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The Journal of Immunology, 2006, 177: 2651-2661.
Copyright © 2006 by The American Association of Immunologists

CCL2 Regulates Angiogenesis via Activation of Ets-1 Transcription Factor1

Svetlana M. Stamatovic*, Richard F. Keep*,{dagger}, Marija Mostarica-Stojkovic§ and Anuska V. Andjelkovic2,*,{ddagger}

* Department of Neurosurgery, {dagger} Molecular and Integrative Physiology and {ddagger} Pathology, University of Michigan, Medical School, Ann Arbor, MI 48109; and § Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Belgrade, Serbia and Montenegro


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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. Activation of Ets-1 by CCL2 further regulated some of Ets-1 target molecules including beta3 integrins. CCL2 induced significant up-regulation of beta3 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Angiogenesis, the formation of new blood vessels from preexisting blood vessels, takes place in many physiological and pathological conditions such as embryo development, ovulation, wound healing, rheumatoid arthritis, diabetic proliferative retinopathy, and tumorigenesis (1, 2, 3, 4, 5). Angiogenesis consists of series of highly ordered and tightly regulated events including degradation of the existing basement membrane, chemotaxis, proliferation, and capillary tube formation (6). Many factors have been found to influence angiogenesis. Some have a stimulatory effect on angiogenesis, including vascular endothelial growth factor (VEGF),3 basic fibroblast growth factor (bFGF), angiopoietin-1, ELR+ CXC chemokines IL-8, NAP-2, ENA-78 and GRO (7, 8, 9, 10, 11, 12, 13, 14). Others, such as endostatin, angiostatin, and ELR– chemokines IFN-{gamma}-inducible protein and monokine induced by IFN-{gamma}, inhibit angiogenesis (13, 14).

Recently, the CC chemokine MCP-1 (CCL2/MCP-1/JE) has been added to the growing list of angiogenic modulators (15, 16, 17, 18). Best known for its role in modulating inflammatory responses by inducing monocyte/macrophage recruitment to sites of inflammation, CCL2 also has a distinct role in angiogenesis. The molecular mechanisms by which CCL2 regulates angiogenesis have still to be fully elucidated. Until very recently, it was generally accepted that CCL2 indirectly stimulates angiogenesis via its chemoattractant effects on monocytes/macrophages, which in turn may release direct-acting angiogenic factors (15, 16, 18). However, a few recent studies have suggested that CCL2 may also exert direct angiogenic effects (17). Support for this hypothesis comes from the fact that endothelial cells express the CCL2 receptor CCR2 (19, 20, 21).

The current study was aimed at identifying critical intracellular signaling molecules that might be involved in CCL2-induced angiogenesis. Attention was focused on Ets-1 because this transcriptional factor is believed to be involved in regulating angiogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
All procedures were performed in strict accordance with the National Institute of Health’s Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of University of Michigan.

Cells

Brain endothelial cell line (bEnd.3) was purchased from American Type Culture Collection. Cells were grown in medium containing DMEM, 10% heat-inactivated FBS, 1x antibiotic/antimycotic, and 2 mM glutamine (all purchased from Invitrogen Life Technologies). Cells from 22 to 25 passages were used for all experiments. Primary cell cultures of brain microvascular endothelial cells were prepared from CCR2 knockout mice or wild-type mice by a method already established in our laboratory and described in detail previously (21).

In vitro angiogenesis assay

This assay was performed using previously described methods (22, 23). Bovine fibrinogen (at final concentration of 2.5 mg/ml; Sigma-Aldrich) was dissolved in DMEM supplemented with aprotinin (200 µg/ml; Sigma-Aldrich) and dispensed into 12-well tissue culture plates. Polymerization was induced by addition of thrombin (25 U/ml; Sigma-Aldrich) for 1 h at 37°C. After that, bEnd.3 cells were seeded onto the fibrin gel at 5 x 104/well and allowed to attach for up to 2 h. The medium was then carefully removed, and fibrin solution, mixed with test agent(s), was added to the cells. Fresh medium with matched test agent(s) was added on top of the generated fibrin gel overlay, and tube formation was assessed 24 h later. The total length of tubular structures was calculated in five different areas using Image J software (National Institutes of Health) (23).

For blocking experiments, bEnd.3 cells were first incubated in the serum-free medium containing PD 98059 (20 µM; Calbiochem) for 1 h or treated with antisense or sense oligonucleotides as described below. They were then trypsinized and plated onto the fibrin gels.

Hemangioma formation in nu/nu mice

Male nude mice (nu/nu; provided by The Jackson Laboratory), 10–12 wk of age, were used. Mice were anesthetized with i.p. ketamine/xylazine (100 mg/kg, 5 mg/kg). They were then inoculated with 0.5 ml of bEnd.3 cell suspension at 5 x 106 cells/ml s.c. in the right femoral flank. Over 3 wk, hemangioma size was measured with caliper three times per week, and hemangioma weight (in grams) was estimated by the following formula: length x width2/2. Experiments were terminated when hemangioma weight reached ~2 g (3 wk postinoculation). The hemangiomas were excised, weighed, photographed, frozen in OCT-embedding medium, and processed for H&E staining and immunohistochemistry. Hemangioma tissue was also taken for RT-PCR analysis.

To establish the contribution of CCL2 to hemangioma growth and angiogenesis, inhibition studies were performed where (nu/nu) mice bearing bEnd.3 cells received either CCL2 antisense (5'-AAGCGTGACAGAGACCTGCATAGTCGTGG-3') phosphorothioate oligonucleotide (5 µM each; Oligos Etc.) into the peritumor (hemangioma) area or a monoclonal anti-CCL2-neutralizing Ab (25 µg/ml/mouse; R&D Systems) i.p. at days 1, 6, 9, 12, 21 after inoculation. Controls received either sense phosphorothioate oligonucleotide (5'-CCACCACTATGCAGGTCTCTGTCACGTTT-3', 5 µM; Oligos Etc.) or vehicle (PBS).

Proliferation assay

Briefly, bEnd.3 cells were cultured in complete medium at a density of 1 x 105 cells/ml on 96-well microplates. After 12 h, the medium was removed and replaced with DMEM containing 0.5% FBS plus murine recombinant CCL2 or VEGF (PreproTech) at different concentrations (12.5–400 ng/ml) for 16 h. Medium alone was added as a negative control. bEnd.3 cell proliferation was measured using a colorimetric BrdU incorporation assay (Cell Proliferation ELISA; Roche) according to the manufacturer’s instructions.

Chemotaxis assay

Chemotaxis assays were performed in BD BioCoat Fibronectin Cell Culture Inserts (BD Biosciences). Briefly, bEnd.3 cells or CCR2–/– mouse brain microvascular endothelial cells (5 x 104 cells in 250 µl of DMEM containing 0.5% FBS) were added to the 3.0-µm insert of the chemotaxis plates. Serial 2-fold dilutions of recombinant mouse CCL2 (range 0.8 to 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. The cells were labeled with Calcein-AM (Molecular Probes) and allowed to migrate across a membrane insert for 22 h. The intensity of fluorescence was measured with a fluorescence plate reader (Applied Biosystems; CytoFlour 4000 plate reader) configured to read at excitation/emission wavelengths of 485/530 nm. The number of migrated cells was estimated from standard curve.

For blocking experiments, in addition to CCL2, medium was supplemented with 10 µg/ml neutralizing goat IgG anti-mouse CCL2 Ab (or the nonspecific isotype-matched control Ab; both obtained from R&D Systems).

cDNA array

At different times after CCL2 treatment (0–16 h), bEnd.3 cells were harvested, and total RNA was prepared using TRIzol reagents (Invitrogen Life Technologies). The procedure for biotinylated cDNA probe synthesis was done using the AmpoLabeling-LPR kit (SuperArray Bioscience) according to the manufacturer’s instructions. The resulting cDNA probe was hybridized to GEArray Q Series mouse angiogenesis gene array (SuperArray Bioscience) according to the manufacturer’s instructions. The relative expression level of each gene was analyzed using a software package provided by SuperArray (SuperArray Bioscience).

EMSA

Nuclear extracts were prepared from bEnd.3 cells using a Nuclear Extract Kit (Active Motif) according to the manufacturer’s instructions. The protein concentration was determined using the Bio-Rad protein assay. EMSA was performed with the Panomics EMSA kit (Panomics) according to manufacturer’s protocol. In brief, Ets-1-binding reactions were conducted with 5 µg of nuclear proteins and 10 ng of biotinylated double-stranded oligonucleotide probe containing the DNA-binding motif for Ets-1 (provided by Panomics). For competition experiments and supershift assays, excess of the corresponding cold (unlabelled) probe or ant-Ets-1 Ab (2 µg/ml; Santa Cruz Biotechnology) was added to the binding reactions and incubated for 5 min before addition of the labeled probes. Reactions were subjected to electrophoresis on 6% polyacrylamide gel in 0.5x Tris-borate-ethylene diamine tetraacetic acid (TBE) buffer followed by electroblotting onto nylon membranes (Biodyne B membrane) and UV cross-linking. Biotinylated oligonucleotides were then detected by probing with streptavidin conjugated to HRP and visualized by ECL and autoradiography.

Transfection

Ets-1 antisense and sense phosphorothioate oligonucleotides were purchased from Oligos Etc. The sequences used were as follows: sense, 5'-ATGAAGGCGGCCGTCGATCT-3'; and antisense, 5'-AGATCGACGGCCGCCTTCAT-3'. Briefly, when bEnd.3 cell culture reached ~90% confluence, the cells were washed once with serum-reduced OptiMEM I medium. The transfection of oligonucleotides was then performed with OptiMEM I medium containing 500 nM oligonucleotides and 12 µg/ml lipofectamine (Invitrogen Life Technologies). Cells were incubated with the transfection medium for 4 h at 37°C, 5% CO2, before changing back to normal medium. After an additional incubation for 20 h, cells were used in blocking experiments. Efficiency of transfection was established by Western blot analysis and EMSA.

Western blotting

Cells were lysed in radioimmunoprecipitation assay buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and 2 mM sodium orthovanadate) containing protease inhibitors (10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM EDTA, and 1 mM PMSF). The protein content was determined using Pierce protein assay kit. Equal amounts of protein were electrophoretically separated by SDS-PAGE on 7.5 or 10% gels and transferred to Trans-Blot nitrocellulose membrane (Bio-Rad). Immunoblotting was performed using rabbit anti-Ets-1 Ab (Santa Cruz Biotechnology), anti- Ets-1 phospho-(T38) Ab (Novus Biological), anti-phospho-ERK1/2 Ab (Cell Signaling Technology), or hamster anti-mouse CD61 (integrin beta3 chain) Ab (BD Biosciences). Immunoreactive proteins were visualized using an ECL detection kit (Pierce). Autoradiographic images underwent semiquantitative densitometry analysis using National Institutes of Health image software package (version 1.63). After probing for pERK1/2 or phospho-Ets-1 to examine ERK activation status or level of phosphorylated Ets-1, immunoblots were stripped using Restore Western Blot Stripping Buffer (Pierce Biotechnology) and reprobed with rabbit anti-ERK1/2 Ab (Cell Signaling Technology) or anti-Ets-1 Ab.

MAPK assays

In vitro MAPK assay was performed using a MAPK assay kit (Upstate Biotechnology) according to the manufacturer’s recommendations.

Immunofluorescence microscopy and histopathology

Immunofluorescence staining was performed on 6-µm-thick cryosections of bEnd.3 cell-induced hemangiomas. The sections were preincubated in blocking solution (PBS containing 2% BSA and 0.5% Tween 20) for 1 h and then incubated with goat anti-mouse CCL2 Ab (R&D Systems), rat anti-mouse CD31 Ab (BD Biosciences), or rat anti-mouse F4/80 Ab (Serotec). After overnight incubation at 4°C, the sections were washed and incubated with secondary Ab conjugated to FITC or Texas Red for 1 h at room temperature. Sections were then analyzed by confocal microscopy (Zeiss; LSM 510). Some sections from each hemangioma were stained directly with H&E for standard histological examination.

RT-PCR

Total RNA was isolated from bEnd.3 cells or hemangioma using the TRIzol reagent (Invitrogen Life Technologies). Briefly, 2 µg from each sample was reverse-transcribed into cDNA using an Invitrogen Life Technologies cDNA Synthesis Kit, following the manufacturer’s protocol. Ets-1 cDNA was amplified as a 510-bp fragment by PCR with the forward primer 5' -TACCCTTCCGTCATTCTCC-3' and reverse primer 5'- TTTTTCCTCTTTCCCCATC-3'. beta3-integrin cDNA was amplified as 430 bp with the forward primer 5'-GGGGACTGCCTGTGTGACTC-3' and reverse primer 5'-CTTTTCGGTCGTGGATGGTG-3. Samples were standardized using primers specific to cDNA encoding mouse beta-actin. A total of 40 cycles for Ets-1 and 30 cycles for beta3 integrin were applied. The PCR cycles included 1-min denaturation at 94°C, 1-min annealing at 55°C, and 1-min extension at 72°C, except for the first cycle, which had 2-min denaturation, and the last cycle with 10-min elongation. The PCR products were resolved using electrophoresis on a 2% agarose gel in 1x TBE buffer (Tris-HCl/EDTA/boric acid; pH 8). The gel was stained with ethidium bromide and photographed.

ELISA

Supernatants collected from in vitro angiogenic assays in fibrin gel were assayed for VEGF level by ELISA, using the mouse VEGF Quantikine kit (R&D Systems).

Statistics

Statistical analyses were performed using commercially available software (Stat-View; SAS Institute). One-way ANOVA was used to compare the mean responses among the experimental groups. The Dunnett’s test was used to determine significance between groups.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CCL2 as potent angiogenic factor

To examine the potential angiogenic effect of CCL2, the expression and effect of CCL2 on hemangioma development in vivo was examined. Hemangiomas offer a unique model to study angiogenesis. They are a primary tumor of microvasculature in which angiogenesis is initially excessive followed by inhibition and regression of the newly formed blood vessels. Injection of 5 x 106 Polyoma middle T-transformed endothelial cells (bEnd.3 cells) s.c. in the right femoral flank of nu/nu mice was sufficient to induce hemangioma formation over 21 days. As shown in Fig. 1A, the hemangiomas were cystic vascular structures with high levels of CCL2 in infiltrated macrophages (F4/80+ cells) and endothelial cells (CD31+ cells). To establish that CCL2 plays a significant role in hemangioma formation, on days 1, 6, 9, 12, and 21 after bEnd.3 injection mice received neutralizing anti-CCL2 Ab or CCL2 antisense phosphorothioate oligonucleotide. Inhibition of CCL2 activity/synthesis markedly reduced hemangioma size (Fig. 1B). Thus, CCL2 can exert a potent effect on hemangioma formation and angiogenesis.


Figure 1
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  		FIGURE 1. CCL2 contributes to angiogenesis in vivo and induces angiogenesis in vitro. A, Hemangiomas 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 fluorescence 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 µm. B, Effect of inhibiting CCL2 activity using neutralizing Ab to CCL2 (25 µg/ml, i.p.) or CCL2 antisense phosphorothioate oligonucleotide (5 µM) on hemangioma growth. Photographs show hemangiomas at day 21. The graph shows the time course of hemangioma weight. Values are mean ± SD; n = 6; *, p < 0.01; **, p < 0.001 vs nontreated bEnd.3 cells.

 
The effect of CCL2 on angiogenesis was further examined in vitro using a fibrin gel angiogenesis assay and bEnd.3 cells. CCL2 significantly enhanced the formation of tube-like structures compared with controls (vehicle-treated cells; Fig. 2). The angiogenic response of bEnd 3 cells to CCL2 was dose dependent. After 24-h stimulation with CCL2, the greatest increase in tube-like formation was observed at the concentration of 100 ng/ml. This increase in tube formation was comparable to that observed after stimulation with VEGF, a known potent angiogenic factor (Fig. 2).


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

 
If CCL2 acts as a direct angiogenic factor, then it is expected that endothelial cells possess CCR2, the receptor for CCL2. As already stated, CCL2 exerts its biological effects on endothelial cells through CCR2 (19, 20, 21). Fig. 3A shows that there is a low, constitutive expression of the CCL2 and CCR2 transcripts in bEnd.3 cells. However, CCL2 treatment of bEnd.3 cells for 4 h induced a significant increase in CCR2 expression. This increase in CCR2 expression was comparable to that observed after stimulation of bEnd 3 cells with TNF-{alpha} (10 ng/ml).


Figure 3
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  		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-{alpha} (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 ± 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 Ab (goat IgG). The receptor dependence of CCL2-induced proliferation was examined by performing the proliferation assay on brain endothelial cells prepared from mice phenotype CCR2–/–. Data represent mean ± SD from five independent experiments. *, p > 0.05; **, p < 0.001. C, CCL2 increases 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 from mice phenotype CCR2–/–. Values represent means ± SD from five independent experiments. **, p < 0.001 vs nontreated controls.

 
Analyzing the hemangioma tissue, we also found a significant amount of CCL2 and CCR2 mRNA expression, the level of which was diminished by applying CCL2 antisense phosphorothioate oligonucleotide (Fig. 3A).

Conditioned media were collected from bEnd 3 cells seeded on the fibrin gel after 0–24 h of treatment with CCL2 and analyzed for VEGF by ELISA. We found that the amount of VEGF was not significantly different between control (nontreated bEnd3 cells) and CCL-2-treated samples (data not shown).

CCL2 also induced proliferation of bEnd.3 cell in a concentration-dependent manner (Fig. 3B). Quantification of incorporated BrdU in bEnd.3 cells indicated peak proliferation at CCL2 concentration of 50 ng/ml (p < 0.01 vs control). This proliferative response was comparable to that observed with VEGF (50 ng/ml). This strongly suggests that CCL2 has a potent mitogenic effect on bEnd.3 cells. The proliferative effect of CCL2 was blocked in the presence of CCL2-neutralizing Ab, and it was also CCR2 dependent. In experiments where brain endothelial cells were prepared from mice genotype CCR2–/–, CCL2 was not able to induce a proliferative response (Fig. 3B).

Stimulation of endothelial cell motility is a common feature of angiogenic factors. As shown in Fig. 3C, CCL2 also displays chemotactic activity for endothelial cells. CCL2 induced directional bEnd.3 cell migration (chemotaxis) in a concentration-dependent manner (0.78–400 ng/ml). Maximal migration was obtained with 100 ng/ml CCL2. CCL2-induced bEnd.3 cell chemotaxis was similar to that found with VEGF (Fig. 3C). Inhibiting CCL2 with an anti-CCL2-neutralizing Ab completely blocked CCL2-enhanced cell migration. In addition, brain endothelial cell migration toward the CCL2 was CCR2 dependent. In the range of concentrations tested (0.78–400 ng/ml), CCL2 did not elicit migration of brain endothelial cells prepared from CCR2–/– mice. Thus, the chemotactic response of brain endothelial cells to CCL2 stimulation is linked to the expression of CCR2 in these cells.

Taken together, these results indicate that CCL2 may act as a potent direct angiogenic factor. In subsequent experiments to investigate the mechanisms underlying such angiogenic activity, 100 ng/ml CCL2 was used because this induced the peak angiogenic response.

Mechanisms of CCL2-induced angiogenesis

To identify potential mechanisms of CCL2-induced angiogenesis, a series of gene array experiments were performed to examine transcript abundance in endothelial cells exposed to CCL2. Table I shows changes in angiogenic factor RNA expression after treatment with CCL2 for 4 and 16 h. Values in the columns for every listed gene represent expression as a percentage of an internal control (beta-actin expression) present on every membrane. In addition, the expression of certain genes in cells treated with CCL2 for 4 and 16 h is compared with control (untreated) cells in the column labeled percentage of fold increase. Only genes where expression was increased 1.5-fold or greater were taken as up-regulated genes. From the table, it is evident that CCL2 induced expression of the following: 1) transcription factors closely associated with angiogenesis (Ets-1, SMAD); 2) growth factors and their receptors (TGF{alpha}, TGFbeta, ephrin B2, ephrin B4, as well as receptors for the VEGF-KDR, neuropilin, angiopoietin Tie1, Tie2, TGF{alpha}R1, TGF{alpha}R2; and 3) adhesion molecules, particularly integrins subunits {alpha}5, {alpha}v, and beta3 (CD61), then PECAM-1 and VCAM-1; and 4) matrix proteins, proteases, and protease inhibitors (collagen 18a1 and fibronectin, Adamts1, urokinase, MMP9, thrombospondin 1, plasminogen inhibitor 1) and other factors (NOS3, cox1, and cox2).


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  		Table I. Angiogenic factor RNA expression

 

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  		Table IA. Continued

 
To elucidate possible mechanisms that might be involved in CCL2-induced angiogenesis, we correlated up-regulated genes with specific transcription factor expression. The results showed that the Ets-1 transcription factor was highly expressed after CCL2 treatment as were some of the genes that are Ets-1 dependent, such as integrins beta3 subunits (CD61), {alpha}v subunits, urokinase (uPA), MMP9, the VEGF receptor KDR, and the angiopoietin receptors Tie1, Tie2. Due to this, further studies focused on the activity of Ets-1 during CCL2-induced angiogenesis.

CCL2-induced angiogenesis through activation of Ets-1 transcription factor

To begin to investigate whether Ets-1 plays a critical role in CCL2-induced angiogenesis, Ets-1 mRNA and protein expression as well as Ets-1 activation (phosphorylation) and Ets-1 DNA-binding activity were analyzed during treatment of bEnd.3 cells with CCL2. Cells were exposed to 100 ng/ml CCL2 for the varying time periods (1–16 h). CCL2 induced significant up-regulation of Ets-1 mRNA within 2 h. This up-regulation peaked at 4 h and persisted up to 16 h (Fig. 4A). Up-regulation of Ets-1 mRNA was followed by increased Ets-1 protein expression (peak at 8 h; Fig. 4A). Besides up-regulation of Ets-1 mRNA and proteins, CCL2 also induced brief phosphorylation Ets-1 (on the threonine 38 residue) observed here in time period 0–4 h (Fig. 4B). This change in phosphorylation status is considered as a trigger for transfer of Ets-1 to nucleus and binding to specific DNA sequences (24). Therefore, we analyzed nuclear extracts for Ets-1-binding activity. As shown Fig. 4C, treatment of bEnd.3 cells with CCL2 induced an increased Ets-1-binding activity over time period 0–4 h. Specificity of the Ets-1-binding activity was tested by 1) competition with homologous DNA and 2) by supershift assay, and those studies verified that CCL2 induced specific Ets-1-binding activity (Fig. 4C).


Figure 4
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  		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, beta-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 µg/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 no effect. Scale bar, 100 µm. Values represent means ± SD from three independent experiments. **, p < 0.001 vs CCL2-treated cells.

 
To confirm that Ets-1 transcription factor plays a critical role in CCL2-induced angiogenesis, bEnd.3 cells were transfected with Ets-1 sense, mismatched, or antisense phosphorothioate oligonucleotides. To determine transfection efficiencies, phosphorothioate oligonucleotides were end-labeled with FITC and transfected into bEnd.3 cells. Homogenous staining of the nuclei with pronounced accumulation in the nucleoli was noted after 6 h in 50% of bEnd.3 cells, and this was used as the transfection time (data not shown). In addition, to test whether antisense oligonucleotide effectively blocked the DNA-binding activity of Ets-1, gel mobility shift assays were performed, which showed that binding activity of Ets-1 was reduced (>50%) in bEnd.3 cells transfected with antisense oligonucleotides (data not shown).

Reducing Ets-1 activity with antisense oligonucleotide significantly blocked CCL2-induced angiogenesis in the fibrin gel assay compared with cells where Ets-1 was not blocked or bEnd.3 cells transfected with sense Ets-1 oligonucleotide (Fig. 4C). These sets of experiments indicate that Ets-1 could be a critical transcription factor for CCL2-induced angiogenesis.

CCL2-regulated Ets-1 target molecule beta3 integrin

Ets-1 transcription factors are denoted as important angiogenic switch molecules that change quiescent endothelial cells to an angiogenic phenotype (24, 25, 26). One of the factors regulated by Ets-1 is beta3 integrin, a critical factor in the process of angiogenesis. Our initial gene array analysis showed an up-regulation of this integrin in bEnd.3 cells after CCL2 treatment (Table I). Further analysis with PCR and Western blot demonstrated that CCL2 induced an increase in beta3-integrin mRNA and protein expression between 4 and 16 h (Fig. 5, A and B). This expression was regulated at the transcription and translation levels. Inhibition of transcription by actinomycin B or inhibition of translation by cyclohexamide significantly decreased beta3-integrin mRNA and protein expression, respectively (data not shown).


Figure 5
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  		FIGURE 5. Effect of CCL2 on beta3 integrin mRNA and protein expression in bEnd.3 cells. CCL2 induced significant up-regulation of beta3 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, beta-actin, and represent means ± SD from three independent experiments. **, p < 0.001 vs control. Blocking Ets-1 activity using phosphorothioated antisense Ets-1 oligonucleotide significantly diminished 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 ± SD from three independent experiments; **, p < 0.001 vs control.

 
Blocking Ets-1 activity with Ets-1 antisense oligonucleotide significantly reduced integrin beta3 mRNA and protein expression after CCL2 treatment. In contrast, transfection of bEnd.3 cells with sense Ets-1 oligonucleotide did not diminish beta3 mRNA and protein expression. Thus, increased expression of beta3 integrin with CCL2 treatment results from Ets-1 activation, and CCL2 probably exerts its angiogenic activity through beta3-integrin function.

CCL2 activates Ets-1 transcription factors through ERK1/2 activation

Phosphorylation of threonine 38 residues on Ets-1 can play a role in mediating its transcriptional activity in response to different factors (26, 27, 28). To investigate the role of phosphorylation in CCL2-induced Ets-1 activation, we examined whether CCL2 induces activation of ERK1/2 in bEnd.3 cells. Then, through the inactivation of ERK1/2 activity, the contribution of ERK1/2 in CCL2-induced Ets-1 activation was evaluated. As shown in Fig. 6A, CCL2 treatment of bEnd.3 cells induced activation of ERK1/2, with the peak activity after 10 min based on an in vitro kinase assay and by Western blot analysis for phosphorylated ERK1/2. Inhibition of ERK1/2 activity with PD98059 significantly reduced DNA-binding activity of Ets-1 in bEnd.3 cells stimulated by CCL2 as well as phosphorylation of Ets-1 (Fig. 6B). However, PD98059 also reduced CCL2-induced up-regulation of Ets-1 mRNA and protein, indicating that ERK1/2 may modulate activation of Ets-1 by modifying Ets-1 turnover as well as through phosphorylation.


Figure 6
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  		FIGURE 6. Participation of ERK1/2 in Ets-1 activation by CCL2. A, ERK1/2 phosphorylation (Western blot) and activity (MAPK assay) were examined in bEnd.3 cells treated with CCL2 (100 ng/ml) for 1 h. Robust phosphorylation and ERK1/2 activation was found by 10 min and persisted for 30 min. Values represent means ± SD from three independent experiments. **, p < 0.001 vs control (nontreated) cells. B, Inhibition of ERK1/2 activation (using inhibitor PD98059) greatly diminished CCL2-induced Ets-1 binding activity as evaluated by EMSA. Treatment with Ets-1 antisense oligonucleotide also blocked Ets-1 binding activity. C, As well as diminishin g Ets-1 binding activity, PD98059 also significantly reduced CCL2-induced Ets-1 mRNA, protein expression in bEnd.3 cells and phosphorylation of Ets-1 threonine 38 residues. Values represent means ± SD from three independent experiments and are expressed as a ratio to the housekeeping gene, beta-actin. **, p < 0.001 vs CCL2-treated cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
CCL2 has been considered as a major factor in facilitating angiogenesis through its recruitment of macrophages to sites of wound injury or peritumor areas and the subsequent release of angiogenic factors by those macrophages (15, 16, 17, 18, 29). However, some recent studies have indicated that CCL2 might also have direct effects on angiogenesis (17, 30). The current study supports this latter concept with evidence that CCL2 promotes some steps of the angiogenic process (endothelial cell proliferation, migration, and tubule formation). This study also helps to elucidate the molecular mechanisms underlying CCL2-induced angiogenesis. In particular, it indicates the following: 1) Ets-1 transcription factor is a critical factor of CCL2-induced angiogenesis; 2) via activation of Ets-1, CCL2 regulates expression of other crucial angiogenic factors, such as integrin beta3 subunits; and 3) CCL2 regulates Ets-1 activation through MAPK (ERK1/2).

Before discussing these results and their implications further, it is necessary to clarify the use of polyoma middle T-transformed brain endothelial cells (bEnd.3 cells) in our study and the importance of the angiogenic effect of CCL2 on these cells. Our results indicate that End.3 cells are a good source for developing hemangiomas. Hemangiomas represent a powerful model to study in vivo angiogenesis for the several reasons: the angiogenic process is extremely potent; the rapid proliferation hemangioma is associated with macrophage infiltration, indicating an important role for the microenvironment in angiogenesis; and hemangiomas are commonly encountered in humans, providing important clinical relevance. Inoculation with polyoma middle T-transformed endothelial cells is also considered as a good autocrine model of angiogenesis and hemangioma (31, 32). We felt that using bEnd.3 cells to evaluate CCL2-induced angiogenesis manner would have several advantages. 1) Due to the fact that the hemangiomas highly express CCL2, investigating the angiogenic effects of CCL2 would give us additional information about hemangioma evolution. 2) Phenotypically, bEnd.3 cells are very similar to brain endothelial cells. 3) The cells can be used to study the process of angiogenesis in vitro. 4) The cells can be pharmacologically and genetically manipulated in vitro for later injection, providing a unique model with which to study the influence of tumor cell-derived signals that regulate angiogenesis.

CCL2 is known to participate in angiogenic events under many conditions (15, 16, 17, 18). For example, monocyte recruitment by CCL2 is a critical event in the neovascularization that occurs in chronic inflammatory conditions such as rheumatoid arthritis, psoriasis, atherosclerosis, and different types of tumors (29, 30, 31, 33, 34, 35, 36, 37, 38). Despite an established correlation between CCL2 levels, infiltrating macrophages, and angiogenesis, several models of angiogenesis indicate that CCL2 can promote an angiogenic phenotype in endothelial cells by a direct endothelial effect as well as indirectly via recruiting macrophages. The possibility of such a direct effect is supported by the fact that endothelial cells are known to express CCR2, the sole receptor for CCL2 (19, 20, 21). Although our RT-PCR analysis showed that bEnd.3 cells express low levels of CCR2 mRNA under resting conditions, the fact that CCL2 in vitro regulated bEnd.3 cell migration in a dose-dependent manner as well the fact that adding anti-neutralizing CCL2 Ab diminishes CCL2-induced chemotaxis of bEnd.3 cells, support findings about presence of functional active CCR2. We believe that low level of expression of CCR2 mRNA on bEnd.3 cells under resting conditions might be the result of specific in vitro conditions that have already been shown to be critical for down-regulation of CCR2 in monocytes/macrophages (39).

The molecular mechanisms underlying chemokine-induced angiogenesis have not been extensively investigated. Only a few studies have indicated that chemokines (particularly CXCL8) can switch endothelial cells to an angiogenic phenotype, regulating several angiogenic factors such as MMP1 and MMP9 (40, 41). Our gene array analysis, for the first time, offers a detailed analysis of the angiogenic factors regulated by CCL2, offering insight into how this chemokine can directly regulate angiogenesis. Our results indicate that Ets-1 transcription factor is a critical factor in that process.

The proto-oncogene c-Ets-1 encodes the prototypic member of a novel family of transcription factors, the Ets proteins. Ets transcription factors bind via an 80-aa C-terminal domain to a GGA (A/T) consensus sequence called the Ets binding site or PEA3 element (24, 27), and it is presented in the promoters of many genes involved in cellular proliferation, differentiation, development, hematopoiesis, apoptosis, metastasis, tissue remodeling, and angiogenesis (24, 27). For example, there are Ets binding sites in the genes for collagenase-1 (MMP-1), stromelysin-1 (MMP-3), MMP9, uPA, VEGFR1 (Flt1), VEGFR2 (KDR), and integrin chain expression (beta3, {alpha}v, and beta4) that control their transcription (25, 26, 42, 43, 44). In vivo, Ets-1 expression has been associated with new blood vessel formation under both physiological and pathophysiological conditions, such as chronic inflammatory reactions and tumor-associated angiogenesis (28, 45, 46). Several angiogenic factors like VEGF, angiotensin II, TGFbeta, and acidic fibroblast growth factor use Ets-1 to regulate angiogenesis (46, 47, 48, 49). Our study indicates that CCL2 directly regulates angiogenesis through activation of Ets-1 transcription factor and up-regulation of Ets-1-regulated angiogenic molecules. In this manner, CCL2 regulated processes associated with brain endothelial cells proliferation, migration, and tubular formation (tubule formation was reduced 95% after treatment with Ets-1 antisense oligonucleotide). These data support a physiological role for Ets-1 in initiation and or/propagation of sprouting and capillary formation.

Among the Ets-1-dependent genes activated during CCL2-induced angiogenesis, beta3 subunits integrins (CD61) may have an important role (25, 26, 44). Our study clearly shows that CCL2, through Ets-1 activation, regulated beta3 expression (mRNA and protein) on bEnd.3 cells. These results strongly suggest that CCL2 may contribute to angiogenesis by inducing synthesis and expression of beta3 integrin in endothelial cells. We cannot, however, exclude the possibility that CCL2, like VEGF, might also act synergistically with beta3 integrin in regulating the complex process of angiogenesis and that this might further enhance the effects of CCL2 on microvascular formation (50).

CCL2 may regulate Ets-1 activation at several levels. Phosphorylation of Ets-1 on the threonine 38 positions by ERK1/2 strongly increases Ets-1 DNA-binding activity (51, 52). Thus, hepatocyte growth factor/scatter factor induces scattering and morphogenesis of epithelial cells through phosphorylation and activation of Ets-1 as the result of MAPK pathway activation (53, 54). In addition, a study by Watanabe et al. (47) on retinal endothelial cells showed that VEGF and hypoxia-induced Ets-1 mRNA expression and that this was diminished after MEK/ERK1/2 blockade. That study indicated two very important points: 1) MEK/ERK1/2 induced phosphorylation of Ets-1 and enhanced its DNA-binding activity to certain genes; and 2) phosphorylated Ets-1 could bind to its own promoter and regulate its own presence and activity in cells via this feedback mechanism (47). These increases in Ets-1 mRNA level were considered to be associated with the transformation of brain endothelial cells into the angiogenic phenotype. Our results concur with these findings. CCL2 induced increased binding activity of Ets-1 through activation of ERK1/2 pathway, and inhibition of this signal (through the specific inhibitor PD 98059) diminished Ets-1 activity and expression of beta3 integrin. At the same time, CCL2 also induced an increase in Ets-1 mRNA and protein levels, and this event was also ERK1/2 dependent. ERK1/2 activation is critical for the induction of an angiogenic phenotype in bEnd.3 cells by CCL2.

Although our study has focused on CCL2 regulation of angiogenesis via Ets-1, a recent study by Zhan et al. (46) found that Ets-1 could regulate CCL2 expression in smooth muscle cells during inflammatory vascular remodeling. If this is also true in brain endothelial cells, CCL2 up-regulation may result in Ets-1 activation and further CCL2 up-regulation in an autocrine fashion. This possibility requires further investigation.

What is the significance of the findings in the current study? In our opinion, this study offers new insight into the mechanisms underlying angiogenesis and pathological disorders associated with abnormal angiogenesis. It also opens new possibilities for antiangiogenic therapy. In terms of mechanism, this study provides for the first time a piece of evidence on how chemokines (CCL2, in particular) can directly regulate angiogenesis and expression of angiogenic factors. Considering that some other chemokines (e.g., CXCL8) also regulate Ets-1-dependent molecules (MMP2 and MMP9) via interaction with their own endothelial receptors, we would like to underscore the fact that the described pattern of modulation of angiogenesis by CCL2 may also apply to other chemokines with angiogenic properties (e.g., CXCL8 or CXCL12) and may be potentially unique for chemokine action. In addition, we would also like to highlight that, in our opinion, CCL2 is a modulator of angiogenic processes, enhancing this process either through direct action on endothelial cells or by modulating the activity of other essential angiogenic factors (e.g., VEGF). CCL2 obviously has a complex and bidirectional relationship with VEGF, and their activity should be considered as a synchronized cooperative activity of two cytokines/growth angiogenic factors. In terms of therapy, understanding the mechanism of action of different angiogenic factors may enable multiple approaches (and combination approaches) to block angiogenesis. The manipulation of chemokine function may have merit as a new therapeutic approach. Although further experimental validation is needed, these findings open up a new direction for future development of therapeutic strategies in treating angiogenesis and angiogenic-related disorders such as hemangioma.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grant NS 044907 (to A.V.A.) from the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Anuska V. Andjelkovic, Department of Neurosurgery and Pathology, University of Michigan, MI 48109. E-mail address: anuskaa{at}umich.edu Back

3 Abbreviations used in this paper: VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor; uPA, urokinase. Back

Received for publication December 29, 2005. Accepted for publication May 23, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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