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Expression and regulation of thrombospondin-1 by human endometrial stromal cells
Expression and regulation of thrombospondin-1 by human endometrial stromal cells Thrombospondin-1 (TSP-1) production was modulated by EGF, IFN-␥, and in vitro decidualization. It is suggested that TSP-1 may contribute to the regulation of neovascularization in the endometrium and gestational tissues. (Fertil Steril威 2005;83:1056 –9. ©2005 by American Society for Reproductive Medicine.)
Abundant angiogenesis is necessary for proliferation and differentiation during embryonic implantation and placentation. Vascular permeability changes throughout the menstrual cycle; the endometrium becomes thick in the proliferative phase and then becomes edematous in the secretory phase. Although these angiogenic events may be regulated by several factors, the detailed mechanisms of vascular growth, development, and permeability in the endometrium are unknown. Thrombospondin (TSP) is characterized as a secreted glycoprotein produced from platelets in response to activation by thrombin; TSP has been produced by a variety of cells in culture. TSP has been identified as an inhibitor of angiogenesis because it was shown to be controlled by a tumor suppressor gene (1). The inhibitory role of TSP in angiogenesis is also supported by its presence adjacent to mature quiescent vessels and its absence from actively growing sprouts, both in vivo (2) and in vitro (3). The specific mechanisms of angiogenic suppression are not understood; the process is further complicated by interaction of TSP-1 with a variety of extracellular macromolecules and growth factors. In a previous study, it was reported that TSP-1 was produced in the human endometrium (4). Although TSP-1 gene expression is reportedly expressed in human endometrium (4), little is known about the regulation of TSP-1 in endometrial stromal cells (ESC). The purpose of this study was to clarify the effects of endothelial growth factor (EGF), interferon-␥ (IFN-␥), and in vitro decidualization on TSP-1 expression by endometrial stromal cells (ESC). Normal endometrial specimens were obtained from 11 premenopausal patients who had undergone hysterectomies for subserous and intramural myomas. All of the specimens were identified as being in the midsecretory phase (days 19 Received December 9, 2003; revised and accepted September 21, 2004. Supported by a Grant-in Aid for Specific Research (no.15591766, Y.K. and no. 13671733, I. M.) from the Ministry of Education, Science, and Culture of Japan. Reprint requests: Yasushi Kawano, M.D., Department of Obstetrics and Gynecology, Oita University, Faculty of Medicine, Idaigaoka 1-1, Hasama, Oita 879-5593, Japan (FAX: 81-97-549-5087; E-mail: [email protected]).
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to 21 of the menstrual cycle) on the basis of standard histologic criteria. This study was approved by the institutional review board of Oita University. Normal ESC were separated from epithelial glands by digesting the tissue fragments with collagenase, as previously described elsewhere (5) with slight modifications. Decidualization was induced by incubating subconfluent cells in media containing 100 nM of medroxyprogesterone (MPA) and 0.5 mM of dibutyryl-cyclic adenosine monophosphate (db-cAMP). The supernatant was replaced with fresh culture medium containing db-cAMP (0.5 mM) and MPA (1 and 100 nM). Phase-contrast microscopy was used to verify morphologic changes associated with differentiation in vitro in response to db-cAMP and MPA. The concentrations of prolactin (PRL) in conditioned medium were measured by a microparticle enzyme immunoassay using materials provided by Abbott Laboratories (North Chicago, IL). To investigate the expression of TSP-1 mRNA by ESC, the total cellular RNA in ESC was isolated by TriZol (GIBCOBRL, Grand Island, NY). The RNA within the gel was transferred by Northern blot onto Hybond-N nylon filters (Amersham, Buckinghamshire, United Kingdom). The filters were hybridized at 45°C under high stringency with 32P-labeled TSP-1 and glyceroaldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probes synthesized by the random primer method. Autoradiography was performed for 7 days at ⫺80°C with intensifying screens. These experiments were performed in triplicate and were repeated three times. To investigate the production of TSP-1 by ESC, the supernatant was replaced with fresh culture medium that contained EGF and IFN-␥ for 24 hours and was collected. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed; samples were separated, and proteins were transferred onto polyvinylidene diflouride membranes (Millipore, Bedford, MA). Then, the membranes were incubated with 1:1000-diluted antibody (human TSP-1 antibody, mouse monoclonal IgG; NeoMarkers, Fremont, CA). After washing samples with three changes of TBS with 0.1% Tween 20, the membranes were incubated with 1:2000-diluted peroxidase-conjugated antibody (anti-mouse immunoglobulin ␥ or chain; ICN Phar-
Fertility and Sterility姞 Vol. 83, No. 4, April 2005 Copyright ©2005 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/05/$30.00 doi:10.1016/j.fertnstert.2004.09.035
FIGURE 1 Expression of mRNA for thrombospondin-1 (TSP-1) in endometrial stromal cells (ESC) after 8 hours of stimulation with various amounts of (A) interferon-␥ and (B) endothelial growth factor (EGF). (C) Production of protein for TSP-1 in ESC after 24 hours of stimulation with IFN-␥ and EGF. (D) Expression of mRNA for TSP-1 in ESC after 7 days of stimulation with db-cAMP and MPA. GAPDH mRNA are provided as internal controls.
Kawano. TSP-1 expression by endometrial cells. Fertil Steril 2005.
maceuticals, Aurora, OH). After samples were washed with four changes of TBS with 0.1% Tween 20, Lumi GLO from the phototope-HRP Western-detection kit (New England BioLabs, Beverly, MA) was added to the blotted membranes. Membranes were then covered with plastic wrap and exposed x-ray film (Amersham) for 1 to 2 minutes. To evaluate the effects of IFN-␥ and EGF, we examined TSP-1 mRNA expression by Northern blot analysis. The regulation of TSP-1 mRNA expression incubated with various concentrations of IFN-␥ and EGF is shown in Figure Fertility and Sterility姞
1A and B. A comparison with controls revealed that TSP-1 mRNA expression was enhanced in a dose-dependent manner by treatment with IFN-␥. However, TSP-1 mRNA expression was inhibited in a dose-dependent manner by treatment with EGF, as compared with controls. The GAPDH mRNA are given as internal controls. To evaluate the effects of EGF and IFN-␥ on ESC, we examined TSP-1 protein production. The supernatants were collected from confluent cultures of ESC, and we performed a Western immunoblot analysis with TSP-1 protein production. The regulation of TSP-1 protein incubated with 1057
IFN-␥ (10 U/mL) and EGF (1 nM) is shown in Figure 1C. The intensity of the band of TSP-1 protein treated with IFN-␥ increased compared with that of controls. However, the intensity of the bands of TSP-1 treated with EGF decreased compared with that of controls. To evaluate the regulation of TSP-1 in the decidualization of ESC in vitro, we examined TSP-1 mRNA expression. When PRL production was stimulated by db-cAMP (0.5 mM) and MPA (100 nM), it statistically significantly increased compared with control (P⬍.01) (data not shown). Expression of TSP-1 mRNA incubated with cAMP (0.5 mM) and MPA (100 nM) is shown in Figure 1D. The increase in intensity of TSP-1 mRNA expression by decidualized ESC was much greater than that of controls. Recent studies have demonstrated that TSP-1 is produced in several organs, including the uterus (4, 6) and platelets and vessels (7, 8). The female reproductive tissues—in particular, the endometrium of menstruating primates—are exceptional in the adult with respect to the high degree of angiogenesis accompanying the monthly development of these tissues. In the first study of TSP-1 in the human endometrium (4), it was shown that the main expression was in the stroma during the secretory phase. Expression of TSP-1 mRNA has been demonstrated in the secretory phase as compared with the proliferative phase. The human endometrium undergoes cyclic, hormonally dependent changes during proliferation, differentiation, sloughing, and repair. Although it has been demonstrated that P can increase TSP-1 mRNA expression by ESC in vitro, elucidation of the details of its production and regulation requires further study. Moreover, P causes an induction of TSP-1 mRNA in isolated ESC, reflected in a 3.8fold increase (4). It is suggested that two P-responsive elements are involved in the production of the human TSP-1 gene. Production of TSP-1 mRNA was somewhat inhibited by exogenous EGF in a dose-dependent manner. On the other hand, TSP-1 mRNA was enhanced in a dose-dependent manner by treatment with IFN-␥. These observations suggested that TSP-1 expression may be regulated by EGF and IFN-␥ in vitro. Endothelial growth factor influences the human endometrial cycle, endometrial-trophoblastic interactions, and endometrial tissue regeneration in the absence of implantation. A marked change in cellular localization of EGF and EGF receptors in the human placenta has been observed between 5 and 6 weeks of gestation during the first trimester; at 4 to 5 weeks, EGF and EGF receptors were almost entirely limited to the cytotrophoblasts, and between 6 and 12 weeks they were found mainly in the syncytiotrophoblasts. The EGF receptor has been found in both glandular epithelium and stromal cells (9). Lymphoid cells isolated from the endometrial cavity may also produce IFN-␥. It has been demonstrated that 1058
Kawano et al.
Correspondence
IFN-␥ secreted from T cells in the endometrium may suppress endometrial proliferation and lead to differentiation (10). According to our data, TSP-1 produced by ESC could be transcriptionally regulated by IFN-␥. On the basis of these observations, it is possible that angiogenesis in the endometrium might be regulated by IFN-␥. It is also suggested that IFN-␥ might be an important factor that modulates the proliferation and differentiation of the human endometrium, resulting in implantation and/or the maintenance of pregnancy. In previous reports, IFN-␥ has been shown to up-regulate the production of several cytokines by ESC. We have previously demonstrated that IFN-␥ down-regulates the production of vascular endothelial growth factor (VEGF), an angiogenic factor (5). Although this well-characterized culture system is suitable for the evaluation of the effect of a single molecule on ESC, the cytokines or growth factors in the endometrium may be more complicated, and the function of ESC may be directly affected by other surrounding cell populations (endometrial epithelial cells, leukocytes, endothelial cells, etc.). Therefore, it is suggested that the regulation of TSP-1 may be further modified in the presence of other cell populations. Perturbations in TSP-1 expression may contribute to many vascular pathologies of the reproductive tract. The presence of TSP-1 in the human endometrium and increased expression of TSP-1 in secretory tissue suggests that menstrual disorders such as dysfunctional uterine bleeding or decreased endometrial receptivity may be a reflection of aberrant TSP-1 expression. In conclusion, our data suggest that TSP-1 protein production and TSP-1 mRNA levels regulated by EGF and IFN-␥ by ESC, EGF, and IFN-␥ may contribute to a regulation of cell growth or proliferation of tissues. This may, in part, be control due to antiangiogenic growth factors such as TSP-1. Yasushi Kawano, M.D. Satomi Nakamura, M.D. Kaei Nasu, M.D. Junichiro Fukuda, M.D. Hisashi Narahara, M.D. Isao Miyakawa, M.D. Department of Obstetrics and Gynecology, Oita University, Faculty of Medicine, Hasama, Oita, Japan REFERENCES 1. Rastinejad F, Polverini PJ, Bouck NP. Regulation of the activity of a new inhibitor of angiogenesis by a cancer suppressor gene. Cell 1989;56:345–55. 2. O’Shea KS, Dixit VM. Unique distribution of extracellular matrix component thrombospondin in the developing mouse embryo. J Cell Biol 1988;107:2737– 48. 3. Iruela-Arispe ML, Bornstein P, Sage H. Thrombospondin exerts an
Vol. 83, No. 4, April 2005
antiangiogenic effect on cord formation by endothelial cells in vitro. Proc Natl Acad Sci USA 1991;88:5026 –30. 4. Iruela-Arispe ML, Porter P, Bornstein P, Sage EH. Thrombospondin-1, an inhibitor of angiogenesis, is regulated by progesterone in the human endometrium. J Clin Invest 1996;97:403–12. 5. Kawano Y, Matsui N, Kamihigashi S, Narahara H, Miyakawa I. Effects of interferon-gamma on secretion of vascular endothelial growth factor by endometrial stromal cells. Am J Reprod Immunol 2000;43:47–52. 6. Morimoto T, Head JR, MacDonald PC, Casey ML. Thrombospondin-1 expression in human myometrium before and during pregnancy, before and during labor, and in human myometrial cells in culture. Biol Reprod 1998;59:862–70.
Fertility and Sterility姞
7. Mosher DF. Physiology of thrombospondin. Annu Rev Med 1990; 41:85–97. 8. Bornstein P, Sage EH. Thrombospondins. Methods Enzymol 1994; 245:62– 85. 9. Prentice A, Thomas EJ, Weddell A, McGill A, Randall BJ, Horne CH. Epidermal growth factor receptor expression in normal endometrium and endometriosis: an immunohistochemical study. Br J Obstet Gynaecol 1992;99:395– 8. 10. Tabibzadeh S, Sun XZ, Kong QF, Kasnic G, Miller J, Satyaswaroop PG. Induction of a polarized micro-environment by human T cells and interferon-gamma in three-dimensional spheroid cultures of human endometrial epithelial cells. Hum Reprod 1993;8:182–92.
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Abundant angiogenesis is necessary for proliferation and differentiation during embryonic implantation and placentation. Vascular permeability changes throughout the menstrual cycle; the endometrium becomes thick in the proliferative phase and then becomes edematous in the secretory phase. Although these angiogenic events may be regulated by several factors, the detailed mechanisms of vascular growth, development, and permeability in the endometrium are unknown. Thrombospondin (TSP) is characterized as a secreted glycoprotein produced from platelets in response to activation by thrombin; TSP has been produced by a variety of cells in culture. TSP has been identified as an inhibitor of angiogenesis because it was shown to be controlled by a tumor suppressor gene (1). The inhibitory role of TSP in angiogenesis is also supported by its presence adjacent to mature quiescent vessels and its absence from actively growing sprouts, both in vivo (2) and in vitro (3). The specific mechanisms of angiogenic suppression are not understood; the process is further complicated by interaction of TSP-1 with a variety of extracellular macromolecules and growth factors. In a previous study, it was reported that TSP-1 was produced in the human endometrium (4). Although TSP-1 gene expression is reportedly expressed in human endometrium (4), little is known about the regulation of TSP-1 in endometrial stromal cells (ESC). The purpose of this study was to clarify the effects of endothelial growth factor (EGF), interferon-␥ (IFN-␥), and in vitro decidualization on TSP-1 expression by endometrial stromal cells (ESC). Normal endometrial specimens were obtained from 11 premenopausal patients who had undergone hysterectomies for subserous and intramural myomas. All of the specimens were identified as being in the midsecretory phase (days 19 Received December 9, 2003; revised and accepted September 21, 2004. Supported by a Grant-in Aid for Specific Research (no.15591766, Y.K. and no. 13671733, I. M.) from the Ministry of Education, Science, and Culture of Japan. Reprint requests: Yasushi Kawano, M.D., Department of Obstetrics and Gynecology, Oita University, Faculty of Medicine, Idaigaoka 1-1, Hasama, Oita 879-5593, Japan (FAX: 81-97-549-5087; E-mail: [email protected]).
1056
to 21 of the menstrual cycle) on the basis of standard histologic criteria. This study was approved by the institutional review board of Oita University. Normal ESC were separated from epithelial glands by digesting the tissue fragments with collagenase, as previously described elsewhere (5) with slight modifications. Decidualization was induced by incubating subconfluent cells in media containing 100 nM of medroxyprogesterone (MPA) and 0.5 mM of dibutyryl-cyclic adenosine monophosphate (db-cAMP). The supernatant was replaced with fresh culture medium containing db-cAMP (0.5 mM) and MPA (1 and 100 nM). Phase-contrast microscopy was used to verify morphologic changes associated with differentiation in vitro in response to db-cAMP and MPA. The concentrations of prolactin (PRL) in conditioned medium were measured by a microparticle enzyme immunoassay using materials provided by Abbott Laboratories (North Chicago, IL). To investigate the expression of TSP-1 mRNA by ESC, the total cellular RNA in ESC was isolated by TriZol (GIBCOBRL, Grand Island, NY). The RNA within the gel was transferred by Northern blot onto Hybond-N nylon filters (Amersham, Buckinghamshire, United Kingdom). The filters were hybridized at 45°C under high stringency with 32P-labeled TSP-1 and glyceroaldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probes synthesized by the random primer method. Autoradiography was performed for 7 days at ⫺80°C with intensifying screens. These experiments were performed in triplicate and were repeated three times. To investigate the production of TSP-1 by ESC, the supernatant was replaced with fresh culture medium that contained EGF and IFN-␥ for 24 hours and was collected. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed; samples were separated, and proteins were transferred onto polyvinylidene diflouride membranes (Millipore, Bedford, MA). Then, the membranes were incubated with 1:1000-diluted antibody (human TSP-1 antibody, mouse monoclonal IgG; NeoMarkers, Fremont, CA). After washing samples with three changes of TBS with 0.1% Tween 20, the membranes were incubated with 1:2000-diluted peroxidase-conjugated antibody (anti-mouse immunoglobulin ␥ or chain; ICN Phar-
Fertility and Sterility姞 Vol. 83, No. 4, April 2005 Copyright ©2005 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/05/$30.00 doi:10.1016/j.fertnstert.2004.09.035
FIGURE 1 Expression of mRNA for thrombospondin-1 (TSP-1) in endometrial stromal cells (ESC) after 8 hours of stimulation with various amounts of (A) interferon-␥ and (B) endothelial growth factor (EGF). (C) Production of protein for TSP-1 in ESC after 24 hours of stimulation with IFN-␥ and EGF. (D) Expression of mRNA for TSP-1 in ESC after 7 days of stimulation with db-cAMP and MPA. GAPDH mRNA are provided as internal controls.
Kawano. TSP-1 expression by endometrial cells. Fertil Steril 2005.
maceuticals, Aurora, OH). After samples were washed with four changes of TBS with 0.1% Tween 20, Lumi GLO from the phototope-HRP Western-detection kit (New England BioLabs, Beverly, MA) was added to the blotted membranes. Membranes were then covered with plastic wrap and exposed x-ray film (Amersham) for 1 to 2 minutes. To evaluate the effects of IFN-␥ and EGF, we examined TSP-1 mRNA expression by Northern blot analysis. The regulation of TSP-1 mRNA expression incubated with various concentrations of IFN-␥ and EGF is shown in Figure Fertility and Sterility姞
1A and B. A comparison with controls revealed that TSP-1 mRNA expression was enhanced in a dose-dependent manner by treatment with IFN-␥. However, TSP-1 mRNA expression was inhibited in a dose-dependent manner by treatment with EGF, as compared with controls. The GAPDH mRNA are given as internal controls. To evaluate the effects of EGF and IFN-␥ on ESC, we examined TSP-1 protein production. The supernatants were collected from confluent cultures of ESC, and we performed a Western immunoblot analysis with TSP-1 protein production. The regulation of TSP-1 protein incubated with 1057
IFN-␥ (10 U/mL) and EGF (1 nM) is shown in Figure 1C. The intensity of the band of TSP-1 protein treated with IFN-␥ increased compared with that of controls. However, the intensity of the bands of TSP-1 treated with EGF decreased compared with that of controls. To evaluate the regulation of TSP-1 in the decidualization of ESC in vitro, we examined TSP-1 mRNA expression. When PRL production was stimulated by db-cAMP (0.5 mM) and MPA (100 nM), it statistically significantly increased compared with control (P⬍.01) (data not shown). Expression of TSP-1 mRNA incubated with cAMP (0.5 mM) and MPA (100 nM) is shown in Figure 1D. The increase in intensity of TSP-1 mRNA expression by decidualized ESC was much greater than that of controls. Recent studies have demonstrated that TSP-1 is produced in several organs, including the uterus (4, 6) and platelets and vessels (7, 8). The female reproductive tissues—in particular, the endometrium of menstruating primates—are exceptional in the adult with respect to the high degree of angiogenesis accompanying the monthly development of these tissues. In the first study of TSP-1 in the human endometrium (4), it was shown that the main expression was in the stroma during the secretory phase. Expression of TSP-1 mRNA has been demonstrated in the secretory phase as compared with the proliferative phase. The human endometrium undergoes cyclic, hormonally dependent changes during proliferation, differentiation, sloughing, and repair. Although it has been demonstrated that P can increase TSP-1 mRNA expression by ESC in vitro, elucidation of the details of its production and regulation requires further study. Moreover, P causes an induction of TSP-1 mRNA in isolated ESC, reflected in a 3.8fold increase (4). It is suggested that two P-responsive elements are involved in the production of the human TSP-1 gene. Production of TSP-1 mRNA was somewhat inhibited by exogenous EGF in a dose-dependent manner. On the other hand, TSP-1 mRNA was enhanced in a dose-dependent manner by treatment with IFN-␥. These observations suggested that TSP-1 expression may be regulated by EGF and IFN-␥ in vitro. Endothelial growth factor influences the human endometrial cycle, endometrial-trophoblastic interactions, and endometrial tissue regeneration in the absence of implantation. A marked change in cellular localization of EGF and EGF receptors in the human placenta has been observed between 5 and 6 weeks of gestation during the first trimester; at 4 to 5 weeks, EGF and EGF receptors were almost entirely limited to the cytotrophoblasts, and between 6 and 12 weeks they were found mainly in the syncytiotrophoblasts. The EGF receptor has been found in both glandular epithelium and stromal cells (9). Lymphoid cells isolated from the endometrial cavity may also produce IFN-␥. It has been demonstrated that 1058
Kawano et al.
Correspondence
IFN-␥ secreted from T cells in the endometrium may suppress endometrial proliferation and lead to differentiation (10). According to our data, TSP-1 produced by ESC could be transcriptionally regulated by IFN-␥. On the basis of these observations, it is possible that angiogenesis in the endometrium might be regulated by IFN-␥. It is also suggested that IFN-␥ might be an important factor that modulates the proliferation and differentiation of the human endometrium, resulting in implantation and/or the maintenance of pregnancy. In previous reports, IFN-␥ has been shown to up-regulate the production of several cytokines by ESC. We have previously demonstrated that IFN-␥ down-regulates the production of vascular endothelial growth factor (VEGF), an angiogenic factor (5). Although this well-characterized culture system is suitable for the evaluation of the effect of a single molecule on ESC, the cytokines or growth factors in the endometrium may be more complicated, and the function of ESC may be directly affected by other surrounding cell populations (endometrial epithelial cells, leukocytes, endothelial cells, etc.). Therefore, it is suggested that the regulation of TSP-1 may be further modified in the presence of other cell populations. Perturbations in TSP-1 expression may contribute to many vascular pathologies of the reproductive tract. The presence of TSP-1 in the human endometrium and increased expression of TSP-1 in secretory tissue suggests that menstrual disorders such as dysfunctional uterine bleeding or decreased endometrial receptivity may be a reflection of aberrant TSP-1 expression. In conclusion, our data suggest that TSP-1 protein production and TSP-1 mRNA levels regulated by EGF and IFN-␥ by ESC, EGF, and IFN-␥ may contribute to a regulation of cell growth or proliferation of tissues. This may, in part, be control due to antiangiogenic growth factors such as TSP-1. Yasushi Kawano, M.D. Satomi Nakamura, M.D. Kaei Nasu, M.D. Junichiro Fukuda, M.D. Hisashi Narahara, M.D. Isao Miyakawa, M.D. Department of Obstetrics and Gynecology, Oita University, Faculty of Medicine, Hasama, Oita, Japan REFERENCES 1. Rastinejad F, Polverini PJ, Bouck NP. Regulation of the activity of a new inhibitor of angiogenesis by a cancer suppressor gene. Cell 1989;56:345–55. 2. O’Shea KS, Dixit VM. Unique distribution of extracellular matrix component thrombospondin in the developing mouse embryo. J Cell Biol 1988;107:2737– 48. 3. Iruela-Arispe ML, Bornstein P, Sage H. Thrombospondin exerts an
Vol. 83, No. 4, April 2005
antiangiogenic effect on cord formation by endothelial cells in vitro. Proc Natl Acad Sci USA 1991;88:5026 –30. 4. Iruela-Arispe ML, Porter P, Bornstein P, Sage EH. Thrombospondin-1, an inhibitor of angiogenesis, is regulated by progesterone in the human endometrium. J Clin Invest 1996;97:403–12. 5. Kawano Y, Matsui N, Kamihigashi S, Narahara H, Miyakawa I. Effects of interferon-gamma on secretion of vascular endothelial growth factor by endometrial stromal cells. Am J Reprod Immunol 2000;43:47–52. 6. Morimoto T, Head JR, MacDonald PC, Casey ML. Thrombospondin-1 expression in human myometrium before and during pregnancy, before and during labor, and in human myometrial cells in culture. Biol Reprod 1998;59:862–70.
Fertility and Sterility姞
7. Mosher DF. Physiology of thrombospondin. Annu Rev Med 1990; 41:85–97. 8. Bornstein P, Sage EH. Thrombospondins. Methods Enzymol 1994; 245:62– 85. 9. Prentice A, Thomas EJ, Weddell A, McGill A, Randall BJ, Horne CH. Epidermal growth factor receptor expression in normal endometrium and endometriosis: an immunohistochemical study. Br J Obstet Gynaecol 1992;99:395– 8. 10. Tabibzadeh S, Sun XZ, Kong QF, Kasnic G, Miller J, Satyaswaroop PG. Induction of a polarized micro-environment by human T cells and interferon-gamma in three-dimensional spheroid cultures of human endometrial epithelial cells. Hum Reprod 1993;8:182–92.
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