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• W2004100557 abstract "Hypoxia downregulated the concentration of endostatin in the culture media of human endometrial stromal cells but did not affect the messenger (m)RNA expression of collagen XVIII. Both mRNA and protein expression of vascular endothelial growth factor were upregulated in a hypoxic condition. Hypoxia downregulated the concentration of endostatin in the culture media of human endometrial stromal cells but did not affect the messenger (m)RNA expression of collagen XVIII. Both mRNA and protein expression of vascular endothelial growth factor were upregulated in a hypoxic condition. Human endometrial development during the menstrual cycle shows unique cycle-specific changes in vascularization. Rapid vascular growth is required to support the proliferation and repair of the endometrium during the menstrual cycle and provides a richly vascularized, receptive endometrium for implantation and placentation. These complex processes of angiogenesis are presumably tightly regulated by multiple system controls that can be switched on and off within a short period (1Torry R.J. Rongish B.J. Angiogenesis in the uterus: potential regulation and relation to tumor angiogenesis.Am J Reprod Immunol. 1992; 27: 171-179Crossref PubMed Scopus (75) Google Scholar, 2Goodger-Macpherson A.M. Rodgers P.A. Blood vessel growth in the endometrium.Microcirculation. 1995; 2: 329-343Crossref PubMed Scopus (36) Google Scholar).Several angiogenic stimulators, including vascular endothelial growth factor (VEGF) (3Li X.F. Gregory J. Ahmed A. Immunolocalisation of vascular endothelial growth factor in human endometrium.Growth Factors. 1994; 11: 277-282Crossref PubMed Scopus (118) Google Scholar, 4Kawano Y. Matsui N. Kamihigashi S. Narahara H. Miyakawa I. Effects of interferon-γ on secretion of vascular endothelial growth factor by endometrial stromal cells.Am J Reprod Immunol. 2000; 43: 47-52Crossref PubMed Google Scholar), basic fibroblast growth factor (5Rusnati M. Casarotti G. Pecorelli S. Ragnotti G. Presta M. Basic fibroblast growth factor in ovulatory cycle and postmenopausal human endometrium.Growth Factors. 1990; 3: 299-307Crossref PubMed Scopus (55) Google Scholar), and hepatocyte growth factor (6Nasu K. Sugano T. Matsui N. Narahara H. Kawano Y. Miyakawa I. Expression of hepatocyte growth factor in cultured endometrial stromal cells is induced through a protein kinase C-dependent pathway.Biol Reprod. 1999; 60: 1183-1187Crossref PubMed Scopus (21) Google Scholar), have been demonstrated to be present in the human endometrium. Angiogenic inhibitors, such as thrombospondin-1 (7Iruela-Arispe M.L. Porter P. Bornstein P. Sage E.H. Thrombospondin-1, an inhibitor of angiogenesis is regulated by progesterone in the human endometrium.J Clin Invest. 1996; 97: 403-412Crossref PubMed Scopus (133) Google Scholar), have also been reported to be expressed in the endometrium.Endostatin is a 20-kd fragment of C-terminal globular domain of collagen XVIII that shows a strong ability to inhibit angiogenesis (8O'Reilly M.S. Boehm T. Shing Y. Fukai N. Vasios G. Lane W.S et al.Endostatin: an endogenous inhibitor of angiogenesis and tumor growth.Cell. 1997; 88: 277-285Abstract Full Text Full Text PDF PubMed Scopus (4210) Google Scholar, 9Dhanabal M. Ramchandran R. Waterman M.J. Lu H. Knebelmann B. Segal M et al.Endostatin induces endothelial cell apoptosis.J Biol Chem. 1999; 274: 11721-11726Crossref PubMed Scopus (586) Google Scholar, 10Dhanabal M. Volk R. Ramchandran R. Simons M. Sukhatme V.P. Cloning, expression, and in vitro activity of human endostatin.Biochem Biophys Res Commun. 1999; 258: 345-352Crossref PubMed Scopus (197) Google Scholar). Collagen XVIII is localized in the basement membrane of blood vessels (11Muragaki Y. Timmons S. Griffith C.M. Oh S.P. Fadel B. Quertermous T et al.Mouse Col18α1 is expressed in a tissue-specific manner as three alternative variants and is localized in basement membrane zones.Proc Natl Acad Sci U S A. 1995; 92: 8763-8767Crossref PubMed Scopus (170) Google Scholar, 12Nasu K. Fujisawa K. Nishida Y. Kai S. Sugano T. Miyakawa I. Tateishi Y. Expression of collagen XVIII mRNA and protein in human umbilical vein and placenta.Reprod Fertil Dev. 2003; 15: 107-114Crossref PubMed Google Scholar). Fragments of collagen XVIII longer than endostatin do not inhibit endothelial cell proliferation (8O'Reilly M.S. Boehm T. Shing Y. Fukai N. Vasios G. Lane W.S et al.Endostatin: an endogenous inhibitor of angiogenesis and tumor growth.Cell. 1997; 88: 277-285Abstract Full Text Full Text PDF PubMed Scopus (4210) Google Scholar). Recently, Chang et al. (13Chang Z. Choon A. Friedl A. Endostatin binds to blood vessels in situ independent of heparan sulfate and does not compete for fibroblast growth factor-2 binding.Am J Pathol. 1999; 155: 71-76Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) demonstrated that endostatin bound to uterine blood vessels, suggesting the possible target of this protein during cyclic endometrial neovascularization.Hypoxia in the endometrium is a physiologic event in the perimenstrual period. Vasoconstriction during menstruation leads to endometrial hypoxia, a possible stimulus for angiogenesis. Desquamated endometrium is influenced by hypoxia, which has been shown to be a strong inducer of VEGF expression in endometrial stromal cells (14Popovici R.M. Irwin J.C. Giaccia A.J. Giudice L.C. Hypoxia and cAMP stimulate vascular endothelial growth factor (VEGF) in human endometrial stromal cells: potential relevance to menstruation and endometrial regeneration.J Clin Endocrinol Metab. 1999; 84: 2245-2248Crossref PubMed Google Scholar, 15Sharkey A.M. Day K. McPherson A. Malik S. Licence D. Smith S.K et al.Vascular endothelial growth factor expression in human endometrium is regulated by hypoxia.J Clin Endocrinol Metab. 2000; 85: 402-409Crossref PubMed Scopus (203) Google Scholar). It is suggested that the effect of hypoxia on VEGF production by endometrial stromal cells might have physiologic relevance during the process of menstruation and in postmenstrual endometrial repair and angiogenesis. In the present study, we evaluated the effects of hypoxia on the expression of collagen XVIII/endostatin and VEGF in cultured human endometrial stromal cells.Normal endometrial specimens were obtained from 20 premenopausal patients (aged 37–43 years) who had undergone hysterectomies for intramural leiomyomas. All the patients had regular menstrual cycles, were multiparous, and were considered to be healthy with the exception of uterine leiomyoma. None had received hormonal therapy before the operation. Ten specimens were diagnosed as being from the late proliferative phase and 10 as being from the mid- to late secretory phase on the basis of standard histologic criteria. All the endometrial specimens were verified to be normal on routine histologic examinations. Normal endometrial stromal cells were isolated and cultured in Roswell Park Memorial Institute 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco-BRL, Gaithersburg, MD), streptomycin (100 U/mL) (Gibco-BRL), and penicillin (100 U/mL) (Gibco-BRL) at 37°C in an atmosphere of 5% CO2 in air at 100% humidity, as previously described (6Nasu K. Sugano T. Matsui N. Narahara H. Kawano Y. Miyakawa I. Expression of hepatocyte growth factor in cultured endometrial stromal cells is induced through a protein kinase C-dependent pathway.Biol Reprod. 1999; 60: 1183-1187Crossref PubMed Scopus (21) Google Scholar). After three passages with standard methods of trypsinization, the cells, which 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), were used for the experiments. This study was approved by the institutional review board of Oita University.To study the release of endostatin and VEGF by endometrial stromal cells, confluent cells cultured on six-well culture plates (Corning, New York, NY) were used. The supernatant was replaced with 1.5 mL of fresh culture medium and further cultured for 24 hours under hypoxic (4% O2, 5% CO2, and 91% N2) or normoxic (20% O2, 5% CO2, and 75% N2) conditions. The supernatant was then collected and stored at −70°C until assay. Isolated cells from each individual patient (n = 20) were used per experiment, and each experiment was performed in triplicate. All experiments were performed in the presence of 10% heat-inactivated FBS. The concentration of endostatin and VEGF was determined in each supernatant with commercially available ELISA kits (Cytimmune Sciences, College Park, MD, and R&D systems, Minneapolis, MN, respectively), as described previously (4Kawano Y. Matsui N. Kamihigashi S. Narahara H. Miyakawa I. Effects of interferon-γ on secretion of vascular endothelial growth factor by endometrial stromal cells.Am J Reprod Immunol. 2000; 43: 47-52Crossref PubMed Google Scholar, 12Nasu K. Fujisawa K. Nishida Y. Kai S. Sugano T. Miyakawa I. Tateishi Y. Expression of collagen XVIII mRNA and protein in human umbilical vein and placenta.Reprod Fertil Dev. 2003; 15: 107-114Crossref PubMed Google Scholar). The sensitivities of the assays for endostatin and VEGF were 1.95 ng/mL and 15.0 pg/mL, respectively. The concentrations of endostatin and VEGF in the culture medium without cells were below the level of detection.To study the expression of collagen XVIII and VEGF messenger (m)RNA in endometrial stromal cells, confluent cells plated on 10-cm culture dishes (Corning) were used for Northern blot analysis. Endometrial stromal cells were further cultured under hypoxic or normoxic conditions for 12 hours. Complementary DNA probes for collagen XVIII and VEGF were prepared, and Northern blotting was performed as previously described (4Kawano Y. Matsui N. Kamihigashi S. Narahara H. Miyakawa I. Effects of interferon-γ on secretion of vascular endothelial growth factor by endometrial stromal cells.Am J Reprod Immunol. 2000; 43: 47-52Crossref PubMed Google Scholar, 12Nasu K. Fujisawa K. Nishida Y. Kai S. Sugano T. Miyakawa I. Tateishi Y. Expression of collagen XVIII mRNA and protein in human umbilical vein and placenta.Reprod Fertil Dev. 2003; 15: 107-114Crossref PubMed Google Scholar). The expression of mRNA for β-actin was also examined as an internal control. The relative levels of collagen XVIII and VEGF mRNA were determined by image analysis of the autoradiograms with a public domain (National Institutes of Health) imaging program (version 1.61; NIH, Bethesda, MD). The results were expressed as the ratio of collagen XVIII and VEGF mRNA signals to the corresponding β-actin mRNA signals.Data are presented as mean ± SD and were analyzed by Student's t-test with commercial software (StatView 4.5; Abacus Concepts, Berkeley, CA). P values of <.05 were considered statistically significant.In 5 of 10 experiments involving endometrial stromal cells from the proliferative phase and 4 of 10 experiments involving endometrial stromal cells from the secretory phase, endostatin was detected in the supernatant of endometrial stromal cells incubated in a normoxic condition. The production of endostatin by endometrial stromal cells isolated from these 9 patients was significantly inhibited in the hypoxic condition (Fig. 1A). The concentrations of endostatin in the supernatant of endometrial stromal cells from the other 11 patients were below the level of detection in both normoxic and hypoxic conditions. However, the secretion of VEGF in the supernatant of endometrial stromal cells cultured in a normoxic condition was detected in all 20 experiments. Hypoxia significantly stimulated the secretion of VEGF (Fig. 1B).Messenger RNAs for collagen XVIII and VEGF were constitutively expressed in endometrial stromal cells cultured in a normoxic condition (Fig. 1C). Hypoxia significantly stimulated the expression of VEGF mRNA but did not affect the expression of collagen XVIII mRNA in all experiments (n = 20) (Fig. 1D).During the perimenstrual period, menstrual bleeding from arterioles occurs after the vasoconstriction of the distal segments of the coiled spiral arteries in the endometrium, resulting in diffuse necrosis, inflammation, and vascular thrombosis. Once the endometrium is shed, angiogenesis must be initiated (1Torry R.J. Rongish B.J. Angiogenesis in the uterus: potential regulation and relation to tumor angiogenesis.Am J Reprod Immunol. 1992; 27: 171-179Crossref PubMed Scopus (75) Google Scholar, 2Goodger-Macpherson A.M. Rodgers P.A. Blood vessel growth in the endometrium.Microcirculation. 1995; 2: 329-343Crossref PubMed Scopus (36) Google Scholar). The endothelial cells are renewed from degenerated coiled arteries in the basalis, migrate, and form microvessel tubes in the endometria during the early proliferative phase (16Au C.L. Rodgers P.A. Immunohistochemical staining of von Willebrand factor in human endometrium during normal menstrual cycle.Hum Reprod. 1993; 8: 17-23Crossref PubMed Google Scholar). Au and Rodgers (16Au C.L. Rodgers P.A. Immunohistochemical staining of von Willebrand factor in human endometrium during normal menstrual cycle.Hum Reprod. 1993; 8: 17-23Crossref PubMed Google Scholar) showed that angiogenic activity was weakest during the menstrual phase, which was followed by a rapid increase in the early proliferative phase, to peak at mid-cycle.Torry and Torry (17Torry D.S. Torry R.J. Angiogenesis and the expression of vascular endothelial growth factor in endometrium and placenta.Am J Reprod Immunol. 1997; 37: 21-29Crossref PubMed Scopus (146) Google Scholar) also observed that endometrial VEGF expression increased from the early proliferative phase to the late secretory phase, presumably owing to increased VEGF production by glandular epithelial cells. Thus, it is suggested that increased endothelial cell proliferation in the mid-proliferative to the late secretory phases associated with the expansion and coiling of the spiral arteries and arterioles coincides with those periods associated with increased endometrial VEGF expression (3Li X.F. Gregory J. Ahmed A. Immunolocalisation of vascular endothelial growth factor in human endometrium.Growth Factors. 1994; 11: 277-282Crossref PubMed Scopus (118) Google Scholar, 18Shifren J.L. Tseng J.F. Zaloudek C.J. Ryan I.P. Meng Y.G. Ferrara N et al.Ovarian steroid regulation of vascular endothelial growth factor in the human endometrium: implications for angiogenesis during the menstrual cycle and in the pathogenesis of endometriosis.J Clin Endocrinol Metab. 1996; 81: 3112-3118Crossref PubMed Scopus (628) Google Scholar).As demonstrated in the present study, endometrial stromal cells are a source of much of the endostatin precursor pool and are able to produce endostatin under normoxic conditions. Because the production of endostatin in normoxic conditions was observed in 9 of 20 experiments, the different cell populations of endometrial stromal cells might be selected during isolation of the cells. Endometrial stromal cells could be divided into two groups: endostatin-producing and endostatin-nonproducing under normoxic conditions. Because the endometrial tissues were obtained from patients with leiomyomas, which might affect the endometrial functions, it is also suggested that the variability of endostatin production by endometrial stromal cells might be influenced by the presence of leiomyomas. It has been demonstrated that endostatin binds to uterine blood vessels (13Chang Z. Choon A. Friedl A. Endostatin binds to blood vessels in situ independent of heparan sulfate and does not compete for fibroblast growth factor-2 binding.Am J Pathol. 1999; 155: 71-76Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) and inhibits angiogenesis (9Dhanabal M. Ramchandran R. Waterman M.J. Lu H. Knebelmann B. Segal M et al.Endostatin induces endothelial cell apoptosis.J Biol Chem. 1999; 274: 11721-11726Crossref PubMed Scopus (586) Google Scholar, 10Dhanabal M. Volk R. Ramchandran R. Simons M. Sukhatme V.P. Cloning, expression, and in vitro activity of human endostatin.Biochem Biophys Res Commun. 1999; 258: 345-352Crossref PubMed Scopus (197) Google Scholar). Taking into account our present findings, it is suggested that endometrial stromal cells can control physiologic angiogenesis in the endometrium by regulating the production of both angiogenic inducers and inhibitors.Popovici et al. (14Popovici R.M. Irwin J.C. Giaccia A.J. Giudice L.C. Hypoxia and cAMP stimulate vascular endothelial growth factor (VEGF) in human endometrial stromal cells: potential relevance to menstruation and endometrial regeneration.J Clin Endocrinol Metab. 1999; 84: 2245-2248Crossref PubMed Google Scholar) and Sharkey et al. (15Sharkey A.M. Day K. McPherson A. Malik S. Licence D. Smith S.K et al.Vascular endothelial growth factor expression in human endometrium is regulated by hypoxia.J Clin Endocrinol Metab. 2000; 85: 402-409Crossref PubMed Scopus (203) Google Scholar) reported that hypoxia stimulated VEGF mRNA expression in endometrial stromal cells, which is consistent with our data. They suggested that the effect of hypoxia on VEGF production by endometrial stromal cells might have physiologic relevance during the process of menstruation and in postmenstrual endometrial repair and angiogenesis. Recently, Wu et al. (19Wu P. Yonekura H. Li H. Nozaki I. Tomono Y. Naito I et al.Hypoxia down-regulates endostatin production by human microvascular endothelial cells and pericytes.Biochem Biophys Res Commun. 2001; 288: 1149-1154Crossref PubMed Scopus (40) Google Scholar) demonstrated that hypoxia downregulates endostatin production by microvascular endothelial cells and pericytes by means of posttranscriptional mechanisms. Our present findings are consistent with their results, suggesting that similar mechanisms might be involved in the inhibition of endostatin release in these cellsIt is considered that, in normal menstrual cycles, the angiogenic status of the endometrium frequently changes and that the production of endostatin and VEGF should be tightly controlled. When the process of angiogenesis begins in the cyclic endometrium, the production of angiogenic stimulators should be enhanced and that of angiogenic inhibitors should be suppressed. Once new blood vessels have been formed, they might no longer need angiogenic stimulators, such as VEGF, and might rapidly turn on the production of angiogenic inhibitors, such as endostatin, to control over-neovascularization. Although the precise mechanisms of the inhibition of endostatin production under a hypoxic condition are still unclear, our present findings suggest that oxygen tension might be one of the regulators of these mechanisms.Further investigations are required to confirm a link between these in vitro experimental data and clinical phenomena in vivo. However, knowledge of the stimulators and inhibitors of endometrial angiogenesis will have immediate impact in the development of strategies for inhibiting or augmenting the growth of blood vessels in normal and pathologic human reproductive processes. In addition, understanding the role of oxygen tension on the regulation of these factors might inform the mechanism of the regulation of physiologic angiogenesis in normal tissues. Human endometrial development during the menstrual cycle shows unique cycle-specific changes in vascularization. Rapid vascular growth is required to support the proliferation and repair of the endometrium during the menstrual cycle and provides a richly vascularized, receptive endometrium for implantation and placentation. These complex processes of angiogenesis are presumably tightly regulated by multiple system controls that can be switched on and off within a short period (1Torry R.J. Rongish B.J. Angiogenesis in the uterus: potential regulation and relation to tumor angiogenesis.Am J Reprod Immunol. 1992; 27: 171-179Crossref PubMed Scopus (75) Google Scholar, 2Goodger-Macpherson A.M. Rodgers P.A. Blood vessel growth in the endometrium.Microcirculation. 1995; 2: 329-343Crossref PubMed Scopus (36) Google Scholar). Several angiogenic stimulators, including vascular endothelial growth factor (VEGF) (3Li X.F. Gregory J. Ahmed A. Immunolocalisation of vascular endothelial growth factor in human endometrium.Growth Factors. 1994; 11: 277-282Crossref PubMed Scopus (118) Google Scholar, 4Kawano Y. Matsui N. Kamihigashi S. Narahara H. Miyakawa I. Effects of interferon-γ on secretion of vascular endothelial growth factor by endometrial stromal cells.Am J Reprod Immunol. 2000; 43: 47-52Crossref PubMed Google Scholar), basic fibroblast growth factor (5Rusnati M. Casarotti G. Pecorelli S. Ragnotti G. Presta M. Basic fibroblast growth factor in ovulatory cycle and postmenopausal human endometrium.Growth Factors. 1990; 3: 299-307Crossref PubMed Scopus (55) Google Scholar), and hepatocyte growth factor (6Nasu K. Sugano T. Matsui N. Narahara H. Kawano Y. Miyakawa I. Expression of hepatocyte growth factor in cultured endometrial stromal cells is induced through a protein kinase C-dependent pathway.Biol Reprod. 1999; 60: 1183-1187Crossref PubMed Scopus (21) Google Scholar), have been demonstrated to be present in the human endometrium. Angiogenic inhibitors, such as thrombospondin-1 (7Iruela-Arispe M.L. Porter P. Bornstein P. Sage E.H. Thrombospondin-1, an inhibitor of angiogenesis is regulated by progesterone in the human endometrium.J Clin Invest. 1996; 97: 403-412Crossref PubMed Scopus (133) Google Scholar), have also been reported to be expressed in the endometrium. Endostatin is a 20-kd fragment of C-terminal globular domain of collagen XVIII that shows a strong ability to inhibit angiogenesis (8O'Reilly M.S. Boehm T. Shing Y. Fukai N. Vasios G. Lane W.S et al.Endostatin: an endogenous inhibitor of angiogenesis and tumor growth.Cell. 1997; 88: 277-285Abstract Full Text Full Text PDF PubMed Scopus (4210) Google Scholar, 9Dhanabal M. Ramchandran R. Waterman M.J. Lu H. Knebelmann B. Segal M et al.Endostatin induces endothelial cell apoptosis.J Biol Chem. 1999; 274: 11721-11726Crossref PubMed Scopus (586) Google Scholar, 10Dhanabal M. Volk R. Ramchandran R. Simons M. Sukhatme V.P. Cloning, expression, and in vitro activity of human endostatin.Biochem Biophys Res Commun. 1999; 258: 345-352Crossref PubMed Scopus (197) Google Scholar). Collagen XVIII is localized in the basement membrane of blood vessels (11Muragaki Y. Timmons S. Griffith C.M. Oh S.P. Fadel B. Quertermous T et al.Mouse Col18α1 is expressed in a tissue-specific manner as three alternative variants and is localized in basement membrane zones.Proc Natl Acad Sci U S A. 1995; 92: 8763-8767Crossref PubMed Scopus (170) Google Scholar, 12Nasu K. Fujisawa K. Nishida Y. Kai S. Sugano T. Miyakawa I. Tateishi Y. Expression of collagen XVIII mRNA and protein in human umbilical vein and placenta.Reprod Fertil Dev. 2003; 15: 107-114Crossref PubMed Google Scholar). Fragments of collagen XVIII longer than endostatin do not inhibit endothelial cell proliferation (8O'Reilly M.S. Boehm T. Shing Y. Fukai N. Vasios G. Lane W.S et al.Endostatin: an endogenous inhibitor of angiogenesis and tumor growth.Cell. 1997; 88: 277-285Abstract Full Text Full Text PDF PubMed Scopus (4210) Google Scholar). Recently, Chang et al. (13Chang Z. Choon A. Friedl A. Endostatin binds to blood vessels in situ independent of heparan sulfate and does not compete for fibroblast growth factor-2 binding.Am J Pathol. 1999; 155: 71-76Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) demonstrated that endostatin bound to uterine blood vessels, suggesting the possible target of this protein during cyclic endometrial neovascularization. Hypoxia in the endometrium is a physiologic event in the perimenstrual period. Vasoconstriction during menstruation leads to endometrial hypoxia, a possible stimulus for angiogenesis. Desquamated endometrium is influenced by hypoxia, which has been shown to be a strong inducer of VEGF expression in endometrial stromal cells (14Popovici R.M. Irwin J.C. Giaccia A.J. Giudice L.C. Hypoxia and cAMP stimulate vascular endothelial growth factor (VEGF) in human endometrial stromal cells: potential relevance to menstruation and endometrial regeneration.J Clin Endocrinol Metab. 1999; 84: 2245-2248Crossref PubMed Google Scholar, 15Sharkey A.M. Day K. McPherson A. Malik S. Licence D. Smith S.K et al.Vascular endothelial growth factor expression in human endometrium is regulated by hypoxia.J Clin Endocrinol Metab. 2000; 85: 402-409Crossref PubMed Scopus (203) Google Scholar). It is suggested that the effect of hypoxia on VEGF production by endometrial stromal cells might have physiologic relevance during the process of menstruation and in postmenstrual endometrial repair and angiogenesis. In the present study, we evaluated the effects of hypoxia on the expression of collagen XVIII/endostatin and VEGF in cultured human endometrial stromal cells. Normal endometrial specimens were obtained from 20 premenopausal patients (aged 37–43 years) who had undergone hysterectomies for intramural leiomyomas. All the patients had regular menstrual cycles, were multiparous, and were considered to be healthy with the exception of uterine leiomyoma. None had received hormonal therapy before the operation. Ten specimens were diagnosed as being from the late proliferative phase and 10 as being from the mid- to late secretory phase on the basis of standard histologic criteria. All the endometrial specimens were verified to be normal on routine histologic examinations. Normal endometrial stromal cells were isolated and cultured in Roswell Park Memorial Institute 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco-BRL, Gaithersburg, MD), streptomycin (100 U/mL) (Gibco-BRL), and penicillin (100 U/mL) (Gibco-BRL) at 37°C in an atmosphere of 5% CO2 in air at 100% humidity, as previously described (6Nasu K. Sugano T. Matsui N. Narahara H. Kawano Y. Miyakawa I. Expression of hepatocyte growth factor in cultured endometrial stromal cells is induced through a protein kinase C-dependent pathway.Biol Reprod. 1999; 60: 1183-1187Crossref PubMed Scopus (21) Google Scholar). After three passages with standard methods of trypsinization, the cells, which 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), were used for the experiments. This study was approved by the institutional review board of Oita University. To study the release of endostatin and VEGF by endometrial stromal cells, confluent cells cultured on six-well culture plates (Corning, New York, NY) were used. The supernatant was replaced with 1.5 mL of fresh culture medium and further cultured for 24 hours under hypoxic (4% O2, 5% CO2, and 91% N2) or normoxic (20% O2, 5% CO2, and 75% N2) conditions. The supernatant was then collected and stored at −70°C until assay. Isolated cells from each individual patient (n = 20) were used per experiment, and each experiment was performed in triplicate. All experiments were performed in the presence of 10% heat-inactivated FBS. The concentration of endostatin and VEGF was determined in each supernatant with commercially available ELISA kits (Cytimmune Sciences, College Park, MD, and R&D systems, Minneapolis, MN, respectively), as described previously (4Kawano Y. Matsui N. Kamihigashi S. Narahara H. Miyakawa I. Effects of interferon-γ on secretion of vascular endothelial growth factor by endometrial stromal cells.Am J Reprod Immunol. 2000; 43: 47-52Crossref PubMed Google Scholar, 12Nasu K. Fujisawa K. Nishida Y. Kai S. Sugano T. Miyakawa I. Tateishi Y. Expression of collagen XVIII mRNA and protein in human umbilical vein and placenta.Reprod Fertil Dev. 2003; 15: 107-114Crossref PubMed Google Scholar). The sensitivities of the assays for endostatin and VEGF were 1.95 ng/mL and 15.0 pg/mL, respectively. The concentrations of endostatin and VEGF in the culture medium without cells were below the level of detection. To study the expression of collagen XVIII and VEGF messenger (m)RNA in endometrial stromal cells, confluent cells plated on 10-cm culture dishes (Corning) were used for Northern blot analysis. Endometrial stromal cells were further cultured under hypoxic or normoxic conditions for 12 hours. Complementary DNA probes for collagen XVIII and VEGF were prepared, and Northern blotting was performed as previously described (4Kawano Y. Matsui N. Kamihigashi S. Narahara H. Miyakawa I. Effects of interferon-γ on secretion of vascular endothelial growth factor by endometrial stromal cells.Am J Reprod Immunol. 2000; 43: 47-52Crossref PubMed Google Scholar, 12Nasu K. Fujisawa K. Nishida Y. Kai S. Sugano T. Miyakawa I. Tateishi Y. Expression of collagen XVIII mRNA and protein in human umbilical vein and placenta.Reprod Fertil Dev. 2003; 15: 107-114Crossref PubMed Google Scholar). The expression of mRNA for β-actin was also examined as an internal control. The relative levels of collagen XVIII and VEGF mRNA were determined by image analysis of the autoradiograms with a public domain (National Institutes of Health) imaging program (version 1.61; NIH, Bethesda, MD). The results were expressed as the ratio of collagen XVIII and VEGF mRNA signals to the corresponding β-actin mRNA signals. Data are presented as mean ± SD and were analyzed by Student's t-test with commercial software (StatView 4.5; Abacus Concepts, Berkeley, CA). P values of <.05 were considered statistically significant. In 5 of 10 experiments involving endometrial stromal cells from the proliferative phase and 4 of 10 experiments involving endometrial stromal cells from the secretory phase, endostatin was detected in the supernatant of endometrial stromal cells incubated in a normoxic condition. The production of endostatin by endometrial stromal cells isolated from these 9 patients was significantly inhibited in the hypoxic condition (Fig. 1A). The concentrations of endostatin in the supernatant of endometrial stromal cells from the other 11 patients were below the level of detection in both normoxic and hypoxic conditions. However, the secretion of VEGF in the supernatant of endometrial stromal cells cultured in a normoxic condition was detected in all 20 experiments. Hypoxia significantly stimulated the secretion of VEGF (Fig. 1B). Messenger RNAs for collagen XVIII and VEGF were constitutively expressed in endometrial stromal cells cultured in a normoxic condition (Fig. 1C). Hypoxia significantly stimulated the expression of VEGF mRNA but did not affect the expression of collagen XVIII mRNA in all experiments (n = 20) (Fig. 1D). During the perimenstrual period, menstrual bleeding from arterioles occurs after the vasoconstriction of the distal segments of the coiled spiral arteries in the endometrium, resulting in diffuse necrosis, inflammation, and vascular thrombosis. Once the endometrium is shed, angiogenesis must be initiated (1Torry R.J. Rongish B.J. Angiogenesis in the uterus: potential regulation and relation to tumor angiogenesis.Am J Reprod Immunol. 1992; 27: 171-179Crossref PubMed Scopus (75) Google Scholar, 2Goodger-Macpherson A.M. Rodgers P.A. Blood vessel growth in the endometrium.Microcirculation. 1995; 2: 329-343Crossref PubMed Scopus (36) Google Scholar). The endothelial cells are renewed from degenerated coiled arteries in the basalis, migrate, and form microvessel tubes in the endometria during the early proliferative phase (16Au C.L. Rodgers P.A. Immunohistochemical staining of von Willebrand factor in human endometrium during normal menstrual cycle.Hum Reprod. 1993; 8: 17-23Crossref PubMed Google Scholar). Au and Rodgers (16Au C.L. Rodgers P.A. Immunohistochemical staining of von Willebrand factor in human endometrium during normal menstrual cycle.Hum Reprod. 1993; 8: 17-23Crossref PubMed Google Scholar) showed that angiogenic activity was weakest during the menstrual phase, which was followed by a rapid increase in the early proliferative phase, to peak at mid-cycle. Torry and Torry (17Torry D.S. Torry R.J. Angiogenesis and the expression of vascular endothelial growth factor in endometrium and placenta.Am J Reprod Immunol. 1997; 37: 21-29Crossref PubMed Scopus (146) Google Scholar) also observed that endometrial VEGF expression increased from the early proliferative phase to the late secretory phase, presumably owing to increased VEGF production by glandular epithelial cells. Thus, it is suggested that increased endothelial cell proliferation in the mid-proliferative to the late secretory phases associated with the expansion and coiling of the spiral arteries and arterioles coincides with those periods associated with increased endometrial VEGF expression (3Li X.F. Gregory J. Ahmed A. Immunolocalisation of vascular endothelial growth factor in human endometrium.Growth Factors. 1994; 11: 277-282Crossref PubMed Scopus (118) Google Scholar, 18Shifren J.L. Tseng J.F. Zaloudek C.J. Ryan I.P. Meng Y.G. Ferrara N et al.Ovarian steroid regulation of vascular endothelial growth factor in the human endometrium: implications for angiogenesis during the menstrual cycle and in the pathogenesis of endometriosis.J Clin Endocrinol Metab. 1996; 81: 3112-3118Crossref PubMed Scopus (628) Google Scholar). As demonstrated in the present study, endometrial stromal cells are a source of much of the endostatin precursor pool and are able to produce endostatin under normoxic conditions. Because the production of endostatin in normoxic conditions was observed in 9 of 20 experiments, the different cell populations of endometrial stromal cells might be selected during isolation of the cells. Endometrial stromal cells could be divided into two groups: endostatin-producing and endostatin-nonproducing under normoxic conditions. Because the endometrial tissues were obtained from patients with leiomyomas, which might affect the endometrial functions, it is also suggested that the variability of endostatin production by endometrial stromal cells might be influenced by the presence of leiomyomas. It has been demonstrated that endostatin binds to uterine blood vessels (13Chang Z. Choon A. Friedl A. Endostatin binds to blood vessels in situ independent of heparan sulfate and does not compete for fibroblast growth factor-2 binding.Am J Pathol. 1999; 155: 71-76Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) and inhibits angiogenesis (9Dhanabal M. Ramchandran R. Waterman M.J. Lu H. Knebelmann B. Segal M et al.Endostatin induces endothelial cell apoptosis.J Biol Chem. 1999; 274: 11721-11726Crossref PubMed Scopus (586) Google Scholar, 10Dhanabal M. Volk R. Ramchandran R. Simons M. Sukhatme V.P. Cloning, expression, and in vitro activity of human endostatin.Biochem Biophys Res Commun. 1999; 258: 345-352Crossref PubMed Scopus (197) Google Scholar). Taking into account our present findings, it is suggested that endometrial stromal cells can control physiologic angiogenesis in the endometrium by regulating the production of both angiogenic inducers and inhibitors. Popovici et al. (14Popovici R.M. Irwin J.C. Giaccia A.J. Giudice L.C. Hypoxia and cAMP stimulate vascular endothelial growth factor (VEGF) in human endometrial stromal cells: potential relevance to menstruation and endometrial regeneration.J Clin Endocrinol Metab. 1999; 84: 2245-2248Crossref PubMed Google Scholar) and Sharkey et al. (15Sharkey A.M. Day K. McPherson A. Malik S. Licence D. Smith S.K et al.Vascular endothelial growth factor expression in human endometrium is regulated by hypoxia.J Clin Endocrinol Metab. 2000; 85: 402-409Crossref PubMed Scopus (203) Google Scholar) reported that hypoxia stimulated VEGF mRNA expression in endometrial stromal cells, which is consistent with our data. They suggested that the effect of hypoxia on VEGF production by endometrial stromal cells might have physiologic relevance during the process of menstruation and in postmenstrual endometrial repair and angiogenesis. Recently, Wu et al. (19Wu P. Yonekura H. Li H. Nozaki I. Tomono Y. Naito I et al.Hypoxia down-regulates endostatin production by human microvascular endothelial cells and pericytes.Biochem Biophys Res Commun. 2001; 288: 1149-1154Crossref PubMed Scopus (40) Google Scholar) demonstrated that hypoxia downregulates endostatin production by microvascular endothelial cells and pericytes by means of posttranscriptional mechanisms. Our present findings are consistent with their results, suggesting that similar mechanisms might be involved in the inhibition of endostatin release in these cells It is considered that, in normal menstrual cycles, the angiogenic status of the endometrium frequently changes and that the production of endostatin and VEGF should be tightly controlled. When the process of angiogenesis begins in the cyclic endometrium, the production of angiogenic stimulators should be enhanced and that of angiogenic inhibitors should be suppressed. Once new blood vessels have been formed, they might no longer need angiogenic stimulators, such as VEGF, and might rapidly turn on the production of angiogenic inhibitors, such as endostatin, to control over-neovascularization. Although the precise mechanisms of the inhibition of endostatin production under a hypoxic condition are still unclear, our present findings suggest that oxygen tension might be one of the regulators of these mechanisms. Further investigations are required to confirm a link between these in vitro experimental data and clinical phenomena in vivo. However, knowledge of the stimulators and inhibitors of endometrial angiogenesis will have immediate impact in the development of strategies for inhibiting or augmenting the growth of blood vessels in normal and pathologic human reproductive processes. In addition, understanding the role of oxygen tension on the regulation of these factors might inform the mechanism of the regulation of physiologic angiogenesis in normal tissues. The authors thank Dr. Hidekatsu Yoshioka, Department of Biochemistry, Faculty of Medicine, Oita University, Japan for the review of the work." @default.
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