Laboratory of Molecular Medicine, Department of Surgery, Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University Medical Center, Columbus, Ohio 43210
Submitted 9 January 2003 ; accepted in final form 18 May 2004
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ABSTRACT |
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endothelium; vascular; macrophage; redox; angiogenesis
MCP-1 is known to participate in angiogenic events under many conditions. Neovascularization is a significant component of chronic inflammatory conditions such as rheumatoid arthritis, psoriasis, and even atherosclerosis. Monocyte recruitment by MCP-1 is known to contribute to the progression of atherosclerosis (13). Work conducted at the Folkman laboratory (27) has shown that the extent of neovascularization in atherosclerotic lesions correlates highly with the extent of macrophage infiltration. Treatment with antiangiogenic agents reduced the extent of macrophage infiltration and plaque formation in apolipoprotein E/ mice that developed atherosclerotic aortas. MCP-1 also has a significant role in wound healing. Macrophages are essential for normal wound repair (7, 22), which is an angiogenesis-dependent process. In human wounds, there are considerable levels of MCP-1 expression by basal keratinocytes, endothelial cells, and infiltrating mononuclear cells. MCP-1 is expressed almost exclusively in the first 7 days after wounding, but other chemokines for monocytes are not expressed at significant levels (11). Finally, MCP-1 is also implicated in the angiogenic capacity of tumors to support their growth. The correlation between MCP-1 expression and tumor growth has been documented for breast (20, 34, 41), ovarian (14), and bladder cancers (2). An association between the presence of tumor-associated macrophages and poor prognosis has also been reported for several other tumor types (6, 29, 40). It is surmised that the tumor-associated macrophages facilitate angiogenesis and that the consequences of this aggressive growth are reflected in higher mortality rates. Despite the established correlation between MCP-1 levels, tumor-associated macrophages, and mortality, few attempts have been made to demonstrate causality between MCP-1 expression and tumor growth in vivo. Demonstrating the biological significance of MCP-1 expression in a model of angiogenesis may have therapeutic implications for a number of important disease states.
There are two murine models of vascular neoplasms. One uses endothelial cells transformed with the middle T antigen of the murine polyoma virus (4, 26, 38), and the other uses cells derived from a spontaneously arising HE (15, 44). The endothelial cells that are virally transformed are on a mixed major histocompatibility complex (MHC) background (H-2d/H-2b), making them suitable for use only in severe combined immunodeficiency (SCID) mice (4). The EOMA cells derived from the spontaneously arising HE are from the 129/J strain, which is commercially available (now called 129P3/J) with a defined H-2b MHC background. EOMA cells also have been well characterized with regard to endothelial cell phenotype (28), protein expression (10, 30, 46), response to angiogenesis inhibitors (19, 31), and development of the Kasabach-Merritt syndrome (15, 42, 44). The fact that mice into which EOMA cells are injected develop Kasabach-Merritt syndrome is a good indicator of how closely this model mimics the human condition. It was previously demonstrated that EOMA cells express relatively high levels of MCP-1 in vitro (12) and that HE proliferation after EOMA cell injection is associated with macrophage infiltration (3). It is not known how MCP-1 expression affects the biology of EOMA cells. Part of deciphering the role of MCP-1 is determining whether angiogenic effects are mediated directly via an autocrine effect on EOMA cells or indirectly via recruitment of macrophages. In this study, we sought to determine the significance of MCP-1 expression in supporting HE proliferation.
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MATERIALS AND METHODS |
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MCP-1 expression levels for in vitro EOMA samples were determined as previously described (12). For tumor samples, residual blood was removed by rinsing the samples in ice-cold PBS, blotting them onto paper, and incubating them in ACK lysis buffer [8.29 g of NH4Cl, 0.07 g of K2CO3, and 2 ml of 0.5 M Na2EDTA (pH 8) per liter of double-distilled H2O, pH 7.4], 10 ml/sample, for 10 min in a 37°C water bath. The tissue was rinsed in ice-cold PBS, blotted on filter paper, and snap frozen in liquid nitrogen. Frozen samples were ground, and the powder was transferred to an Eppendorf tube and resuspended in homogenization buffer [10 µl of protease inhibitor cocktail (Sigma, St. Louis, MO), 5 µl of PMSF (100 mM), 125 µl of 20% SDS, and 860 µl of PBS] at 100 mg/ml powder of homogenization buffer. The tissue was homogenized on ice four times for 20 s each with 5- to 10-s breaks. The homogenate was centrifuged at 3,500 rpm for 20 min at 4°C. The supernatants were collected and stored at 80°C until ELISA was performed. Bicinchoninic acid protein assay (Pierce, Rockford, IL) was performed according to the manufacturer's instructions to standardize MCP-1 values per milligram of protein.
BrdU Assay
Cell proliferation was assayed using bromodeoxyuridine (BrdU) ELISA (Roche, Indianapolis, IN) according to the manufacturer's instructions. EOMA cells were plated on flat-bottom 96-well tissue culture plates (Becton Dickinson, Franklin Lakes, NJ) at 10,000 cells/cm2 in normal growth medium (NGM) [Dulbecco's modified Eagle's medium (DMEM; Invitrogen/GIBCO, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals, Norcross, GA) and 100 U/ml penicillin-100 µg/ml streptomycin (1% P/S; Invitrogen/GIBCO)]. Incubation conditions were 37°C, 5% CO2, and 100% relative humidity. After cells were allowed to adhere and recover overnight, medium was removed and cells were washed twice with PBS and then twice with very low-serum medium (VLSM; DMEM, 0.2% FBS, and 1% P/S). Cells were allowed to synchronize their cell cycle in VLSM for 32 h. After serum starvation, medium was removed and NGM containing BrdU and challenges was added to wells. Challenges consisted of goat anti-mouse MCP-1 neutralizing antibody (AF-479-NA; R&D Systems, Minneapolis, MN) at 500 and 50 ng/106 cells in NGM, isotype control antibody (goat IgG AB-108-C; R&D Systems) at 500 and 50 ng/106 cells in NGM, NGM alone (reference control), mouse MCP-1 protein (479-JE; R&D Systems) at 500, 1,000, and 2,000 ng/106 cells in NGM, and VLSM alone (negative control). After overnight incubation for 16 h, cell growth was arrested, cells were fixed, and BrdU incorporation was assayed via colorimetric detection using a plate reader (model ELx808; Bio-Tek Instruments, Winooski, VT) at 450 nm.
Transwell Migration Assays
EOMA migration. RAW 264.7 macrophage cells (American Type Culture Collection, Manassas, VA) were seeded (2.77 x 106 cells/0.6 ml) in NGM (DMEM, 10% FBS, and 1% P/S) in 24-well tissue culture plates and incubated for 1 h at 37°C and 5% CO2 to allow RAW cells to adhere. Wells not seeded with RAW cells had an equal volume of NGM added to them. Wells were rinsed with PBS, and 0.6 ml/well LSM (DMEM, 0.5% FBS, and 1% P/S) was added. Transwell inserts with 8-µm pore size (Costar, Corning, NY) were equilibrated by incubation in LSM for 12 h at 37°C and 5% CO2 and placed in the 24-well plates, and then EOMA cells (105 cells/100 µl LSM) were seeded in the upper chamber. Where indicated, 100 ng/ml recombinant murine VEGF, 25 ng/ml recombinant murine MCP-1, 250 ng/ml goat anti-mouse MCP-1 monoclonal antibody, or 250 ng/ml goat IgG isotype control antibody were included in the LSM containing the EOMA cells at the time of seeding.
RAW cell migration. EOMA cells (3 x 105 cells/600 µl NGM) were seeded in the lower chamber of the 24-well plates, incubated for 1 h at 37°C and 5% CO2 to allow adherence, and rinsed with PBS, and then 600 µl of low-serum medium (LSM) were added. Equilibrated transwell inserts were placed and seeded with RAW cells (5 x 105 cells/100 µl LSM). Where indicated, 25 ng/ml recombinant murine MCP-1, 250 ng/ml goat anti-mouse MCP-1 monoclonal antibody, or 250 ng/ml goat IgG isotype control antibody were included in the LSM containing the EOMA cells or LSM alone at the time of lower well seeding. Migration was measured after incubation at 37°C and 5% CO2 for 5 h. Cells on the upper surface of the transwell membrane were removed by rubbing with a sterile cotton swab, and cells on the lower surface were fixed and visualized using the Hema 3 stain set (Fisher Diagnostics, Middletown, VA). Stained membranes were digitally imaged while overlaid on a hemocytometer, and the number of cells per square millimeter was determined at three different locations on each membrane.
In Vitro Assay for Sprout Formation by EOMA Cells
Collagen gels were prepared by adding 0.25 ml of collagen solution [25 µl of 10x PBS, 137.41 µl of 0.02 N acetic acid, 4.55 mg/ml solubilized type 1 rat-tail collagen solution (Upstate Biotechnology, Waltham, MA), 0.023 µl of 1 M NaOH, and 87.577 µl of H2O] to each well of a 24-well plate and were incubated at 37°C for 30 min. Gels were washed thoroughly with PBS and then equilibrated for 4 h with 1 ml NGM/well. EOMA cells (1 ml; 1.6 x 105/ml) were seeded onto each gel in NGM and incubated at 37°C and 5% CO2 until they were 80% confluent. Treatments were performed in LSM. Cells were treated for 48 h with either 1 ml LSM alone, 100 ng/ml recombinant murine VEGF (Biovision, Mountain View, CA), 2.5 or 100 ng/ml recombinant MCP-1 (R&D Systems), 250 ng/ml goat anti-mouse MCP-1 monoclonal antibody (R&D Systems), 250 ng/ml goat IgG isotype control antibody (R&D Systems), or 1 ml RAW conditioned medium. RAW conditioned medium was prepared by incubating 106 RAW cells/ml in LSM for 16 h. Collected medium was centrifuged at 1,200 rpm for 7 min at 4°C, and the supernatant was applied to collagen gels.
Peritoneal Macrophage Collection
Macrophages were obtained by intraperitoneal injection of 1.0 ml of 3% Brewer thioglycolate (Fisher Scientific Products, Pittsburgh, PA) as described previously (25). C57Bl/6 SCID mice (Jackson Laboratories, Bar Harbor, ME) were used to obtain a pure population of macrophages from the peritoneal exudate, which was collected 4 days after thioglycolate injection. Macrophages were collected by injecting 10 ml of PBS and aspirating the peritoneal fluid back into the syringe using a 19-gauge needle. Cells were spun down, counted using a hemocytometer, tested by trypan blue exclusion for viability, and resuspended in PBS at 2 x 107 cells/ml.
Hemangioendothelioma Production
EOMA cells were prepared for injection by harvesting them from culture with trypsin-EDTA, washing them three times in PBS, and loading them into a 1-ml tuberculin syringe. Mice (68 wk old) received 5 x 106 EOMA cells (5 x 107 cells/ml PBS) by subcutaneous injection in the dorsal midline. Where indicated, mice received 2.5 µg of goat anti-mouse MCP-1 monoclonal antibody (0.5 µg/106 cells), goat IgG isotype control antibody (0.5 µg/106 cells), or 5 x 105 peritoneal macrophages, all of which were contained within a 25-µl aliquot that was added to the EOMA cell suspensions in the syringes. MCP-1/ mice (gift of Dr. Barrett Rollins, Dana-Farber/Harvard Cancer Center, Boston, MA) were generated by targeted disruption of MCP-1 gene as described by Lu et al. (23). These mice are on a C57Bl/6 MHC background, so wild-type C57Bl/6b mice were used as controls. All mice were euthanized 7 days after injection for HE specimen harvest.
Immunohistochemistry
Tissue specimens were snap frozen in optimum cutting temperature (OCT) compound (Miles, Elkhart, IN), supercooled in isopentane, and stored in liquid nitrogen. Frozen tissues were sectioned at 6-µm thickness, fixed for 5 min in acetone at 4°C, and stained using routine immunoperoxidase methods. The primary antibody used for macrophage identification was F4/80, a rat anti-mouse IgG2b monoclonal antibody (Serotec, Raleigh, NC) used at 1:50 dilution. The secondary antibody used was mouse anti-rat IgG2b alkaline phosphatase-conjugated antibody. Control sections were generated using a rat IgG2 isotype control antibody to test primary antibody specificity and rat serum to test secondary antibody specificity. Tissue sections were counterstained with hematoxylin.
Statistical Analyses
For in vitro experiments, data are reported as means ± SD of three experiments, with each sample run in triplicate. Means were compared using an independent samples t-test, and P < 0.05 was considered statistically significant. For in vivo data, statistical analysis was done using multiple Fisher's exact tests, with P values adjusted using the step-down Bonferroni method of Holm. Comparisons between treatment groups were performed within the same mouse strain, and P < 0.05 was considered statistically significant.
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RESULTS |
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In the collagen gel assay, there was no spontaneous induction of sprouting from basal levels of MCP-1 expression, as shown in the untreated EOMA cells (Fig. 4E). Sprout formation was clearly triggered by VEGF, macrophage-conditioned media, and MCP-1 (Fig. 4, AD). Treatment with MCP-1 neutralizing antibody or IgG control antibody had no effect on sprouting (Fig. 4, F and G). While augmenting MCP-1 levels did induce a sprout response, it suggests that contributions from basal levels of MCP-1 expression toward sprout formation are minimal and that angiogenic responses may be enhanced by macrophage recruitment based on the levels of sprouting seen with macrophage-conditioned media.
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DISCUSSION |
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Our results indicate that MCP-1 promotes HE proliferation by recruiting macrophages to stimulate proangiogenic behaviors such as sprout formation in vitro. The concept of a positive feedback loop in the stimulation of angiogenic behavior in endothelial cells is supported by the fact that endothelial cells are known to express CCR2, the sole receptor for MCP-1 (35, 45). On the basis of reverse transcription-polymerase chain reaction results with sequencing of the product, EOMA cells express CCR2 as well (data not shown). Thus they have the capacity to respond directly to MCP-1. Our in vitro observations suggest that the direct effects of MCP-1 on EOMA angiogenic responses are minimal. However, the effects of MCP-1 on EOMA angiogenic responses are enhanced in the presence of macrophages as shown by the ability of MCP-1 to stimulate endothelial cell migration in vitro and the consistent presence of macrophage infiltration in HE lesions in vivo.
It is important to note that our findings demonstrating a critical role for MCP-1 in HE proliferation are likely to have broad significance beyond the specific experimental model used in the current study. Salcedo et al. (35) evaluated the role of MCP-1 in breast cancer by injecting a human breast carcinoma cell line into SCID mice. Those mice treated with neutralizing antibody to MCP-1 demonstrated increased survival and decreased volume of lung metastases compared with control mice. Taken together, our results and those of Salcedo et al. highlight the potential of MCP-1-neutralizing approaches to limit tumor formation in vivo. The present results constitute the first in vivo evidence demonstrating a complete response for any neoplasm, and specifically a vascular proliferative lesion, to anti-MCP-1 therapy in mice with intact immune systems. It is becoming clear that multiple approaches are required to block angiogenesis (24), and manipulation of chemokine function may have merit as a new therapeutic approach (5). These results support the concept of antiangiogenic strategies that go beyond a focus on endothelial cells.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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