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Original Studies |
Laboratoire d’Endocrinologie de la Reproduction, Centre de Recherche, Pavillon Saint-François d’Assise, Centre Hospitalier Universitaire de Québec, Université Laval, Québec, Canada G1L 3L5
Address all correspondence and requests for reprints to: Dr. Ali Akoum, Laboratoire d’Endocrinologie de la Reproduction, Centre de Recherche, Hôpital Saint-François d’Assise, 10 rue de l’Espinay, Local D0–711, Québec, Canada G1L 3L5. E-mail: ali.akoum{at}crsfa.ulaval.ca
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the course of the normal menstrual cycle, the endometrium undergoes profuse increases in the length, branching, and coiling of spiral arteries (10). However, more aggressive angiogenesis may occur in the peritoneal cavity of women with endometriosis (11); the establishment of the explant not only depends on the maintenance of the blood vessels within the ectopic endometrium but also on the growth of new blood vessels, through the peritoneum, to sustain the foreign endometrium (12). Active endometriotic explants have pronounced vascularization both within and around the tissue (13).
Several angiogenic factors have been identified, including acidic and basic fibroblast growth factors, platelet-derived endothelial cell growth factor, and vascular endothelial cell growth factor (VEGF) (14, 15, 16). The identification of the mediators of angiogenesis involved in endometriosis and their origins will be instrumental to elucidating the pathogenesis of the disease. In the peritoneal fluid, which bathes the peritoneal and ovarian surface where most endometriotic lesions occur, an elevated angiogenic activity was found in women with endometriosis, compared with normal women (17). Higher peritoneal concentrations of VEGF were also demonstrated in women with moderate to severe endometriosis than in women without the disease (18, 19). Peritoneal and resident macrophages found in endometriotic lesions seemed to be the major sources of VEGF secretion (19). However, we also believe that endometrial cells capable of aberrant implantation and growth in ectopic locations produce angiogenic factors that ensure their survival and development. In the present study, we show that immortalized endometriotic epithelial cells produce factors that promote human endothelial cell proliferation, and reveal a similar activity in the culture medium of primary endometriotic cell cultures. Of the mitogenic fractions isolated from the conditioned medium of the endometriotic cell line, one contained macrophage migration inhibitory factor (MIF), which displayed a potent mitogenic activity for endothelial cells and seemed to be a major mediator of the mitogenic activity released by immortalized and primary endometriotic cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Endometriotic biopsies were collected, at laparoscopy, from three women who had given an informed consent for a research protocol approved by the Saint-François d’Assise Hospital Ethics Committee on Human Research. These women were between 32 and 39 yr old, consulted for infertility and/or pelvic pain, had no endometrial hyperplasia or neoplasia, and had not received any antiinflammatory or hormonal medication during a period of at least 3 months before surgery. One woman had revised American Fertility Society stage I endometriosis, and two had stage II. Endometriotic biopsies were from brownish lesions and peritoneal red vesicles. As soon as they were collected, the biopsies were placed, at 4 C, in sterile HBSS containing 100 U/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin and were transported to the laboratory. The biopsy specimens were examined histologically, and the diagnosis of endometriosis was confirmed, in all cases, by the identification of both glandular and stromal endometrial elements.
Culture of primary and immortalized endometriotic cells
Endometriotic tissue was immediately minced into small pieces and dissociated with collagenase, as previously described (see 35). Cells were pelleted by centrifugation (200 x g/10 min); resuspended in DMEM-F12 containing 10 µg/mL insulin, 5 µg/mL transferrin, 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, and 2.5 µg/mL Amphotericin B (Life Technologies, Inc., Burlington, Ontario, Canada); and plated in 100-mm-diameter culture dishes; then grown, at 37 C, in 5% carbon dioxide. Both tadpole-shaped epithelial and fibroblast-like stromal cells were present in culture, as identified morphologically by light microscopy and immunocytochemically with specific monoclonal antibodies to cytokeratins and vimentin, as previously described (see 35). No leukocytes were detected in the endometriotic cells detached from culture dishes and assessed by flow cytometry (data not shown).
Endometriotic cell line Clo03 (20) was cultured in 100-mm-diameter culture dishes and maintained at 37 C, 5% CO2 in the same culture medium described above.
Preparation of conditioned media from endometriotic cell cultures
At confluence, cells were rinsed four times with HEPES buffered saline to eliminate FBS, then incubated for 24 h with M199 medium (Life Technologies, Inc.) (6 mL/Petri dish). The conditioned medium from primary endometriotic cell cultures (PCM) or from endometriotic cell line culture (LCM) was collected afterwards on ice, centrifuged to eliminate cell debris, concentrated and diafiltrated using Amicon’s YM1 membranes (Millipore Corp., Nepean, ON, Canada), aliquoted, and stored at -70 C until biological assay or further purification. The protein content of the samples was measured by Micro BCA Protein Assay Reagent Kit (Pierce Chemical Co., Rockford, IL) and analyzed by SDS-PAGE in an electrophoresis system (Mini-Protean II, Bio-Rad Laboratories, Inc. Ltd, Mississauga, ON, Canada), according to Laemmli et al. (21). Bands were visualized by silver staining.
Purification of angiogenic factors from LCM
The chromatographic system consisted of two 2150 pumps, a gradient controller, and a 2221 integrator (Pharmacia Biotech, Baie d’Urfé, PQ, Canada). A Mono Q HR 5/5 anion exchange column (Pharmacia) was used for high-resolution separation of proteins. Two mg of LCM in 500 µL of 20-mM phosphate buffer (pH 7.0) were loaded using a 2154 high-performance liquid chromatography (HPLC) injection valve with 500-µL loop at a flow rate of 1 mL/min. Column eluates were monitored by ultraviolet absorbance, at 216 nm, using a 2158 Uvicord SD detector. Eight fractions, eluted by a sodium chloride (NaCl) step-wise gradient (0, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 1 M NaCl), were collected separately, concentrated with Amicon’s YM1 membranes (Millipore Corp.), aliquoted, and saved at -70 C until being analyzed by SDS-PAGE and tested for mitogenic activity.
One of the fractions exerting a significant mitogenic action on human endothelial cells (F1, obtained with 0 M NaCl) was selected for further analyses, because it exhibited the lowest number of bands by electrophoresis. F1 was fractionated, using a Microcon-10 microconcentrator (Millipore Corp.), and both the lower part (F1a) and the upper part (F1b) were collected, aliquoted, and saved at -70 C until assay.
Analysis of cell proliferation
Endothelial cells from human coronary arteries (HCAEC, Cell Systems, Kirkland, WA) were cultured according to the supplier’s specifications. Briefly, cells were seeded in 24-well culture plates (4 x 104 cells/well) precoated with attachment factor and feeded with CS-C complete serum-free medium (Cell Systems). The medium was refreshed every 24 h; at 60–70% confluence, cells were rinsed twice with HEPES buffered saline/Ca/Mg and incubated with CS-C mitogen-free medium (Cell Systems) for 24 h at 37 C, 5% CO2. Cells were then exposed, in fresh CS-C mitogen-free medium, to different concentrations of the evaluated substances, or to 10 µg/mL of endothelial cell growth supplement (ECGS) (Cell Systems), which was included as positive control in each experiment. For neutralization experiments, bioactive fractions were incubated, for 1 h at 37 C, with different concentrations of polyclonal goat anti-MIF antibody (10–1000 ng/mL) (R and D Systems, Minneapolis, MN) or with equivalent concentrations of normal goat IgGs (R and D Systems) before being added to endothelial cell cultures. Twelve hours later, 2.5 µCi [3H]-thymidine (SA, 20 Ci/mmol; NEN Life Science Products, Boston, MA) were added to each well, and incubation was continued for 12 h more. The media were then discarded; and cells were washed twice with ice-cold PBS, fixed for 20 min in cold methanol, treated for 30 min at 4 C with 10% trichloroacetic acid, rinsed with cold ethanol, air dried, and dissolved in 0.5 mL of 0.6-N sodium hydroxide. After neutralization with 0.5 mL of 0.6-N chlorhydric acid (HCl), the radioactivity was measured by liquid scintillation spectrometry. Mitogenic activity was expressed as [3H]-thymidine incorporation percentage of control (medium without mitogens).
Amino acid sequence analysis
After purification by HPLC and a Microcon-10 microconcentrator, 20 µg of the bioactive fraction (F1a) were separated by SDS-PAGE as follows: Aliquots were heated to 37 C for 15 min before loading, and 100 mM sodium triglycolate (ICN Biomedicals, Inc., Aurora, OH) was added to the upper reservoir to prevent N-terminal blocking during electrophoresis (22). Proteins were visualized by Coomassie blue R-250 (0.1% solution in 50% methanol), individual bands were cut, and in-gel enzymatic digestion was carried out, for 24 h at 37 C, according to Williams et al. (23) using lysylendopeptidase (Wako Pure Chemical Industries Ltd., Richmond, VA). The resulting peptides were fractionated on a 1090 HPLC system (Hewlett-Packard Co., Palo Alto, CA) using a 1 mm x 25 cm C-18 reverse-phase column (5 µm, 300 pore size) (Vydac, Hesperia, CA) equilibrated with 98% buffer A [trifluoroacetic acid 0.1%] and 2% buffer B (80% acetonitrile, 0.08% trifluoroacetic acid). Peptides were eluted, at 50 µL/min, with a linear gradient of buffer B and were detected by ultraviolet absorbance at 216 mm. Selected peptides were then subjected to N-terminal sequencing by automatic Edman degradation performed on a model 473A pulsed liquid protein sequencer (PE Applied Biosystems, Foster City, CA). Analysis was performed by the Eastern Quebec Proteomics Core Facility (CHUQ, Sainte-Foy, PQ, Canada).
Western blot analysis
Proteins in the F1a fraction (1 µg) and PCM (1 and 10 µg) were separated under reducing conditions in 15% SDS-PAGE, as mentioned earlier, and transferred, during 2 h at 4 C and 250 mA, onto a 0.22-µm nitrocellulose membrane using electrophoretic transfer cell (Trans-Blot, Bio-Rad Laboratories, Inc.). The nitrocellulose membrane was then incubated, for 4 h at room temperature, in a solution containing 5% skimmed milk in 0.1 M Tris buffer, 0.9% NaCl, 0.05% Tween 20, pH 7.2 (TBST blocking solution) and 3% normal rabbit serum. The membrane was then cut into strips and incubated with an affinity-purified polyclonal goat anti-MIF antibody (2 µg/mL in TBST blocking solution) (R and D Systems) or with normal goat immunoglobulins (IgGs) (R and D Systems) at equivalent concentration, overnight at 4 C. Thereafter, the strips were incubated, for 1 h at room temperature, with peroxidase-labeled rabbit antigoat IgG antibodies (Fc specific) diluted 1:10,000 (Sigma, St. Louis, MO), washed, and incubated with chemiluminescence (ECL) reagent (Amersham Pharmacia Biotech, Oakville, Ontario, Canada) for 1 min, air dried, wrapped in a plastic bag, and exposed to X-OMAT AR film (Eastman Kodak Co., Rochester, NY) for 15 sec.
Statistical analysis
Values represent the mean of triplicate measurements. Data were analyzed using one-way ANOVA. A post hoc Tukey’s test was used for multiple comparisons. Differences were considered statistically significant for P values < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The conditioned medium derived from Clo03 cell line, concentrated
by Amicon’s YM1 membranes, stimulated the incorporation of
[3H]-thymidine into the DNA of HCAEC in a
dose-dependent manner. Figure 1
illustrates results from three independent experiments showing that the
level of [3H]-thymidine incorporation in cells
incubated with 1, 10, and 100 ng/mL LCM increased by 151.1 ±
27.6% (P < 0.05), 190.6 ± 27.5%
(P < 0.05), and 216.7 ± 35.4%
(P < 0.05) of control, respectively. In each
experiment, endothelial cell responsiveness to mitogens was evaluated
by including ECGS as positive control, which (as shown in Fig. 1)
increased the level of [3H]-thymidine
incorporation by 290.0 ± 46.0% of control (P <
0.001).
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In the present work, only F1 was considered for further investigations
because electrophoretic analysis of this fraction showed it contained
fewer bands than both the initial material (LCM) (Fig. 2) and the two other bioactive fractions
F2 and F8 (data not shown).
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)] were isolated
from F1 and recovered in the pass-through fraction (F1a). The
biological assay of F1a revealed potent mitogenic activity toward
endothelial cells. Such activity, expressed as
[3H]-thymidine incorporation percentage of
control, was significant at as low as 1 pg/mL (133.7 ± 5.8%;
P < 0.05), increased as a function of F1a concentration in
culture [reaching a maximal level at 0.1 ng/mL (177.3 ± 15.0%;
P < 0.01)[, and gradually decreased afterwards at higher
concentrations: 1 ng/mL (163.5 ± 10.7; P < 0.05) and 10
ng/mL (153.8 ± 9.0%; P < 0.05). Identification and evaluation of MIF mitogenic activity
The in-gel lysylendopeptidase digestion of the 12.6-kDa band of
F1a, followed by HPLC purification and N-terminal sequencing of 11
aminoacids from an internal 1.18-kDa peptide, revealed 100% identity
with the human MIF (hMIF). As shown in Fig. 3A, the 11-aminoacids sequence perfectly
matched that between amino acids 68 and 78 of hMIF, whose reported
molecular mass (12.476 kDa) is very close to that of the 12.6-kDa
protein. Moreover, the 12.6-kDa band was specifically recognized by an
affinity-purified polyclonal goat anti-hMIF antibody, as shown by
Western blot analysis of F1a fraction and PCM (Fig. 3B). This provides
strong evidence for the identification of this protein as human MIF and
demonstrates its secretion by primary ectopic endometrial cell
cultures.
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, the endothelial cell growth-promoting
activity of F1a was significantly inhibited by the antibody
(P < 0.01), whereas nonspecific goat IgGs had no
significant effect. Recombinant MIF was then assessed for its ability
to promote endothelial cell proliferation, and (as illustrated in Fig. 5) the protein displayed dose-dependent
mitogenic action on HCAEC. This effect was statistically significant at
0.1 pg/mL (164 ± 15% of control, P < 0.05) and
reached its highest level at 100 pg/mL (266 ± 23% of control,
P < 0.01).
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We finally assessed whether primary endometriotic cell cultures
release any mitogenic activity for endothelial cells and found that PCM
increased 3H-thymidine incorporation in HCAEC in
a dose-dependent manner. The activity was significantly reduced
(34 ± 5%, P < 0.05) after preincubation of PCM
with anti-MIF antibody (Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Antibodies specific to MIF significantly reduced the mitogenic activity displayed by the F1a fraction and that detected in the culture medium of primary endometriotic cells. Recombinant hMIF (over 97% pure, according to the manufacturer) was intrinsically able to promote endothelial cell proliferation (this in a dose-dependent fashion and detectable at as low as 0.1 pg/mL). These findings reveal that MIF is released by endometriotic cells and acts as a potent mediator of the mitogenic activity for human endothelial cells in vitro. This may have a considerable interest, in view of the key role of angiogenesis in the ectopic growth of endometrial cells and the activity of the implants. Interestingly, the present findings coincide with the recent publication of data showing an important role for MIF in tumor growth-associated angiogenesis in vivo, and in vitro in autocrine regulation of microvascular endothelial cell proliferation (24). This is in keeping with our data, and it further supports the novel role of MIF.
MIF was one of the first reported cytokines. It was originally described as a lymphokine that retained macrophages and inhibited their migration from inflammatory loci (25, 26, 27), and extensive study of its biological functions has been made possible by the recent cloning of mouse and human MIF genes (28). Nonetheless, the precise pathophysiological role of MIF remains undefined, and its receptor has yet to be identified. Recently, MIF was reported as an anterior pituitary-derived hormone that can override the glucocorticoid-mediated suppression of inflammatory and immune responses (29). MIF has also been shown to be expressed abundantly by macrophages (30) and to potentiate the proinflammatory actions of these cells (28, 29, 30). High levels of MIF expression were found in a variety of inflammatory conditions such as atopic dermatitis in the epidermal layer of inflammatory skin lesions (31), immunologically induced glomerulonephritis (32, 33, 34), and rat adjuvant arthritis (35). Available data also indicate that MIF is associated not only with the immune response but also with cell proliferation and differentiation during wound repair (31) and tumor growth (36). MIF synthesis is up-regulated in human metastatic colon cancer (31) and in metastatic human prostatic adenocarcinoma (37), and the interaction between tumor cells and macrophages in the latter disease has been shown to stimulate angiogenesis and tissue proliferation (38). Moreover, MIF was detected in human vascular endothelial cells (31) and was found to be abundantly expressed in human corneal endothelial and epithelial cells (39, 40, 41) after tissue damage, suggesting a possible role in tissue repair and cell growth. These data, along with our results showing MIF as a major growth promoting factor for endothelial cells produced by endometriotic cells, support the hypothesis that MIF is a key autocrine and paracrine mediator involved in neovascularization and tissue remodeling processes.
Endometriosis, defined as the ectopic growth of endometrial tissue, is frequently associated with an immunoinflammatory process observed both eutopically in the endometrium (42, 43, 44, 45) and ectopically in the peritoneal cavity, where the disease frequently develops (46, 47). Elevated numbers of macrophages was detected in the uterine endometrium (44), within endometriotic lesions (48, 49), and in the peritoneal cavity of patients suffering from endometriosis (50, 51, 52). Furthermore, activated peritoneal macrophages were found to be a major source of VEGF, a factor also expressed by tissue macrophages present in endometriotic lesions (19). Therefore, the finding that MIF can be secreted by endometriotic cells strongly suggests that it may contribute to the angiogenic process via at least two different ways: directly, by stimulating endothelial cell proliferation; and indirectly, by accumulating macrophages. These cells, in addition to their ability to produce VEGF and probably other angiogenic factors, represent a major source of MIF itself, as previously reported (30). Further studies are required, however, to evaluate the angiogenic activity of MIF in vivo and its expression in different types of endometriotic lesions. In fact, biochemically active endometriosis lesions (53), such as red vesicles and hemorrhagic lesions, are characterized by increased vascularization both around and within the endometriotic tissue, suggesting active neovascularization or angiogenic processes; whereas pigmented hypovascularized lesions are less active, and white lesions are considered as scars (11, 13).
Other fractions displaying potent mitogenic activity toward endothelial cells were also isolated in the experiments presented in this paper. Further investigations will be required to identify the factor(s) responsible for this activity and to determine the nature of the proteins that were copurified with MIF in the bioactive fraction F1a and their contribution the mitogenic activity observed.
In conclusion, this investigation revealed that MIF is a novel and a potent growth-promoting factor for human endothelial cells. This could be of considerable interest for the understanding of numerous angiogenesis-associated pathologies or inflammatory conditions, including endometriosis and neovascularization processes occurring in tissues such as human endometrium undergoing dynamic changes.
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Acknowledgments |
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Footnotes |
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2 Holds a Wyeth-Ayerst Laboratories, Inc. Canada
fellowship in endocrinology of reproduction.
3 A Chercheur-Boursier Senior of the Fonds de la Recherche en
Santé du Québec.
Received December 3, 1999.
Revised July 31, 2000.
Accepted August 18, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in-gel
) (P < 0.05).