical factor in the process of angiogenesis. Our Phosphorylation of threonine 38 residues on Ets-1 can play a role initial gene array analysis showed an up-regulation of this integrin in mediating its transcriptional activity in response to different fac- in bEnd.3 cells after CCL2 treatment (Table I). Further analysis tors (26–28). To investigate the role of phosphorylation in CCL2- with PCR and Western blot demonstrated that CCL2 induced an induced Ets-1 activation, we examined whether CCL2 induces ac- ␤ increase in 3-integrin mRNA and protein expression between 4 tivation of ERK1/2 in bEnd.3 cells. Then, through the inactivation and 16 h (Fig. 5, A and B). This expression was regulated at the of ERK1/2 activity, the contribution of ERK1/2 in CCL2-induced transcription and translation levels. Inhibition of transcription by Ets-1 activation was evaluated. As shown in Fig. 6A, CCL2 treat- actinomycin B or inhibition of translation by cyclohexamide sig- ment of bEnd.3 cells induced activation of ERK1/2, with the peak ␤ nificantly decreased 3-integrin mRNA and protein expression, re- activity after 10 min based on an in vitro kinase assay and by spectively (data not shown). Western blot analysis for phosphorylated ERK1/2. Inhibition of 2656 MECHANISMS OF CHEMOKINE-INDUCED ANGIOGENESIS

Table I. Angiogenic factor RNA expression

x-Fold x-Fold Genebank Gene Name Control 4 hr Increase 16 hr Increase

NM_009621 Adamts1 1.02 97.13 (95)1 83.6 (82)1 NM_013906 Adamts8 1.38 U83509 Angiopoietin-1 2.88 0.198 (14)2 NM_007426 Angiopoietin2 0.565 U22516 Angiogenin NM_007643 CD36 0.93 11.98 (13)1 NM_009868 Cadherin 5 84.78 100 1.2 83.6 (1) NM_007693 Vasostatin/c Hromogranin A 10.3 92.62 (9)1 48.49 (4.7)1 NM_009929 Procollagen, Type XVIII, ␣1 2.28 0.4 (6)2 44 (19)1 M13926 G-CSF 14.46 0.94 (15)2 NM_010217 Tissue factor 99.43 100 (1) 80.95 (0.8) NM_007901 Edg1 14.38 88.27 (6.1)1 83.63 (6)1 NM_007909 Ephrin A2 0.063 - NM_010109 Ephrin A receptor 0.37 7.88 (21)1 3.5 (9.4)1 NM_010111 Ephrin B2 92.91 100 (1) 83.5 (0.9) NM_010113 EGF 15.45 (15.4)1 NM_007912 EGFR 0.029 NM_007932 Endoglin 36.27 99.17 (2.7)1 83.5 (2.3)1 NM_010144 Ephrin B4 9.27 57.41 (6.2)1 80.9 (9)1 Downloaded from U71126 crb-2 1.76 0.21 (8.4)1 35.92 (20.5)1 NM_011808 c-cts1 39.68 99.82 (2.5)1 83.63 (2)1 NM_010168 Prothrombin kringle-1 U67610 aFGF 1.4 (1.4) NM_030614 FGF16 M30644 bFGF

M30642 FGF4 5.57 1.11 (5)2 (5.5)2 http://www.jimmunol.org/ M92416 FGF6 3.78 (3.8)2 (3.8)2 U58503 FGF7/KGF 8.11 16.93 (2.1)1 (8)2 M33760 FGFR1 (FLG) 100 100 (1) 83.63 (0.8) M81342 FGFR3 33.15 4.42 (7.5)2 10.84 (3)2 NM_008011 FGFR4 3.83 (3.8)1 5.12 (5.1)1 D89628 VEGF-D/FIGF X59397 KDR 4.99 89.87 (18)1 83.6 (17)1 L07297 VEGFR 100 100 (1) 83.63 (0.8) M18194 Fn1 23.84 14.71 (1.6)2 79.3 (3.3)1 J04596 Gro1 0.28 0.015 (0.05) X84046 HGF 2.11 7.52 (3.6)1 3.63 (1.7)1 by guest on October 2, 2021 NM_010431 Hif1a 88.92 99.84 (1.1) 83.63 (1) M31885 ID1 0.3 (0.3) 17.2 (17)1 NM_008321 ID3 NM_010502 IFN␣1 - 19.72 (19.7)1 NM_010510 IFN-b1 1.01 2.31 (2.3)1 K00083 IFNr 3.37 0.79 (4.3)1 NM_010512 IGF-1 0.604 (0.6) 83.6 (84)1 NM_010548 IL-10 NM_016780 CD61 41.08 (41)1 83.6 (84)1 NM_008539 Madh1 3.37 (3.4)1 83.63 (84)1 NM_010784 Midkine NM_008610 Gelatinase A NM_013599 Gelatinase B 0.17 (0.2) 8.89 (9)1 NM_031195 SR-A 12.46 (12.5)1 NM_008713 NOS3 14.77 86.69 (5.9)1 83 (5.7)1 NM_008737 Neuropilin 35.59 (35.6)1 81.1 (81)1 M29464 PDGF ␣ 0.028 (0.03) AF162784 PDGF␤ 0.65 (0.65) NM_011058 PDGFR␣ NM_008809 PDGFR␤ 15.37 (15.4)1 NM_008816 PECAM1 16.82 100 (6)1 83.63 (4.9)1 AB017491 PF4 4.12 (4)1 1.4 NM_008827 Placental growth factor 77.26 (77)1 83.63 (84)1 X02389 PLAU 96.84 (97)1 83.63 (84)1 NM_008969 PTGS1 7.75 (7.8)1 70 (70)1 NM_011198 Cox-2 0.477 4.13 (8.7)1 25.5 (53.5)1 D90225 Pleiotrophin 0.28 (0.3) NM_019765 Restin 0.06 100 (100)1 76.7 (77)1 NM_011333 Scya2 1.15 1.15 39.34 (40)1 NM_009257 Maspin M33960 PAI-1 8.07 29.95 (4)1 42.12 (5)1 X16490 PAI-2 AF017057 PEDF 1.21 1.2 47.05 (47)1 NM_009242 SPARC 76.32 100 1.3 83.63 (1.1)1 NM_009263 Osteopontin 1.86 23.64 (12.7)1 50.5 (27.2)1 The Journal of Immunology 2657

Table I. Continued

x-Fold x-Fold Genebank Gene Name Control 4 hr Increase 16 hr Increase

D13738 Tic-2 0.9 2.83 (3.14)1 48.64 (54)1 U65016 TGF-␣ M13177 TGF␤1 34.49 (34.5)1 82.3 (82.3)1 X57413 TGF ␤2 11.21 (11)1 7.31 (7.3)1 M32745 TGF␤3 1.46 (1.5)1 15.39 (15.4)1 D28526 TGF␤R1(ALK-5) 6.09 (6.1)1 83.18 (83.2)1 NM_009371 TGF␤R2 0.198 86.99 (439)1 83.63 (422)1 AF039601 [beta] glycan 4.12 2.96 (1.5)2 78.94 (19)1 M87276 THBS1 1.68 10.84 (6.4)1 16.78 (10)1 L07803 THBS2 L24434 TIMP2 AF102887 THBS3 X73960 Tie1 24.86 (25)1 78.99 (79)1 NM_011593 TIMP1 1.06 (1) 50.96 (51)1 Mm181969 TIMP2 9.66 79.96 (8.3)1 83.63 (9)1 NM_011607 Tenascin C 1.63 0.969 (0.6) 66.94 (41)1 NM_013693 TNF ␣ 0.866 M84487 VCAM-1 0.09 4.99 (55)1 8.49 (94)1 M95200 VEGF 0.6 0.6 Downloaded from U48800 VEGF-B 0.67 (0.67) 0.7 (0.7) U73620 VEGF-C 0.05 (0.05)

ERK1/2 activity with PD98059 significantly reduced DNA-bind- give us additional information about hemangioma evolution. 2) ing activity of Ets-1 in bEnd.3 cells stimulated by CCL2 as well as Phenotypically, bEnd.3 cells are very similar to brain endothelial http://www.jimmunol.org/ phosphorylation of Ets-1 (Fig. 6B). However, PD98059 also re- cells. 3) The cells can be used to study the process of angiogenesis duced CCL2-induced up-regulation of Ets-1 mRNA and protein, in vitro. 4) The cells can be pharmacologically and genetically indicating that ERK1/2 may modulate activation of Ets-1 by mod- manipulated in vitro for later injection, providing a unique model ifying Ets-1 turnover as well as through phosphorylation. with which to study the influence of tumor cell-derived signals that regulate angiogenesis. Discussion CCL2 is known to participate in angiogenic events under many CCL2 has been considered as a major factor in facilitating angio- conditions (15–18). For example, monocyte recruitment by CCL2 genesis through its recruitment of macrophages to sites of wound is a critical event in the neovascularization that occurs in chronic by guest on October 2, 2021 injury or peritumor areas and the subsequent release of angiogenic inflammatory conditions such as rheumatoid arthritis, psoriasis, factors by those macrophages (15–18, 29). However, some recent atherosclerosis, and different types of tumors (29–31, 33–38). De- studies have indicated that CCL2 might also have direct effects on spite an established correlation between CCL2 levels, infiltrating angiogenesis (17, 30). The current study supports this latter con- macrophages, and angiogenesis, several models of angiogenesis cept with evidence that CCL2 promotes some steps of the angio- indicate that CCL2 can promote an angiogenic phenotype in en- genic process (endothelial cell proliferation, migration, and tubule dothelial cells by a direct endothelial effect as well as indirectly via formation). This study also helps to elucidate the molecular mech- recruiting macrophages. The possibility of such a direct effect is anisms underlying CCL2-induced angiogenesis. In particular, it supported by the fact that endothelial cells are known to express indicates the following: 1) Ets-1 transcription factor is a critical CCR2, the sole receptor for CCL2 (19–21). Although our RT-PCR factor of CCL2-induced angiogenesis; 2) via activation of Ets-1, analysis showed that bEnd.3 cells express low levels of CCR2 CCL2 regulates expression of other crucial angiogenic factors, mRNA under resting conditions, the fact that CCL2 in vitro reg- such as integrin ␤ subunits; and 3) CCL2 regulates Ets-1 activa- 3 ulated bEnd.3 cell migration in a dose-dependent manner as well tion through MAPK (ERK1/2). the fact that adding anti-neutralizing CCL2 Ab diminishes CCL2- Before discussing these results and their implications further, it induced chemotaxis of bEnd.3 cells, support findings about pres- is necessary to clarify the use of polyoma middle T-transformed brain endothelial cells (bEnd.3 cells) in our study and the impor- ence of functional active CCR2. We believe that low level of ex- tance of the angiogenic effect of CCL2 on these cells. Our results pression of CCR2 mRNA on bEnd.3 cells under resting conditions indicate that End.3 cells are a good source for developing heman- might be the result of specific in vitro conditions that have already giomas. Hemangiomas represent a powerful model to study in vivo been shown to be critical for down-regulation of CCR2 in mono- angiogenesis for the several reasons: the angiogenic process is ex- cytes/macrophages (39). tremely potent; the rapid proliferation hemangioma is associated The molecular mechanisms underlying chemokine-induced an- with macrophage infiltration, indicating an important role for the giogenesis have not been extensively investigated. Only a few microenvironment in angiogenesis; and hemangiomas are com- studies have indicated that chemokines (particularly CXCL8) can monly encountered in humans, providing important clinical rele- switch endothelial cells to an angiogenic phenotype, regulating vance. Inoculation with polyoma middle T-transformed endothe- several angiogenic factors such as MMP1 and MMP9 (40, 41). Our lial cells is also considered as a good autocrine model of gene array analysis, for the first time, offers a detailed analysis of angiogenesis and hemangioma (31, 32). We felt that using bEnd.3 the angiogenic factors regulated by CCL2, offering insight into cells to evaluate CCL2-induced angiogenesis manner would have how this chemokine can directly regulate angiogenesis. Our results several advantages. 1) Due to the fact that the hemangiomas highly indicate that Ets-1 transcription factor is a critical factor in that express CCL2, investigating the angiogenic effects of CCL2 would process. 2658 MECHANISMS OF CHEMOKINE-INDUCED ANGIOGENESIS Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 4. CCL2-induced activation of Ets-1 transcription factor. A, CCL2 induced increased expression of Ets-1 mRNA and protein in bEnd.3 cells. bEnd.3 cells were treated with murine recombinant CCL2 (100 ng/ml) for 0–16 h. Representative examples of PCR and Western blot for CCL2 are shown. Levels of Ets-1 mRNA and protein were expressed as a ratio to those of a housekeeping gene, ␤-actin. B, CCL2 also induced phosphorylation of Ets-1 on the threonine 38 position evaluated here by Western blot analysis. C, Ets-1 DNA binding activity was evaluated by EMSA or Supershift assay. Nuclear extracts were prepared from control bEnd.3 cells or cells treated with CCL2 (100 ng/ml) in the presence or absence of homologue DNA (competitor) were separated on 6% gels. In the Supershift assay, polyclonal anti-Ets-1 Ab (2 ␮g/ml) was added. D, Reducing Ets-1 activity using phosphorothioated antisense Ets-1oligonucleotide significantly diminished CCL2-induced tube formation in bEnd.3 cells. Sense Ets-1 phosphorothioated oligonucleotide sequence had .p Ͻ 0.001 vs CCL2-treated cells ,ءء .no effect. Scale bar, 100 ␮m. Values represent means Ϯ SD from three independent experiments

The proto-oncogene c-Ets-1 encodes the prototypic member of a cules. In this manner, CCL2 regulated processes associated with novel family of transcription factors, the Ets proteins. Ets transcription brain endothelial cells proliferation, migration, and tubular for- factors bind via an 80-aa C-terminal domain to a GGA (A/T) con- mation (tubule formation was reduced 95% after treatment with sensus sequence called the Ets binding site or PEA3 element (24, 27), Ets-1 antisense oligonucleotide). These data support a physio- and it is presented in the promoters of many genes involved in cellular logical role for Ets-1 in initiation and or/propagation of sprout- proliferation, differentiation, development, hematopoiesis, apoptosis, ing and capillary formation. metastasis, tissue remodeling, and angiogenesis (24, 27). For exam- Among the Ets-1-dependent genes activated during CCL2-in- ␤ ple, there are Ets binding sites in the genes for collagenase-1 (MMP- duced angiogenesis, 3 subunits integrins (CD61) may have an 1), stromelysin-1 (MMP-3), MMP9, uPA, VEGFR1 (Flt1), VEGFR2 important role (25, 26, 44). Our study clearly shows that CCL2, ␤ ␣ ␤ ␤ (KDR), and integrin chain expression ( 3, v, and 4) that control through Ets-1 activation, regulated 3 expression (mRNA and pro- their transcription (25, 26, 42–44). In vivo, Ets-1 expression has been tein) on bEnd.3 cells. These results strongly suggest that CCL2 associated with new blood vessel formation under both physio- may contribute to angiogenesis by inducing synthesis and expres- ␤ logical and pathophysiological conditions, such as chronic in- sion of 3 integrin in endothelial cells. We cannot, however, ex- flammatory reactions and tumor-associated angiogenesis (28, clude the possibility that CCL2, like VEGF, might also act syn- ␤ 45, 46). Several angiogenic factors like VEGF, angiotensin II, ergistically with 3 integrin in regulating the complex process of TGF␤, and acidic fibroblast growth factor use Ets-1 to regulate angiogenesis and that this might further enhance the effects of angiogenesis (46–49). Our study indicates that CCL2 directly CCL2 on microvascular formation (50). regulates angiogenesis through activation of Ets-1 transcription CCL2 may regulate Ets-1 activation at several levels. Phosphor- factor and up-regulation of Ets-1-regulated angiogenic mole- ylation of Ets-1 on the threonine 38 positions by ERK1/2 strongly The Journal of Immunology 2659

␤ FIGURE 5. Effect of CCL2 on 3 integrin mRNA and protein expression in bEnd.3 cells. CCL2 induced ␤ significant up-regulation of 3 integrin mRNA (A; PCR) and protein expression (B; Western blot) in bEnd.3 cells over 4 to 16 h. Values are expressed relative to the housekeeping gene, ␤-actin, and represent means Ϯ SD p Ͻ 0.001 vs ,ءء .from three independent experiments control. Blocking Ets-1 activity using phosphorothio- ated antisense Ets-1 oligonucleotide significantly dimin- ished Ets-1 mRNA (C; PCR) and protein expression (D; Western blot) during treatment with CCL2. As a control, bEnd.3 cells were transfected with phosphorothioated Ets-1 sense oligonucleotide. Values represent means Ϯ Downloaded from p Ͻ 0.001 ,ءء ;SD from three independent experiments vs control. http://www.jimmunol.org/

increases Ets-1 DNA-binding activity (51, 52). Thus, hepatocyte our opinion, this study offers new insight into the mechanisms growth factor/scatter factor induces scattering and morphogenesis underlying angiogenesis and pathological disorders associated by guest on October 2, 2021 of epithelial cells through phosphorylation and activation of Ets-1 with abnormal angiogenesis. It also opens new possibilities for as the result of MAPK pathway activation (53, 54). In addition, a antiangiogenic therapy. In terms of mechanism, this study provides study by Watanabe et al. (47) on retinal endothelial cells showed for the first time a piece of evidence on how chemokines (CCL2, that VEGF and hypoxia-induced Ets-1 mRNA expression and that in particular) can directly regulate angiogenesis and expression of this was diminished after MEK/ERK1/2 blockade. That study in- angiogenic factors. Considering that some other chemokines (e.g., dicated two very important points: 1) MEK/ERK1/2 induced phos- CXCL8) also regulate Ets-1-dependent molecules (MMP2 and phorylation of Ets-1 and enhanced its DNA-binding activity to MMP9) via interaction with their own endothelial receptors, we would like to underscore the fact that the described pattern of certain genes; and 2) phosphorylated Ets-1 could bind to its own modulation of angiogenesis by CCL2 may also apply to other che- promoter and regulate its own presence and activity in cells via this mokines with angiogenic properties (e.g., CXCL8 or CXCL12) feedback mechanism (47). These increases in Ets-1 mRNA level and may be potentially unique for chemokine action. In addition, were considered to be associated with the transformation of brain we would also like to highlight that, in our opinion, CCL2 is a endothelial cells into the angiogenic phenotype. Our results concur modulator of angiogenic processes, enhancing this process either with these findings. CCL2 induced increased binding activity of through direct action on endothelial cells or by modulating the Ets-1 through activation of ERK1/2 pathway, and inhibition of this activity of other essential angiogenic factors (e.g., VEGF). CCL2 signal (through the specific inhibitor PD 98059) diminished Ets-1 obviously has a complex and bidirectional relationship with ␤ activity and expression of 3 integrin. At the same time, CCL2 VEGF, and their activity should be considered as a synchronized also induced an increase in Ets-1 mRNA and protein levels, and cooperative activity of two cytokines/growth angiogenic factors. In this event was also ERK1/2 dependent. ERK1/2 activation is crit- terms of therapy, understanding the mechanism of action of dif- ical for the induction of an angiogenic phenotype in bEnd.3 cells ferent angiogenic factors may enable multiple approaches (and by CCL2. combination approaches) to block angiogenesis. The manipulation Although our study has focused on CCL2 regulation of angio- of chemokine function may have merit as a new therapeutic ap- genesis via Ets-1, a recent study by Zhan et al. (46) found that proach. Although further experimental validation is needed, these Ets-1 could regulate CCL2 expression in smooth muscle cells dur- findings open up a new direction for future development of ther- ing inflammatory vascular remodeling. If this is also true in brain apeutic strategies in treating angiogenesis and angiogenic-related endothelial cells, CCL2 up-regulation may result in Ets-1 activa- disorders such as hemangioma. tion and further CCL2 up-regulation in an autocrine fashion. This possibility requires further investigation. Disclosures What is the significance of the findings in the current study? In The authors have no financial conflict of interest. 2660 MECHANISMS OF CHEMOKINE-INDUCED ANGIOGENESIS

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