Estrogen Stimulates Homing of Endothelial Progenitor Cells to Endometriotic Lesions
2016; Elsevier BV; Volume: 186; Issue: 8 Linguagem: Inglês
10.1016/j.ajpath.2016.04.004
ISSN1525-2191
AutoresJeannette Rudzitis‐Auth, Anca Nenicu, Ruth M. Nickels, Michael D. Menger, Matthias W. Laschke,
Tópico(s)Endometrial and Cervical Cancer Treatments
ResumoThe incorporation of endothelial progenitor cells (EPCs) into microvessels contributes to the vascularization of endometriotic lesions. Herein, we analyzed whether this vasculogenic process is regulated by estrogen. Estrogen- and vehicle-treated human EPCs were analyzed for migration and tube formation. Endometriotic lesions were induced in irradiated FVB/N mice, which were reconstituted with bone marrow from FVB/N-TgN (Tie2/green fluorescent protein) 287 Sato mice. The animals were treated with 100 μg/kg β-estradiol 17-valerate or vehicle (control) over 7 and 28 days. Lesion growth, cyst formation, homing of green fluorescent protein+/Tie2+ EPCs, vascularization, cell proliferation, and apoptosis were analyzed by high-resolution ultrasonography, caliper measurements, histology, and immunohistochemistry. Numbers of blood circulating EPCs were assessed by flow cytometry. In vitro, estrogen-treated EPCs exhibited a higher migratory and tube-forming capacity when compared with controls. In vivo, numbers of circulating EPCs were not affected by estrogen. However, estrogen significantly increased the number of EPCs incorporated into the lesions' microvasculature, resulting in an improved early vascularization. Estrogen further stimulated the growth of lesions, which exhibited massively dilated glands with a flattened layer of stroma. This was mainly because of an increased glandular secretory activity, whereas cell proliferation and apoptosis were not markedly affected. These findings indicate that vasculogenesis in endometriotic lesions is dependent on estrogen, which adds a novel hormonally regulated mechanism to the complex pathophysiology of endometriosis. The incorporation of endothelial progenitor cells (EPCs) into microvessels contributes to the vascularization of endometriotic lesions. Herein, we analyzed whether this vasculogenic process is regulated by estrogen. Estrogen- and vehicle-treated human EPCs were analyzed for migration and tube formation. Endometriotic lesions were induced in irradiated FVB/N mice, which were reconstituted with bone marrow from FVB/N-TgN (Tie2/green fluorescent protein) 287 Sato mice. The animals were treated with 100 μg/kg β-estradiol 17-valerate or vehicle (control) over 7 and 28 days. Lesion growth, cyst formation, homing of green fluorescent protein+/Tie2+ EPCs, vascularization, cell proliferation, and apoptosis were analyzed by high-resolution ultrasonography, caliper measurements, histology, and immunohistochemistry. Numbers of blood circulating EPCs were assessed by flow cytometry. In vitro, estrogen-treated EPCs exhibited a higher migratory and tube-forming capacity when compared with controls. In vivo, numbers of circulating EPCs were not affected by estrogen. However, estrogen significantly increased the number of EPCs incorporated into the lesions' microvasculature, resulting in an improved early vascularization. Estrogen further stimulated the growth of lesions, which exhibited massively dilated glands with a flattened layer of stroma. This was mainly because of an increased glandular secretory activity, whereas cell proliferation and apoptosis were not markedly affected. These findings indicate that vasculogenesis in endometriotic lesions is dependent on estrogen, which adds a novel hormonally regulated mechanism to the complex pathophysiology of endometriosis. Endometriosis (ie, the presence of endometrial-like tissue outside the uterine cavity) is a frequent gynecological disease of women in the reproductive age, which is particularly characterized by a broad spectrum of pain symptoms.1Giudice L.C. Clinical practice: endometriosis.N Engl J Med. 2010; 362: 2389-2398Crossref PubMed Scopus (1311) Google Scholar, 2Laux-Biehlmann A. d'Hooghe T. Zollner T.M. Menstruation pulls the trigger for inflammation and pain in endometriosis.Trends Pharmacol Sci. 2015; 36: 270-276Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar Because estrogen stimulates the growth of endometriotic lesions,3Zhao Y. Gong P. Chen Y. Nwachukwu J.C. Srinivasan S. Ko C. Bagchi M.K. Taylor R.N. Korach K.S. Nettles K.W. Katzenellenbogen J.A. Katzenellenbogen B.S. Dual suppression of estrogenic and inflammatory activities for targeting of endometriosis.Sci Transl Med. 2015; 7: 271ra9Crossref PubMed Scopus (107) Google Scholar current pharmacological treatment primarily focuses on the temporary induction of a hypoestrogenic state.4Hickey M. Ballard K. Farquhar C. Endometriosis.BMJ. 2014; 348: g1752Crossref PubMed Scopus (137) Google Scholar, 5Vercellini P. Viganò P. Somigliana E. Fedele L. Endometriosis: pathogenesis and treatment.Nat Rev Endocrinol. 2014; 10: 261-275Crossref PubMed Scopus (981) Google Scholar The development of new blood vessels is a major pathogenic factor, which drives the onset and progression of endometriosis. In fact, the survival, spread, and engraftment of ectopic endometrial-like tissue inside the peritoneal cavity are crucially dependent on an adequate vascularization.6Laschke M.W. Menger M.D. In vitro and in vivo approaches to study angiogenesis in the pathophysiology and therapy of endometriosis.Hum Reprod Update. 2007; 13: 331-342Crossref PubMed Scopus (146) Google Scholar, 7Rocha A.L. Reis F.M. Taylor R.N. Angiogenesis and endometriosis.Obstet Gynecol Int. 2013; 2013: 859619Crossref PubMed Google Scholar Accordingly, a continuously growing number of studies suggest the inclusion of anti-angiogenic agents in future treatment concepts.8Laschke M.W. Menger M.D. Anti-angiogenic treatment strategies for the therapy of endometriosis.Hum Reprod Update. 2012; 18: 682-702Crossref PubMed Scopus (124) Google Scholar, 9Edwards A.K. Nakamura D.S. Virani S. Wessels J.M. Tayade C. Animal models for anti-angiogenic therapy in endometriosis.J Reprod Immunol. 2013; 97: 85-94Crossref PubMed Scopus (30) Google Scholar, 10Rudzitis-Auth J. Menger M.D. Laschke M.W. Resveratrol is a potent inhibitor of vascularization and cell proliferation in experimental endometriosis.Hum Reprod. 2013; 28: 1339-1347Crossref PubMed Scopus (60) Google Scholar There are two processes that contribute to the vascularization of endometriotic lesions (ie, angiogenesis and vasculogenesis).11Taylor R.N. Yu J. Torres P.B. Schickedanz A.C. Park J.K. Mueller M.D. Sidell N. Mechanistic and therapeutic implications of angiogenesis in endometriosis.Reprod Sci. 2009; 16: 140-146Crossref PubMed Scopus (161) Google Scholar, 12Laschke M.W. Giebels C. Menger M.D. Vasculogenesis: a new piece of the endometriosis puzzle.Hum Reprod Update. 2011; 17: 628-636Crossref PubMed Scopus (96) Google Scholar Angiogenesis is defined as the stepwise ingrowth of newly developing blood vessels into the lesions from the surrounding host tissue. This process is initiated by the production and release of various angiogenic growth factors from the hypoxic endometrial tissue and results in the development of dense microvascular networks, which finally undergo vascular maturation and remodeling to adapt to the environmental conditions.13Groothuis P.G. Nap A.W. Winterhager E. Grümmer R. Vascular development in endometriosis.Angiogenesis. 2005; 8: 147-156Crossref PubMed Scopus (160) Google Scholar Besides, we and others have recently demonstrated that the vascularization of endometriotic lesions also involves vasculogenesis [ie, the de novo formation of blood vessels from circulating bone marrow–derived endothelial progenitor cells (EPCs), which home to the lesions and are incorporated into the microvascular endothelium].14Becker C.M. Beaudry P. Funakoshi T. Benny O. Zaslavsky A. Zurakowski D. Folkman J. D'Amato R.J. Ryeom S. Circulating endothelial progenitor cells are up-regulated in a mouse model of endometriosis.Am J Pathol. 2011; 178: 1782-1791Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 15Laschke M.W. Giebels C. Nickels R.M. Scheuer C. Menger M.D. Endothelial progenitor cells contribute to the vascularization of endometriotic lesions.Am J Pathol. 2011; 178: 442-450Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar This observation not only adds a novel biological process to the complex pathophysiology of endometriosis, but also offers the possibility to develop novel EPC-based approaches for the future diagnosis and therapy of the disease. A major prerequisite to achieve this goal is the detailed knowledge of the mechanisms that regulate vasculogenesis in endometriosis. These mechanisms, however, remain elusive so far. Accordingly, our aim was to analyze, for the first time, the effect of estrogen on the mobilization and homing of EPCs to endometriotic lesions. We surgically induced endometriotic lesions in a murine green fluorescent protein (GFP)+/GFP− crossover design model, which allowed for the detection of GFP+/Tie2+ EPCs in the microvasculature of GFP− endometriotic lesions in estrogen-stimulated and non-stimulated animals. All experiments were performed according to the German legislation on protection of animals and the NIH Guide for the Care and Use of Laboratory Animals16Committee for the Update of the Guide for the Care and Use of Laboratory Animals; National Research CouncilGuide for the Care and Use of Laboratory Animals.ed 8. National Academies Press, Washington, DC2011Crossref Google Scholar and were approved by the local governmental animal care committee (permission number: 25/2012). For the experiments, 12- to 16-week-old female FVB/N mice and transgenic FVB/N-TgN (Tie2/GFP) 287 Sato mice (Jackson Laboratories, Bar Harbor, ME) with a body weight of 20 to 25 g were used. They were maintained under controlled environmental conditions and had free access to tap water and standard pellet food (Altromin, Lage, Germany). For the generation of GFP+/Tie2+ chimeras, bone marrow from 14 FVB/N-TgN (Tie2/GFP) 287 Sato mice was transplanted into 28 lethally irradiated wild-type FVB/N mice. The recipient mice were placed in a steel container and exposed to a single dose of 8.5 Gy 4 hours before bone marrow transplantation. The donor mice were sacrificed, and the femurs and tibias were flushed with cold phosphate-buffered saline (PBS). Subsequently, the recipient mice received 2 × 107 bone marrow cells resuspended in 300 μL of cold PBS via tail vein injection. Finally, the animals were allowed to recover for 4 weeks to enable the complete reconstitution of the bone marrow before the surgical induction of endometriotic lesions (Figure 1). Endometriotic lesions were surgically induced by suturing uterine tissue samples from 14 wild-type FVB/N donor mice to the abdominal wall of the GFP+/Tie2+ chimeras (Figure 2). Estrus cycling of the donor mice was assessed by cytological analysis of vaginal lavage samples to exclude differences between individual tissue samples because of different sex hormone levels of the animals, which was achieved by pipetting 15 μL of 0.9% saline into the vagina and subsequent transfer of the cell suspension onto a glass slide for examination under a phase contrast microscope (CH-2; Olympus, Hamburg, Germany). Only the animals in the stage of estrus were used as donors.17Rudzitis-Auth J. Körbel C. Scheuer C. Menger M.D. Laschke M.W. Xanthohumol inhibits growth and vascularization of developing endometriotic lesions.Hum Reprod. 2012; 27: 1735-1744Crossref PubMed Scopus (40) Google Scholar For the isolation of the two uterine horns, the donor mice were anesthetized by an i.p. injection of 75 mg/kg body weight ketamine (Pharmacia GmbH, Erlangen, Germany) and 15 mg/kg body weight xylazin (Bayer, Leverkusen, Germany). After midline laparotomy, the uterine horns were carefully excised and transferred to a Petri dish containing Dulbecco's modified Eagle's medium (10% fetal calf serum, 100 U/mL penicillin, 0.1 mg/mL streptomycin; PAA, Cölbe, Germany). The horns were opened longitudinally, and 2-mm tissue samples were removed using a dermal biopsy punch (Stiefel Laboratorium GmbH, Offenbach am Main, Germany). Anesthetized recipient animals were laparotomized, and tissue samples were fixed with the endometrium adjacent to the peritoneum by means of a 6-0 Prolene suture (Ethicon Products, Norderstedt, Germany) to the right and the left side of the abdominal wall.18Körbel C. Menger M.D. Laschke M.W. Size and spatial orientation of uterine tissue transplants on the peritoneum crucially determine the growth and cyst formation of endometriosis-like lesions in mice.Hum Reprod. 2010; 25: 2551-2558Crossref PubMed Scopus (27) Google Scholar The laparotomy was then closed with running 6-0 Prolene muscle and skin sutures. To analyze the effect of estrogen on the homing of EPCs to the endometriotic lesions, the recipient mice were either treated with 100 μg/kg β-estradiol 17-valerate (dissolved in 100 μL corn oil; Sigma-Aldrich, Taufkirchen, Germany) or vehicle (control) by s.c. injection once a week over a period of 7 and 28 days. The fixed uterine tissue samples were repetitively analyzed by means of high-resolution ultrasound imaging. For this purpose, the mice were anesthetized with 2% isoflurane in oxygen and fixed in supine position on a heated stage, which was equipped with ECG electrodes and heart rate display (THM100; Indus Instruments, Houston, TX).18Körbel C. Menger M.D. Laschke M.W. Size and spatial orientation of uterine tissue transplants on the peritoneum crucially determine the growth and cyst formation of endometriosis-like lesions in mice.Hum Reprod. 2010; 25: 2551-2558Crossref PubMed Scopus (27) Google Scholar After chemical depilation of the abdomen (Nair hair removal lotion; Church & Dwight Canada Corp., Mississauga, ON, Canada), ultrasound coupling gel (Aquasonic 100; Parker, NJ) was applied to the skin. Ultrasound imaging of the tissue samples was performed by means of Vevo 770 version 2.3.0 (VisualSonics, Toronto, ON, Canada) high-resolution in vivo microimaging system and a real-time microvisualization 704 Scanhead (VisualSonics) with a center frequency of 40 MHz and a focal depth of 6 mm.19Laschke M.W. Körbel C. Rudzitis-Auth J. Gashaw I. Reinhardt M. Hauff P. Zollner T.M. Menger M.D. High-resolution ultrasound imaging: a novel technique for the noninvasive in vivo analysis of endometriotic lesion and cyst formation in small animal models.Am J Pathol. 2010; 176: 585-593Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 20Nenicu A. Körbel C. Gu Y. Menger M.D. Laschke M.W. Combined blockade of angiotensin II type 1 receptor and activation of peroxisome proliferator-activated receptor-γ by telmisartan effectively inhibits vascularization and growth of murine endometriosis-like lesions.Hum Reprod. 2014; 29: 1011-1024Crossref PubMed Scopus (40) Google Scholar The ultrasound images were analyzed with a three-dimensional reconstruction and analysis software licensed to VisualSonics for distribution with the Vevo 770 high-resolution imaging system. The analyses included the determination of the overall volume of endometriotic lesions as well as of their stromal tissue and cysts (in mm3) by manual image segmentation. Boundaries of endometriotic lesions and their cysts were manually outlined in parallel slices, which were separated by a step size of 200 μm in the three-dimensional ultrasound images. On the basis of the outlined areas, volumes were subsequently computed by the VisualSonics software. Moreover, we calculated the growth of lesions and stromal tissue (in % of the initial lesion and stromal tissue size) and assessed the cyst/lesion ratio (in %). After the last ultrasound analysis at day 28, the animals were anesthetized by i.p. injection of ketamine and xylazine and carefully laparotomized under a stereomicroscope to measure the largest (D1) and perpendicularly aligned diameter (D2) of the endometriotic lesions by means of a digital caliper. The lesion size (S) was then calculated by the following: S = D1 × D2 × π/4.21Becker C.M. Rohwer N. Funakoshi T. Cramer T. Bernhardt W. Birsner A. Folkman J. D'Amato R.J. 2-Methoxyestradiol inhibits hypoxia-inducible factor-1{alpha} and suppresses growth of lesions in a mouse model of endometriosis.Am J Pathol. 2008; 172: 534-544Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar Formalin-fixed specimens of tissue harvested at day 7 and 28 were embedded in paraffin. Sections (3 μm thick) were cut and stained with hematoxylin and eosin according to standard procedures. The sections were examined under a BX60 microscope (Olympus, Hamburg, Germany) to determine the rate of uterine tissue samples (%), which had developed into endometriotic lesions consisting of both endometrial glands and stroma. Only typical lesions were included in the quantitative analyses, whereas simple granuloma, which had developed around the suture material without containing any endometrial glands, was excluded (Figure 3). GFP+ EPCs incorporated into the GFP− microvascular endothelium of endometriotic lesions were detected immunohistochemically using a goat polyclonal anti-GFP antibody (1:50; Rockland Immunochemicals Inc., Limerick, PA) as primary antibody. Subsequently, the tissue sections were incubated with the corresponding secondary biotinylated antibody, followed by avidin-peroxidase (1:50; Sigma-Aldrich). 3-Amino-9-ethylcarbazole (AEC Substrate System; Abcam, Cambridge, UK) was used as a chromogen, and counterstaining was performed with hemalaun. Sections of a uterine horn from a FVB/N-TgN (Tie2/GFP) 287 Sato mouse served as positive staining control. Sections solely incubated with the secondary antibody were used as negative control. The fraction of GFP+ EPCs (in % of all endothelial cells) in endometriotic lesions was assessed by light microscopy (BX60; Olympus). For the immunofluorescence microscopic detection of microvessels within endometriotic lesions, sections were stained with a monoclonal rat anti-mouse antibody against the endothelial cell marker CD31 (1:300; Dianova, Hamburg, Germany). A goat anti-rat IgG cyanine 3 (Cy3) antibody (Dianova) served as a secondary antibody. Cell nuclei were stained with Hoechst 33342 (1:500; Sigma-Aldrich). The microvessel density (mm−2) was measured using a BZ-8000 microscope (Keyence, Osaka, Japan). For the immunohistochemical detection of proliferating and apoptotic cells in the stroma of endometriotic lesions, sections were stained with a rabbit polyclonal antibody against the proliferation marker Ki-67 (1:100; Abcam) and a rabbit polyclonal antibody against the apoptosis marker cleaved caspase-3 (1:100; Cell Signaling, Danvers, MA). A goat anti-rabbit biotinylated antibody (ready-to-use; Abcam) served as secondary antibody, followed by avidin-peroxidase (1:50; Sigma-Aldrich). 3-Amino-9-ethylcarbazole (AEC Substrate System; Abcam) was used as chromogen, and counterstaining was performed with hemalaun. The fraction of proliferating and apoptotic stromal cells (%) was assessed by light microscopy (BX60; Olympus). To assess the expression levels of important estrogen receptor–regulated target genes in endometriotic lesions, we performed a quantitative real-time PCR array (PAMM-005ZA; Qiagen, Hilden, Germany). For this purpose, total RNA was extracted from six pooled frozen vehicle-treated and estrogen-treated endometriotic lesions (one lesion per mouse) at day 7 using the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol and followed by DNase treatment using DNA-free kit (Qiagen). The quantity and integrity of the isolated RNA were assessed with a NanoDrop ND-1000 UV-Vis spectrophotometer (NanoDrop Technologies, Rockland, MA). A total of 260 ng RNA per group was used for first-strand cDNA synthesis reactions using the RT2 First Strand Kit (Qiagen), according to the supplier's instructions. cDNA was subsequently combined with the RT2 SYBR Green ROX qPCR Mastermix (Qiagen) and applied to pathway-specific RT2 Profiler PCR Arrays (96-well format) for mouse estrogen receptor signaling (PAMM-005ZA; Qiagen). An Applied Biosystems StepOnePlus model 7000 device was used with the following cycle conditions: one cycle of 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute. All expression levels were normalized to the housekeeping genes Actb, B2m, Gapdh, Gusb, and Hsp90ab1, which did not vary significantly between the study groups. The cycle threshold (Ct) values were recorded for each of the 96 wells, and expression levels were calculated using a standard equation: [2(−Ct)]. The expression of individual genes was analyzed by calculating the fold-change in expression levels in the estrogen-stimulated group compared with the vehicle-treated control group. This procedure was confirmed by another set of pooled RNA from the same animals and a second set of microarrays to assess the variability associated with the preparation of targets and processing of microarrays. To analyze whether the treatment with β-estradiol 17-valerate increases the number of EPCs in the peripheral blood, we additionally performed flow cytometric analyses of blood samples from the mice at day 7 and 28. After lysis of red blood cells and Fc blockade (CD16/CD32; BD Pharmingen, Heidelberg, Germany), the lymphocyte population was analyzed for the expression of the EPC markers stem cell antigen-1-fluorescein isothiocyanate (BD Pharmingen) and vascular endothelial growth factor receptor (VEGFR)-2-phycoerythrin (BD Pharmingen). Isotype-identical antibodies served as controls (rat IgG2aκ-fluorescein isothiocyanate/-phycoerythrin; BD Pharmingen). Two-color flow cytometric analyses were performed by means of a FACScan (BD Pharmingen). Data were evaluated by the software package CellQuestPro version 3.2 (BD Pharmingen). Endometriotic lesions were analyzed at an early and late phase of development (ie, day 7 and 28 after surgical induction). In a first set of experiments, a total of 48 uterine tissue samples were transplanted in 12 GFP+/Tie2+ bone marrow–reconstituted animals. The mice were randomly divided into two groups (n = 6 each) receiving either 100 μg/kg β-estradiol 17-valerate or vehicle (control). After 7 days, tissue and blood samples were taken and further processed for histology, immunohistochemistry, gene expression analyses, and flow cytometry. In a second set of experiments, a total of 32 uterine tissue samples were transplanted in 16 GFP+/Tie2+ bone marrow–reconstituted animals. The mice were randomly divided into two groups (n = 8 each) receiving either 100 μg/kg β-estradiol 17-valerate or vehicle (control) by s.c. injection once a week over a period of 28 days. To measure the volume of cysts and stromal tissue, ultrasound image analyses of developing endometriotic lesions were performed directly after tissue transplantation (d0) as well as at days 7, 14, 21, and 28. At the end of the experiments, the size of the endometriotic lesions was additionally assessed by means of a digital caliper. Subsequently, tissue and blood samples were taken and further processed for histology, immunohistochemistry, and flow cytometry. In additional 22 GFP+/Tie2+ bone marrow–reconstituted mice, treated with estrogen (n = 11) or vehicle (n = 11) as described above, blood estrogen levels were measured at day 7 and 28. Blood samples were collected from the vena cava of the animals. Serum was prepared and stored at −20°C until the assessment of estrogen levels by means of radioimmunoassay.22Klein R. Schams D. Failing K. Hoffmann B. Investigations on the re-establishment of the positive feedback of oestradiol during anoestrus in the bitch.Reprod Domest Anim. 2003; 38: 13-20Crossref PubMed Scopus (24) Google Scholar In additional experiments, we analyzed in vitro the effect of estrogen on the migratory and tube-forming activity of isolated human EPCs. Heparinized venous blood was drawn from six healthy female volunteers after obtaining their written informed consent and with the approval of the local ethics review board. Mononuclear cells were isolated from the blood samples by density gradient centrifugation (Biocoll; Biochrom GmbH, Berlin, Germany), washed three times with PBS, and seeded onto 6-well plates in endothelial growth medium (EGM)-2 (Lonza, Köln, Germany). Subsequently, the cells were cultured at 37°C and 5% CO2 with daily medium change in the first week. Thereafter, medium was changed every 3 days. After 2 weeks, small cell colonies of late EPCs with cobblestone-like morphology appeared, as previously described.23Fuchs S. Hermanns M.I. Kirkpatrick C.J. Retention of a differentiated endothelial phenotype by outgrowth endothelial cells isolated from human peripheral blood and expanded in long-term cultures.Cell Tissue Res. 2006; 326: 79-92Crossref PubMed Scopus (87) Google Scholar These small colonies were further cultured for 1 week, before they were trypsinized and expanded over several passages. The EPC phenotype of the isolated cells was confirmed by detection of CD45−, CD34+, and VEGFR-2+ using flow cytometry. In addition, the cells were characterized by DiI-acetylated low-density lipoprotein and Ulex Europaeus Agglutinin-1 uptake. The adherent cells in culture were incubated with 2 μg/mL DiI-acetylated low-density lipoprotein (MoBiTec GmbH, Göttingen, Germany) for 2 hours at 37°C and then washed with PBS. After fixation with 2% formalin, the cells were incubated with 10 μg/mL UEA for 2 hours at 37°C. In addition, cell nuclei were stained with 2 μg/mL Hoechst 33342 (Sigma-Aldrich). Finally, the cells were examined by fluorescence microscopy (BZ-8000; Keyence). The migratory activity of human EPCs was assessed by using 24-well chemotaxis chambers and polyvinylpyrrolidone-coated polycarbonate filters with a pore size of 8 μm (BD Biosciences). The upper chambers were loaded with 15 × 104 EPCs in 200 μL growth factor–free endothelial cell growth medium (EBM-2 Basal Medium; Lonza). The lower chambers were loaded with 750 μL EBM-2 supplemented with growth factors (EGM-2 SingleQuots Kit; Lonza) and with vehicle (PBS) or 0.1 to 10 nmol/L β-estradiol (Sigma-Aldrich). After incubation for 18 hours, the membrane was washed with PBS and the upper side of the membrane was wiped gently with a cotton tip. The membrane was then fixed with methanol and stained with Dade Diff-Quick (Dade Diagnostika GmbH, München, Germany). The number of migrated cells was counted in 20 microscopic regions of interest at ×20 magnification (BZ-8000; Keyence) and is given as cells/mm2. All migration assays were performed in triplicate. To study the effect of estrogen on tube formation of human EPCs, 150 μL growth factor–reduced Matrigel (R&D Systems, Wiesbaden, Germany) per well was plated into a 48-well plate. The plate was then incubated at 37°C for 30 minutes to allow the Matrigel to polymerize. Subsequently, 2 × 104 EPCs in 300 μL EGM-2 supplemented with growth factors (EGM-2 SingleQuots Kit; Lonza) and vehicle (PBS) or different concentrations (0.1 to 10 nmol/L) of β-estradiol (Sigma-Aldrich) were cultured on the Matrigel at 37°C in 5% CO2 humidified atmosphere. After 36 hours, tube formation of EPCs was examined under a phase contrast microscope (BZ-8000; Keyence) and quantified by measuring the total tube length (mm) in an observation area of 1 mm2 using ImageJ software version 1.47 (NIH, Bethesda, MD). All tube formation assays were performed in triplicate. Data were first analyzed for normal distribution and equal variance. In case of parametric data, differences between the two experimental groups were assessed by the unpaired Student's t-test. In case of nonparametric data, differences between the two experimental groups were assessed by the Mann-Whitney rank sum test. To test for time effects within each experimental group, analysis of variance for repeated measurements was applied. This was followed by the Dunnett post hoc test (SigmaStat; Jandel Corp., San Rafael, CA). All data are given as means ± SEM. Statistical significance was accepted for P < 0.05. To assess the efficiency of estrogen treatment, we measured in a first set of experiments the estrogen levels at day 7 and 28 in irradiated mice, which were bone marrow reconstituted and either treated with 100 μg/kg β-estradiol 17-valerate or vehicle. We found that the vehicle-treated animals exhibited low serum estrogen levels of 7.3 ± 0.7 pg/mL at day 7 and 6.9 ± 0.6 pg/mL at day 28. Estrogen treatment significantly increased the estrogen serum levels to 14.3 ± 0.7 pg/mL at day 7 and 19.3 ± 2.7 pg/mL at day 28. This significant difference in hormone level indicates that the present experimental setting was suitable to analyze the effect of estrogen on the homing of circulating EPCs into newly developing endometriotic lesions. For the induction of endometriotic lesions, we fixed uterine tissue samples to the abdominal wall of recipient mice. The histological analysis of hematoxylin and eosin–stained sections of these tissue samples at day 28 revealed that they exhibited different histomorphological phenotypes. In both groups, we detected typical endometriotic lesions consisting of endometrial glands, which were surrounded by a vascularized stroma (Figure 3, C–F). Of interest, endometriotic lesions in estrogen-stimulated animals exhibited cyst-like dilated endometrial glands with a flattened layer of endometrial stroma and a markedly increased cyst volume (Figure 3, E and F) when compared with those of vehicle-treated control animals (Figure 3, C and D). In addition, we found that several uterine tissue samples had regressed during the 28-day implantation period. In this case, we solely detected granuloma around the suture material. These granulomas did not contain any endometrial glands and were characterized by the accumulation of inflammatory cells around the suture material (Figure 3, A and B). In comparison to vehicle-treated mice, we detected a lower number of granulomas in estrogen-stimulated animals. Accordingly, 94% ± 6% of uterine tissue samples developed into endometriotic lesions in estrogen-stimulated mice versus 63% ± 16% in
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