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Regulation of contractility of cultured human endometrial stromal cells by tumor necrosis factor-α
European Journal of Obstetrics & Gynecology and Reproductive Biology 138 (2008) 66–70 www.elsevier.com/locate/ejogrb
Regulation of contractility of cultured human endometrial stromal cells by tumor necrosis factor-a Akitoshi Yuge, Kaei Nasu *, Hatsumi Tsusue, Etsuko Ikegami, Masakazu Nishida, Harunobu Matsumoto, Hisashi Narahara Department of Obstetrics and Gynecology, Faculty of Medicine, Oita University, Idaigaoka 1-1, Hasama-machi, Yufu-shi, Oita 879-5593, Japan Received 30 January 2007; received in revised form 12 April 2007; accepted 18 May 2007
Abstract Objective: The aim of this study is to evaluate the involvement of tumor necrosis factor (TNF)-a in endometrial tissue remodeling during the perimenstrual period. Study design: Human endometrial stromal cells (ESCs) were isolated from eight premenopausal patients in the late secretory phase. The effects of TNF-a on the contractility of cultured ESCs were investigated by collagen gel contraction assay. The effects of TNF-a on the proliferation of ESCs were also assessed by a modified methylthiazoletetrazolium assay. Results: TNF-a significantly upregulated the collagen gel contractility of ESCs in a dose-dependent manner. TNF-a did not affect ESC proliferation. Conclusion: The results suggest that TNF-a may promote endometrial tissue repair by stimulating the contraction of the extracellular matrix by ESCs. By regulating ESC function during the perimenstrual period, TNF-a may be involved in the physiological tissue remodeling of the cyclic endometrium. # 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Endometrial stromal cells; Tumor necrosis factor-a; Contractility; Tissue remodeling
1. Introduction The human uterine endometrium is a dynamic organ that undergoes cyclic phases of remarkable periodic growth, remodeling, and breakdown. Unlike in most adult tissues, in the uterine endometrium cyclic growth and tissue remodeling occur throughout the reproductive years. Endometrial tissue remodeling during the menstrual cycle is thought to be regulated by ovarian steroids as well as by various cytokines, neuropeptides, and growth factors that are produced locally and secreted in endocrine, paracrine, as well as autocrine manners [1]. The tissue remodeling events that occur during menstruation share common features with injury and repair events in other tissues, which may occur not only after injury * Corresponding author. Tel.: +81 97 586 5922; fax: +81 97 586 6687. E-mail address: [email protected] (K. Nasu).
but also in association with various pathologies [1]. However, there are important differences between tissues, and a range of features from different models are likely to apply to endometrial repair. There are also aspects of wound healing in other adult tissues that do not appear to apply to the endometrium. These include the development of granulation tissue, which occurs during the healing of most cutaneous wounds, and the formation of blood clots, which provide important mediators for the initiation of repair. In addition, most wounds heal with scarring, and this is generally not seen in human endometrial repair. Tumor necrosis factor (TNF)-a, a 17-kDa polypeptide with immunologic functions similar to those of interleukin (IL)-1 and IL-6 [2], was originally described as a factor produced by macrophages in response to agents such as bacterial lipopolysaccharide. Its major immunologic roles are thought to be the control of inflammatory responses and protection from bacterial infection [3]. In addition to its
0301-2115/$ – see front matter # 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2007.05.010
A. Yuge et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 138 (2008) 66–70
immunologic functions, TNF-a is known to have pleiotrophic effects, which modulate cellular growth, differentiation, and the synthesis of various substances [4,5]. The existence of both TNF-a and its receptors in the various reproductive tissues has been reported. In normal endometrium, it has been demonstrated that TNF-a promotes cell growth in early proliferative stages, facilitates cell death and shedding in late stages, encourages angiogenesis, stimulates the synthesis of remodeling enzymes, induces dyscohesion of epithelial cells [6,7], and inhibits decidualization of ESCs in vitro [8]. Immunohistochemical and in situ hybridization analyses have shown the presence of TNF-a protein and mRNA within both stromal/epithelial cells and leukocytes of the human endometrium [9–14]. TNF-a exerts its biological effects via two cell-surface receptors: TNF receptor type 1 (TNF-RI) (50–60 kDa) and TNF receptor type 2 (TNF-RII) (75–80 kDa). Each receptor is encoded by two independent genes and is capable of mediating distinct intracellular signals [15,16]. The expression of both TNF-RI and TNF-RII has been detected in endometrial stromal cells (ESCs) [17]. It has been demonstrated that most of the biological activities of TNF-a are transduced through TNF-RI [15,16]. In the present study, we investigated the effects of TNF-a on the proliferation and contractility of cultured human ESCs using well-characterized in vitro models [18,19]. We also discussed the role of TNF-a on endometrial tissue remodeling during the menstrual cycle.
2. Materials and methods 2.1. ESC isolation procedure and cell culture conditions Normal endometrial specimens were obtained from eight premenopausal patients (aged 35–41 years) who had undergone hysterectomies for intramural or subserosal leiomyomas. All of the specimens were determined to be from the late secretory phase on the basis of the standard histologic criteria [20]. This study was approved by the institutional review board (IRB) of Oita University, and written informed consent was obtained from all patients. Normal ESCs were separated from epithelial glands by digesting the tissue fragments with collagenase, as previously described [21]. Briefly, the tissue was minced into 2–3 mm pieces and incubated with collagenase (200 U/ml) (GibcoBRL, Gaithersburg, MD, USA) in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco-BRL) with stirring for 30 min at 37 8C. The suspension was filtered through a 150mm wire sieve to remove mucus and undigested tissue. The filtrate was then passed through an 80-mm wire sieve, which allowed the stromal cells to pass through while the intact glands were retained. After washing the cells three times with serum-free DMEM, they were transferred to culture flasks (Corning, New York, NY, USA) at a density of 106 cells/ml in DMEM supplemented with 10% heat-inactivated fetal bovine
67
serum (FBS) (Gibco-BRL), streptomycin (100 U/ml) (GibcoBRL), and penicillin (100 U/ml) (Gibco-BRL). The culture medium was replaced every 4 days. After three passages using standard trypsinization methods, the cells were >99% pure as analyzed by immunocytochemical staining with antibodies to vimentin (V9, Dako, Copenhagen, Denmark), cytokeratin (Dako), factor VIII (Dako), and leukocyte common antigen (2B11 + PD7/26, Dako), and were then used for the experiments. The cultures were incubated at 37 8C in an atmosphere of 5% CO2 in air at 100% humidity. Cells isolated from each individual patient were used for one experiment at a time. Each experiment was performed in triplicate and repeated at least three times. 2.2. Assessment of ESC proliferation after TNF-a treatment ESC proliferation after TNF-a treatment was determined in 96-well plates by a modified methylthiazoletetrazolium (MTT) assay using WST-1 (Roche Diagnostics GmbH, Penzberg, Germany) according to the manufacturer’s protocol. Then, 5 104 ESCs in DMEM supplemented with 10% FBS were distributed into each well of a 96-well flat-bottomed microplate (Corning, New York, NY, USA) and incubated overnight. The medium was then removed and the cells were incubated for 48 h with 200 ml of serumfree DMEM containing various concentrations of TNF-a (0.1–10 ng/ml) (R&D Systems, Minneapolis, MN, USA). Thereafter, 20 ml of WST-1 dye was added to each well, which was further incubated for 4 h. Cell proliferation was evaluated by measuring the absorbance at 540 nm. Data were calculated as the ratio of values obtained for the treated cells and those for the untreated controls. 2.3. Collagen gel contraction assay Cellular collagen gel contraction assays were performed as previously described [18,19]. A sterile solution of acidsoluble collagen type I purified from porcine tendons (Cellmatrix type I-A; Nitta Gelatin Inc., Osaka, Japan) was prepared according to the manufacturer’s instructions. ESCs were embedded in the collagen gel and cultured threedimensionally. Briefly, ESCs were suspended in the collagen solution (3.0 105 cells/ml). The collagen/cell mixture (2 ml/plate) was dispensed into 35-mm culture plates (Corning) coated with 0.2% bovine serum albumin (Sigma); the mixture was allowed to polymerize at 37 8C for 30 min. Immediately after polymerization, 1 ml of DMEM supplemented with 10% FBS containing TNF-a (final concentration: 0.1–10 ng/ml) was added to each plate. After incubation for 48 h, the collagen gels were photographed and the area of the gel surface was measured with the public domain Image Program 1.61 developed at National Institutes of Health (Bethesda, MD, USA). The incubation time was determined by background experiments.
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A. Yuge et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 138 (2008) 66–70
3. Results
Fig. 1. The effect of TNF-a on the cell proliferation of ESCs as assessed by WST-1 assay. ESCs were treated with various concentrations of TNF-a (0.1–10 ng/ml) for 48 h. Thereafter, 20 ml of WST-1 dye was added to each well and further incubated for 4 h. Cell proliferation was evaluated by measuring the absorbance at 540 nm. Representative results are shown.
The effects of TNF-a on ESC proliferation were investigated by use of a modified MTT assay. TNF-a did not affect the proliferation of these cells (Fig. 1). As a preliminary experiment, we evaluated the effects of TNF-a on the collagen gel contraction without the presence of ESCs. TNF-a did not induce the gel contraction in the absence of ESCs. The effects of TNF-a on the collagen gel contraction of ESCs were evaluated. In the presence of FBS, untreated ESCs showed significant collagen gel contractility (54.0% contraction after 48 h compared with 0 h). As shown in Fig. 2, the gel surface areas were significantly reduced in the presence of TNF-a in a dose-dependent manner (37.2% reduction of the gel surface area at a concentration of 10 ng/ ml of TNF-a, p < 0.0001 versus untreated controls). Therefore, it is likely that TNF-a significantly enhanced the collagen gel contractility of ESCs.
2.4. Statistical analysis 4. Discussion Data were presented as means S.D. and were analyzed by the Bonferroni/Dunn test with StatView 4.5 (Abacus Concepts, Berkeley, CA, USA). A p value <0.05 was accepted as statistically significant.
During the secretory phase, the endometrium undergoes changes, including differentiation of its epithelium into secretory glands and the transformation of its stromal
Fig. 2. The effects of TNF-a on the collagen gel contractility of ESCs. ESCs were treated with various concentrations of TNF-a (0.1–10 ng/ml) for 48 h. The collagen gels were photographed (A and B) and the collagen gel contractility was assessed by measuring the gel surface area (C). *p < 0.0001 vs. untreated controls (Bonferroni/Dunn test).
A. Yuge et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 138 (2008) 66–70
mesenchyme into predecidual cells. During the late secretory phase of the cycle, widespread degeneration occurs in the basal lamina supporting the decidualized endometrial cells and the endothelium of blood vessels [22]. There is a very rapid but incomplete degeneration of the functionalis layer, exposing open blood vessels and glands. Endometrial destruction is a consequence of the activity of matrix degrading enzymes on the extracellular matrix (i.e., both the fibrillar matrix and basal lamina) after the withdrawal of steroid hormone support at the end of each menstrual cycle, with the resultant loss of blood vessel integrity and shedding of most of the functional layer [1]. Menstrual bleeding stops by the time tissue destruction have ceased. When substantial endometrial tissue loss occurs during menstruation, ESC ingrowth, extracellular matrix deposition, and angiogenesis, reestablish organ integrity, often in concert with tissue contraction. Regeneration begins in areas where the mouths of the basal glands are free from overlying degenerated tissue. There is simultaneous and progressive epithelial outgrowth from these glands as well as ingrowth from the intact peripheral surface membrane bordering the denuded basalis. The stromal tissue begins to grow only when the endometrial wound is completely re-epithelialized. These events are thought to occur under the influence of increasing estrogen concentrations, and are probably locally regulated by a number of cytokines and other regulatory factors, including TNF-a [1]. Another important event during endometrial wound healing is the contraction of connective tissue, which is carried out by fibroblastic cells. ESCs appear to be responsible for wound contraction. The presence of serum itself induced the contractility of ESCs in three-dimensional culture as shown in our previous reports [18,19]. We have demonstrated that collagen gel contraction is further stimulated by platelet-derived growth factor [18] and by transforming growth factor-b [19]. In the present study, we evaluated the effects of TNF-a with respect to endometrial tissue remodeling during the menstrual cycle, and found that TNF-a could stimulate ESCs to contract the collagen gel matrix. These results are similar to those of previous reports that have examined uterine smooth muscle cells [23]. The contractile activity of ESCs may favor expulsion of the endometrium during menstruation, reduce the size of the endometrial wound defect for eventual wound closure, and reduce the amount of fibroplasia and angiogenesis necessary for the reestablishment of organ integrity. Although the precise mechanisms of TNF-a-induced contractile activity of ESCs are still unknown from the present study, possible candidate mediators of this phenomenon are prostaglandins. Previous studies using immunohistochemistry, Northern blot analysis, and explant cultures have demonstrated the production of TNF-a mRNA and protein by human endometrium [9–14,24]. These reports indicate that ESCs as well as endometrial epithelial cells may be the sources of TNF-a in the endometrium, suggesting that autocrine and
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paracrine mechanisms take place in the induction of contractility of ESCs. Some studies have shown that the endometrium increases TNF-a mRNA and protein production during the secretory and menstrual phases of the menstrual cycle [7,12,24,25], suggesting the roles of TNF-a in tissue remodeling during the perimenstrual period. Taken together, these data suggest the involvement of TNF-a in endometrial tissue remodeling during the perimenstrual period. In summary, we demonstrated that TNF-a stimulates the contractility of ESCs without affecting their proliferation. The present results suggest that TNF-a may contribute to endometrial tissue remodeling during perimenstruation by regulating ESC cellular functions.
Acknowledgements This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (no. 16591672 to K. Nasu).
References [1] Salamonsen LA. Tissue injury and repair in the female human reproductive tract. Reproduction 2003;125:301–11. [2] Dinarello CA. Interleukin-1 and its biologically related cytokines. Adv Immunol 1989;44:153–204. [3] Old LJ. Tumor necrosis factor. Science 1985;230:631–2. [4] Le J, Vilcek J. Biology of disease. Tumor necrosis factor and interleukin 1: cytokines with multiple overlapping biological activities. Lab Invest 1987;56:234–48. [5] Beutler B, Cerami A. The biology of cachectin/TNF-a primary mediator of the host response. Ann Rev Immunol 1989;7:625–55. [6] Hunt JS. Expression and regulation of the tumour necrosis factor-a gene in the female reproductive tract. Reprod Fertil Dev 1993;5: 141–53. [7] Tabibzadeh S, Kong QF, Kapur S, Satyaswaroop PG, Aktories K. Tumour necrosis factor-a-mediated dyscohesion of epithelial cells is associated with discorded expression of cadherin/b-catenin and disassembly of actin filaments. Hum Reprod 1995;10:994–1004. [8] Inoue T, Kanzaki H, Iwai M, et al. Tumour necrosis factor a inhibits in vitro decidualization of endometrial stromal cells. Hum Reprod 1994;9:2411–7. [9] Tabibzadeh S. Ubiquitous expression of TNF-a/cachectin immunoreactivity in human endometrium. Am J Reprod Immunol 1991;26:1–4. [10] Tabibzadeh S. Cytokine regulation of human endometrial function. Ann NY Acad Sci 1991;622:89–97. [11] Hunt JS, Chen H-L, Hu X-L, Tabibzadeh S. Tumour necrosis factor a mRNA and protein in human endometrium. Biol Reprod 1992;47: 141–7. [12] Philippeaux M-M, Piguet PF. Expression of tumor necrosis factor-a and its mRNA in the endometrial mucosa during the menstrual cycle. Am J Pathol 1993;143:480–6. [13] Garcia FU, Chen HL, Yang Y, Pace JL, Hu X-L, Hunt JS. Tumour necrosis factor a mRNA and protein in endometrial tumours: analysis by in situ hybridization and immunocytochemistry. Hum Pathol 1994;25:1324–31. [14] Laird SM, Tuckerman EM, Saravelos H, Li TC. The production of tumour necrosis factor a (TNF-a) by human endometrial stromal cells in culture. Hum Reprod 1996;11:1318–23.
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[15] Engelmann H, Holtmann H, Brakebusch C, et al. Antibodies to a soluble form of a tumor necrosis factor (TNF) receptor have TNF-like activity. J Biol Chem 1990;265:14497–504. [16] Shalaby MR, Sundan A, Loetscher H, Brockhaus M, Lesslauer W, Espevik T. Binding and regulation of cellular functions by monoclonal antibodies against human tumor necrosis factor receptors. J Exp Med 1990;172:1517–20. [17] Nishida M, Nasu K, Fukuda J, Kawano Y, Narahara H, Miyakawa I. Down-regulation of interleukin-1 receptor type 1 expression causes the dysregulated expression of CXC chemokines in endometriotic stromal cells: a possible mechanism for the altered immunological functions in endometriosis. J Clin Endocrinol Metab 2004;89:5094–100. [18] Matsumoto H, Nasu K, Nishida M, Itoh H, Sun B, Miyakawa I. Regulation of proliferation, motility, and contractivity of human endometrial stromal cells by platelet-derived growth factor. J Clin Endocrinol Metab 2005;90:3560–7. [19] Nasu K, Nishida M, Matsumoto H, et al. Regulation of proliferation, motility, and contractivity of human endometrial stromal cells by transforming growth factor-b isoforms. Fertil Steril 2005;2(Suppl. 84):1114–23.
[20] Noyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Fertil Steril 1950;1:3–25. [21] Nasu K, Matsui N, Narahara H, Tanaka Y, Miyakawa I. Effects of interferon-g on cytokine production by endometrial stromal cells. Hum Reprod 1998;13:2598–601. [22] Roberts DK, Parmley TH, Walker NJ, Horbeit DV. Ultrastructure of the microvasculature in the human endometrium throughout the normal menstrual cycle. Am J Obstet Gynecol 1992;166:1393–406. [23] Ivanov V, Roomi W, Kalinovsky T, Niedzwiecki A, Rath M. Natural nutrient mixture effectively reduces collagen matrix contraction driven by human uterine smooth muscle cells. J Obstet Gynaecol Res 2006;32:23–31. [24] Tabibzadeh S, Zupi E, Babaknia A, Liu R, Marconi D, Romanini C. Site and menstrual cycle-dependent expression of proteins of the tumour necrosis factor (TNF) receptor family, and BCL-2 oncogene and phase specific production of TNF-a in human endometrium. Hum Reprod 1995;10:277–86. [25] Tabibzadeh S. The signals and molecular pathways involved in human menstruation, a unique process of tissue destruction and remodeling. Mol Hum Reprod 1996;2:77–92.
Regulation of contractility of cultured human endometrial stromal cells by tumor necrosis factor-a Akitoshi Yuge, Kaei Nasu *, Hatsumi Tsusue, Etsuko Ikegami, Masakazu Nishida, Harunobu Matsumoto, Hisashi Narahara Department of Obstetrics and Gynecology, Faculty of Medicine, Oita University, Idaigaoka 1-1, Hasama-machi, Yufu-shi, Oita 879-5593, Japan Received 30 January 2007; received in revised form 12 April 2007; accepted 18 May 2007
Abstract Objective: The aim of this study is to evaluate the involvement of tumor necrosis factor (TNF)-a in endometrial tissue remodeling during the perimenstrual period. Study design: Human endometrial stromal cells (ESCs) were isolated from eight premenopausal patients in the late secretory phase. The effects of TNF-a on the contractility of cultured ESCs were investigated by collagen gel contraction assay. The effects of TNF-a on the proliferation of ESCs were also assessed by a modified methylthiazoletetrazolium assay. Results: TNF-a significantly upregulated the collagen gel contractility of ESCs in a dose-dependent manner. TNF-a did not affect ESC proliferation. Conclusion: The results suggest that TNF-a may promote endometrial tissue repair by stimulating the contraction of the extracellular matrix by ESCs. By regulating ESC function during the perimenstrual period, TNF-a may be involved in the physiological tissue remodeling of the cyclic endometrium. # 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Endometrial stromal cells; Tumor necrosis factor-a; Contractility; Tissue remodeling
1. Introduction The human uterine endometrium is a dynamic organ that undergoes cyclic phases of remarkable periodic growth, remodeling, and breakdown. Unlike in most adult tissues, in the uterine endometrium cyclic growth and tissue remodeling occur throughout the reproductive years. Endometrial tissue remodeling during the menstrual cycle is thought to be regulated by ovarian steroids as well as by various cytokines, neuropeptides, and growth factors that are produced locally and secreted in endocrine, paracrine, as well as autocrine manners [1]. The tissue remodeling events that occur during menstruation share common features with injury and repair events in other tissues, which may occur not only after injury * Corresponding author. Tel.: +81 97 586 5922; fax: +81 97 586 6687. E-mail address: [email protected] (K. Nasu).
but also in association with various pathologies [1]. However, there are important differences between tissues, and a range of features from different models are likely to apply to endometrial repair. There are also aspects of wound healing in other adult tissues that do not appear to apply to the endometrium. These include the development of granulation tissue, which occurs during the healing of most cutaneous wounds, and the formation of blood clots, which provide important mediators for the initiation of repair. In addition, most wounds heal with scarring, and this is generally not seen in human endometrial repair. Tumor necrosis factor (TNF)-a, a 17-kDa polypeptide with immunologic functions similar to those of interleukin (IL)-1 and IL-6 [2], was originally described as a factor produced by macrophages in response to agents such as bacterial lipopolysaccharide. Its major immunologic roles are thought to be the control of inflammatory responses and protection from bacterial infection [3]. In addition to its
0301-2115/$ – see front matter # 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2007.05.010
A. Yuge et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 138 (2008) 66–70
immunologic functions, TNF-a is known to have pleiotrophic effects, which modulate cellular growth, differentiation, and the synthesis of various substances [4,5]. The existence of both TNF-a and its receptors in the various reproductive tissues has been reported. In normal endometrium, it has been demonstrated that TNF-a promotes cell growth in early proliferative stages, facilitates cell death and shedding in late stages, encourages angiogenesis, stimulates the synthesis of remodeling enzymes, induces dyscohesion of epithelial cells [6,7], and inhibits decidualization of ESCs in vitro [8]. Immunohistochemical and in situ hybridization analyses have shown the presence of TNF-a protein and mRNA within both stromal/epithelial cells and leukocytes of the human endometrium [9–14]. TNF-a exerts its biological effects via two cell-surface receptors: TNF receptor type 1 (TNF-RI) (50–60 kDa) and TNF receptor type 2 (TNF-RII) (75–80 kDa). Each receptor is encoded by two independent genes and is capable of mediating distinct intracellular signals [15,16]. The expression of both TNF-RI and TNF-RII has been detected in endometrial stromal cells (ESCs) [17]. It has been demonstrated that most of the biological activities of TNF-a are transduced through TNF-RI [15,16]. In the present study, we investigated the effects of TNF-a on the proliferation and contractility of cultured human ESCs using well-characterized in vitro models [18,19]. We also discussed the role of TNF-a on endometrial tissue remodeling during the menstrual cycle.
2. Materials and methods 2.1. ESC isolation procedure and cell culture conditions Normal endometrial specimens were obtained from eight premenopausal patients (aged 35–41 years) who had undergone hysterectomies for intramural or subserosal leiomyomas. All of the specimens were determined to be from the late secretory phase on the basis of the standard histologic criteria [20]. This study was approved by the institutional review board (IRB) of Oita University, and written informed consent was obtained from all patients. Normal ESCs were separated from epithelial glands by digesting the tissue fragments with collagenase, as previously described [21]. Briefly, the tissue was minced into 2–3 mm pieces and incubated with collagenase (200 U/ml) (GibcoBRL, Gaithersburg, MD, USA) in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco-BRL) with stirring for 30 min at 37 8C. The suspension was filtered through a 150mm wire sieve to remove mucus and undigested tissue. The filtrate was then passed through an 80-mm wire sieve, which allowed the stromal cells to pass through while the intact glands were retained. After washing the cells three times with serum-free DMEM, they were transferred to culture flasks (Corning, New York, NY, USA) at a density of 106 cells/ml in DMEM supplemented with 10% heat-inactivated fetal bovine
67
serum (FBS) (Gibco-BRL), streptomycin (100 U/ml) (GibcoBRL), and penicillin (100 U/ml) (Gibco-BRL). The culture medium was replaced every 4 days. After three passages using standard trypsinization methods, the cells were >99% pure as analyzed by immunocytochemical staining with antibodies to vimentin (V9, Dako, Copenhagen, Denmark), cytokeratin (Dako), factor VIII (Dako), and leukocyte common antigen (2B11 + PD7/26, Dako), and were then used for the experiments. The cultures were incubated at 37 8C in an atmosphere of 5% CO2 in air at 100% humidity. Cells isolated from each individual patient were used for one experiment at a time. Each experiment was performed in triplicate and repeated at least three times. 2.2. Assessment of ESC proliferation after TNF-a treatment ESC proliferation after TNF-a treatment was determined in 96-well plates by a modified methylthiazoletetrazolium (MTT) assay using WST-1 (Roche Diagnostics GmbH, Penzberg, Germany) according to the manufacturer’s protocol. Then, 5 104 ESCs in DMEM supplemented with 10% FBS were distributed into each well of a 96-well flat-bottomed microplate (Corning, New York, NY, USA) and incubated overnight. The medium was then removed and the cells were incubated for 48 h with 200 ml of serumfree DMEM containing various concentrations of TNF-a (0.1–10 ng/ml) (R&D Systems, Minneapolis, MN, USA). Thereafter, 20 ml of WST-1 dye was added to each well, which was further incubated for 4 h. Cell proliferation was evaluated by measuring the absorbance at 540 nm. Data were calculated as the ratio of values obtained for the treated cells and those for the untreated controls. 2.3. Collagen gel contraction assay Cellular collagen gel contraction assays were performed as previously described [18,19]. A sterile solution of acidsoluble collagen type I purified from porcine tendons (Cellmatrix type I-A; Nitta Gelatin Inc., Osaka, Japan) was prepared according to the manufacturer’s instructions. ESCs were embedded in the collagen gel and cultured threedimensionally. Briefly, ESCs were suspended in the collagen solution (3.0 105 cells/ml). The collagen/cell mixture (2 ml/plate) was dispensed into 35-mm culture plates (Corning) coated with 0.2% bovine serum albumin (Sigma); the mixture was allowed to polymerize at 37 8C for 30 min. Immediately after polymerization, 1 ml of DMEM supplemented with 10% FBS containing TNF-a (final concentration: 0.1–10 ng/ml) was added to each plate. After incubation for 48 h, the collagen gels were photographed and the area of the gel surface was measured with the public domain Image Program 1.61 developed at National Institutes of Health (Bethesda, MD, USA). The incubation time was determined by background experiments.
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A. Yuge et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 138 (2008) 66–70
3. Results
Fig. 1. The effect of TNF-a on the cell proliferation of ESCs as assessed by WST-1 assay. ESCs were treated with various concentrations of TNF-a (0.1–10 ng/ml) for 48 h. Thereafter, 20 ml of WST-1 dye was added to each well and further incubated for 4 h. Cell proliferation was evaluated by measuring the absorbance at 540 nm. Representative results are shown.
The effects of TNF-a on ESC proliferation were investigated by use of a modified MTT assay. TNF-a did not affect the proliferation of these cells (Fig. 1). As a preliminary experiment, we evaluated the effects of TNF-a on the collagen gel contraction without the presence of ESCs. TNF-a did not induce the gel contraction in the absence of ESCs. The effects of TNF-a on the collagen gel contraction of ESCs were evaluated. In the presence of FBS, untreated ESCs showed significant collagen gel contractility (54.0% contraction after 48 h compared with 0 h). As shown in Fig. 2, the gel surface areas were significantly reduced in the presence of TNF-a in a dose-dependent manner (37.2% reduction of the gel surface area at a concentration of 10 ng/ ml of TNF-a, p < 0.0001 versus untreated controls). Therefore, it is likely that TNF-a significantly enhanced the collagen gel contractility of ESCs.
2.4. Statistical analysis 4. Discussion Data were presented as means S.D. and were analyzed by the Bonferroni/Dunn test with StatView 4.5 (Abacus Concepts, Berkeley, CA, USA). A p value <0.05 was accepted as statistically significant.
During the secretory phase, the endometrium undergoes changes, including differentiation of its epithelium into secretory glands and the transformation of its stromal
Fig. 2. The effects of TNF-a on the collagen gel contractility of ESCs. ESCs were treated with various concentrations of TNF-a (0.1–10 ng/ml) for 48 h. The collagen gels were photographed (A and B) and the collagen gel contractility was assessed by measuring the gel surface area (C). *p < 0.0001 vs. untreated controls (Bonferroni/Dunn test).
A. Yuge et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 138 (2008) 66–70
mesenchyme into predecidual cells. During the late secretory phase of the cycle, widespread degeneration occurs in the basal lamina supporting the decidualized endometrial cells and the endothelium of blood vessels [22]. There is a very rapid but incomplete degeneration of the functionalis layer, exposing open blood vessels and glands. Endometrial destruction is a consequence of the activity of matrix degrading enzymes on the extracellular matrix (i.e., both the fibrillar matrix and basal lamina) after the withdrawal of steroid hormone support at the end of each menstrual cycle, with the resultant loss of blood vessel integrity and shedding of most of the functional layer [1]. Menstrual bleeding stops by the time tissue destruction have ceased. When substantial endometrial tissue loss occurs during menstruation, ESC ingrowth, extracellular matrix deposition, and angiogenesis, reestablish organ integrity, often in concert with tissue contraction. Regeneration begins in areas where the mouths of the basal glands are free from overlying degenerated tissue. There is simultaneous and progressive epithelial outgrowth from these glands as well as ingrowth from the intact peripheral surface membrane bordering the denuded basalis. The stromal tissue begins to grow only when the endometrial wound is completely re-epithelialized. These events are thought to occur under the influence of increasing estrogen concentrations, and are probably locally regulated by a number of cytokines and other regulatory factors, including TNF-a [1]. Another important event during endometrial wound healing is the contraction of connective tissue, which is carried out by fibroblastic cells. ESCs appear to be responsible for wound contraction. The presence of serum itself induced the contractility of ESCs in three-dimensional culture as shown in our previous reports [18,19]. We have demonstrated that collagen gel contraction is further stimulated by platelet-derived growth factor [18] and by transforming growth factor-b [19]. In the present study, we evaluated the effects of TNF-a with respect to endometrial tissue remodeling during the menstrual cycle, and found that TNF-a could stimulate ESCs to contract the collagen gel matrix. These results are similar to those of previous reports that have examined uterine smooth muscle cells [23]. The contractile activity of ESCs may favor expulsion of the endometrium during menstruation, reduce the size of the endometrial wound defect for eventual wound closure, and reduce the amount of fibroplasia and angiogenesis necessary for the reestablishment of organ integrity. Although the precise mechanisms of TNF-a-induced contractile activity of ESCs are still unknown from the present study, possible candidate mediators of this phenomenon are prostaglandins. Previous studies using immunohistochemistry, Northern blot analysis, and explant cultures have demonstrated the production of TNF-a mRNA and protein by human endometrium [9–14,24]. These reports indicate that ESCs as well as endometrial epithelial cells may be the sources of TNF-a in the endometrium, suggesting that autocrine and
69
paracrine mechanisms take place in the induction of contractility of ESCs. Some studies have shown that the endometrium increases TNF-a mRNA and protein production during the secretory and menstrual phases of the menstrual cycle [7,12,24,25], suggesting the roles of TNF-a in tissue remodeling during the perimenstrual period. Taken together, these data suggest the involvement of TNF-a in endometrial tissue remodeling during the perimenstrual period. In summary, we demonstrated that TNF-a stimulates the contractility of ESCs without affecting their proliferation. The present results suggest that TNF-a may contribute to endometrial tissue remodeling during perimenstruation by regulating ESC cellular functions.
Acknowledgements This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (no. 16591672 to K. Nasu).
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