| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Department of Obstetrics and Gynecology, Yale University School of Medicine (U.A.K, J.L., O.G.-K., Y.S., A.A.), New Haven, Connecticut 06520-8063; and Departments of Histology and Embryology (U.A.K., Y.S., R.D.) and Medical Biology and Genetics (O.G.-K.), Akdeniz University School of Medicine, Antalya 07070, Turkey
Address all correspondence and requests for reprints to: Dr. Aydin Arici, Section of Reproductive Endocrinology, Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Connecticut 06520-8063. E-mail: aydin.arici{at}yale.edu.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, and but did not express progesterone (P4) receptors A and B in vivo and in vitro. Estradiol (E2; 10–10–10–8 M) and P4 (10–12–10–8 M), alone or in combination, induced HEEC proliferation compared with control values after 48 h of treatment (P < 0.05). Furthermore, after 8 d of treatment, there were significantly more angiogenic patterns in E2 (10–8 M), P4 (10–10 M), and E2 plus P4 (10–8 and 10–10 M) treatment groups compared with the control group (angiogenic scores, 2.95 ± 0.16, 3.26 ± 0.16, 3.06 ± 0.17, and 1.93 ± 0.15, respectively; P < 0.01). In conclusion, our results suggest that there are direct effects of E2 and P4 on HEEC and provide a new understanding of the physiological role of sex steroids in the regulation of endometrial events such as angiogenesis. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Angiogenesis is a process of new microvessels emerging from existing blood vessels. Physiological angiogenesis takes place during placental development after the vasculogenic stage and during fetal development. Physiological angiogenesis rarely occurs in postnatal life, except for wound healing and in tissues of the human female reproductive tract, such as corpus luteum and endometrium, influenced by steroid-dependent growth and/or regression (3, 4, 5). However, pathological angiogenesis occurs in many abnormal tissue growths, such as malignancy and endometriosis (6).
Endothelial cell proliferation varies throughout the menstrual cycle independently from the stage of the cycle (5). However, the necessity for new vessel growth and its regression in the human endometrium varies spatially and temporally in relation to the menstrual cycle phase. Differently from classical angiogenesis (7, 8), endometrial angiogenesis is likely to occur through a process of elongation and expansion of preexisting blood vessels (9) and may be subdivided into three distinct stages: during menstruation with the aim of vascular bed refurbishment, during the proliferative phase with the aim of vascular supply, and during the secretory phase with the aim of spiral arteriole growth and coiling (9). Therefore, in contrast to most vascular beds, which keep a persistent structure throughout life, the endometrial vascular network grows and regresses during the menstrual cycle and provides a dynamic model for the study of physiological angiogenesis.
Albeit circulating estrogen and progesterone predominantly regulate the overall control of endometrial growth and regression, the direct and indirect roles of sex steroids in endometrial angiogenesis are less clear. Moreover, there are conflicting reports on the expression of estrogen or progesterone (P4) receptors (ER and PR, respectively) in human endometrial endothelium both in vivo and in vitro (10, 11, 12, 13).
To date, only a few studies have developed a technique for the isolation and characterization of endothelial cells of the endometrium (10, 14, 15, 16). The limitations of culturing human endometrial endothelial cells (HEEC) for in vitro studies have restricted our understanding of their physiological functions as well as their pathological role in vascular endothelium-related endometrial diseases such as endometriosis, abnormal endometrial bleeding, endometrial vascular thrombosis, and endometrial malignancy. Our hypothesis was set up to understand the direct role of ovarian steroids in endometrial endothelial cell angiogenesis, such as proliferation and vascularization. To investigate the endometrial angiogenic response of estrogen and P4 in an in vitro model, we have developed a reliable method for the isolation of HEEC. This allowed us to investigate the regulation of angiogenesis by sex steroids.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Endometrial tissues were obtained from human uteri after hysterectomy and from endometrial biopsies, which were conducted for benign diseases other than endometrial disease. Informed consent in writing was obtained from each patient before surgery; consent forms and protocols were approved by the human investigation committee of Yale University. The mean age of the patients was 38.3 yr (range, 28–44 yr). The patients underwent surgery for leiomyomata (n = 4) and voluntary sterilization by tubal ligation (n = 4). The day of the menstrual cycle was established from the patient’s menstrual history and was verified by histological examination of the endometrium (four of them were from midproliferative, two were late proliferative, and two were from early secretory). The tissues were placed in Hanks’ balanced salt solution and transported to the laboratory for endometrial endothelial cell isolation and long-term culture. Each experimental setup was repeated on at least three occasions using cells obtained from different patients.
Isolation and culture of human endometrial stromal and glandular cells
Endometrial stromal and glandular cells were separated and maintained in monolayer culture as described previously with minor modifications (17). Briefly, endometrial tissue was minced with a sterile stainless surgical blade and digested by incubation of tissue minces in Hanks’ balanced salt solution (Sigma-Aldrich Corp., St. Louis, MO) that contained HEPES (25 mmol), penicillin (200 U/ml), streptomycin (200 mg/ml), collagenase H (1 mg/ml, 15 U/mg; Roche, Mannheim, Germany), and deoxyribonuclease (0.1 mg/ml, 1500 U/mg; Roche) for 45–60 min at 37 C with agitation every 5 min using a 20-ml syringe. Collagenase H is an enzyme mixture that is used for the disaggregation of tissues and the isolation of endothelial cells from various sources (18). The dispersed endometrial cells were separated by filtration through a wire sieve (73-µm diameter pore; Sigma-Aldrich Corp.). The endometrial glands (largely undispersed) were retained by the sieve, whereas the dispersed stromal and endothelial cells passed through the sieve into the filtrate.
The stromal cells were plated in Ham’s F-12/DMEM (1:1 vol/vol; Sigma-Aldrich Corp.) and fetal bovine serum (FBS; 10%, vol/vol; Invitrogen Life Technologies, Inc., Gaithersburg, MD). Cells were plated in plastic flasks (75 cm2; Falcon, BD Biosciences Franklin Lakes, NJ), maintained at 37 C in a humidified atmosphere (5% CO2 in air), and allowed to attach to the flask. On the following day of the culture, the medium was changed to remove unattached cells, dead cells, and erythrocytes.
Preparation of microbead-conjugated anti-CD105-coated petri dishes
CD105 (endoglin) is a glycoprotein, and its expression is highly restricted to endothelium in all tissues except bone marrow (19). A microbead-conjugated anti-CD105 (20 µl/ml; Miltenyi Biotech, Auburn, CA) solution was prepared in 50 mM Tris-Cl, pH 9.5. After adding the anti-CD105 microbead solution to petri dishes, the outside bottom surface of the petri dishes was enforced with a magnet to enhance and stabilize the antibody binding to dishes. Petri dishes (60-mm diameter; Falcon) were then incubated with the Tris-Cl solution (3 ml for each petri dish) for 2 h at 37 C. Thereafter, the solution was removed, and each dish was rinsed three times with 0.15 M NaCl. Dishes were then incubated with BSA [0.1% (w/v) in PBS] for 30 min at room temperature. The BSA solution was discarded before adding the cells.
Isolation of endometrial endothelial cells
On the second day of the endometrial stromal/endothelial cell culture (
60–80% confluence), cells were washed with PBS harvested by standard methods of trypsinization and were centrifuged at 1800 rpm for 5 min. The supernatant was discarded, and pellet was suspended in 2 ml (/107 cells) cell dilution buffer (PBS, pH 7.2, supplemented with 0.5% BSA and 2 mM EDTA). Thereafter, cells were filtered through a 30-µm pore size nylon mesh (Miltenyi Biotech) to prevent cells from clumping. After applying the filtered cell suspension to the dishes, the dishes were placed on a shaker, and CD105-positive cells were allowed to attach to antibody-covered beads with gentle agitation for 7–10 min at 8–10 C. Afterward, unattached cells were discarded, and dishes were rinsed several times with cell dilution buffer. Finally, the remaining attached cells were cultured with EGM MV-Microvascular endothelial cell medium, supplemented with SingleQuots containing growth factors, cytokines, and endothelial growth supplements (Bulletkits, CambrexClonetics, Baltimore, MD). The medium was replaced every 2 d. The HEEC were grown to 80–90% confluence in a 37 C in a 95% air/5% CO2 incubator. Cells were harvested using EDTA, trypsin-EDTA, and trypsin inhibitor solutions (Cambrex-Clonetics). Cells were then split 1:4 for passaging to 60-mm culture dishes (Falcon) or to four-well chamber slides (Falcon). Immunocytochemistry of endothelial cells, glandular cells, and leukocyte-specific markers was carried out in the second passage for cellular characterization. Second passage HEEC were also cultured in 12-well culture plates coated with or without growth factor-reduced Matrigel (BD Biosciences, Bedford, MA), and their appearances were recorded microscopically every 24 h for morphology while growing to confluence.
When cells reached 70–80% confluence, culture medium was switched for 24 h to a medium that contained 2% charcoal-stripped, steroid-depleted FBS. All experiments were then carried out in a phenol-red free medium containing 2% charcoal-stripped, steroid-depleted FBS.
Immunocytochemistry and immunohistochemistry
HEEC were grown to preconfluence on four-chamber slides. Chamber slides were fixed in cold methanol (–20 C) for 10 min. After several washings in distilled water and then three times for 10 min each time in PBS (pH 7.4), endogenous peroxidase activity was quenched by 3% H2O2 (0.6 ml H2O2 and 5.4 ml methanol) for 10 min and rinsed in 0.05% Tween 20 in PBS, pH 7.4 (PBS-T). Slides were then incubated with 5% normal horse serum or 5% normal goat serum for 30 min and subsequently with mouse monoclonal antibodies against CD31 (1:100; Novocastra Laboratories Ltd., Newcastle, UK), CD146 (P1H12 antibody; 5 µg/ml; Chemicon International, Temecula, CA), ER (1:80; Novocastra Laboratories Ltd.), ERß (developed against recombinant protein encoding amino acids 1–153 of human ERß expressed in Escherichia coli used as immunogen; 3 µg/ml; GeneTex, Inc., San Antonio, TX) (20), PR (ready to use; Lab Vision Corp., Fremont, CA; and DakoCytomation, Carpinteria, CA), and rabbit polyclonal von Willebrand factor (vWF) antibody (1:1000; Sigma-Aldrich Corp.) for 60 min at room temperature. In negative control slides, equivalent isotype antibodies or normal rabbit IgG were used instead of primary antibodies. After several rinses in PBS, biotinylated secondary antibodies (horse antimouse IgG or goat antirabbit IgG, Vector Laboratories, Inc., Burlingame, CA) were applied for 30 min. After several PBS rinses, culture slides were incubated with streptavidin-peroxidase complex for 30 min (Vector Laboratories). Subsequently, slides were rinsed several times in PBS and then incubated with 3-amino-9-ethyl-carbazole (BioGenex, San Ramon, CA) for 10 min. Slides were lightly counterstained with hematoxylin before permanent mounting.
Double immunohistochemistry staining
After the initial immunostaining of endometrial tissue sections for PR, the same sections were incubated with anti-CD31 antibody for 1 h at room temperature for double immunostaining using the streptavidin-alkaline phosphatase technique. After several rinses in PBS, horse biotinylated antimouse IgG was applied to sections for 30 min. After several PBS rinses, tissue sections were incubated with streptavidin-alkaline phosphatase complex for 30 min (Lab Vision Corp.). Subsequently, slides were rinsed several times in PBS, then incubated with Fast Red (Vector Laboratories, Inc.) for 10 min. Slides were lightly counterstained with hematoxylin before permanent mounting.
Western blot analysis
Total protein from second and third passage HEEC was extracted in a lysis buffer [50 mM HEPES (pH 7.4), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2-6H2O, 1 mM EGTA, 100 mM NaF, 10 mM sodium pyrophosphate and protease inhibitors, 1 mM Na3VO4, 10 mg/ml leupeptin, 10 mg/ml aprotinin, and 4 mM phenylmethylsulfonylfluoride]. The protein concentration was determined by a detergent-compatible protein assay (Bio-Rad Laboratories, Hercules, CA). Samples (20 µg) were loaded, electrophoretically separated by sodium dodecyl sulfate-polyacrylamide gel using 7.5% Tris-HCl Ready Gels (Bio-Rad Laboratories), and electroblotted onto Hybond ECL nitrocellulose membrane (Amersham Biosciences, Little Chalfont, UK). The membrane was blocked with 5% nonfat dry milk in PBS-T buffer for 1 h to reduce nonspecific binding. Then the membrane was incubated for 1 h with monoclonal antibodies against ER (Novocastra Laboratories Ltd. and GeneTex, Inc.), ERß (GeneTex, Inc.), and PR (Neomarkers, Fremont, CA). After several rinses with PBS-T for 15 min once and 10 min twice, the membrane was incubated for 1 h with peroxidase-labeled secondary antibodies (Vector Laboratories, Inc.), diluted at 1:10,000, and subsequently washed with PBS-T for 15 min once and 10 min twice. The immunoblot was developed using a chemiluminescent kit (NEN, Boston, MA).
Cell proliferation assay
HEEC (2 x 104/well) were seeded in 96-well culture plates. HEEC proliferation in 96-well culture plates was determined by a colorimetric assay using the CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay kit (Promega Corp., Madison, WI). This kit determines the number of viable cells. The CellTiter 96 AQueous Assay is composed of solutions of a novel tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS)] and an electron coupling reagent (phenazine methosulfate). MTS is bioreduced by cells into a formazan product that is soluble in tissue culture medium. The absorbance of the formazan at 490 nm can be measured directly in 96-well assay plates without additional processing. The conversion of MTS into aqueous, soluble formazan was accomplished by dehydrogenase enzymes found in metabolically active cells. The quantity of formazan product, as measured by the amount of 490 nm absorbance, was directly proportional to the number of living cells in culture. The first column of each 96-well plate did not contain any cells and was used as a blank. Consecutive columns were treated with various concentrations of estradiol (E2), P4, E2 combined with P4, and phenol red-free DMEM containing 2% FBS, alone as the control group. Cell proliferation was evaluated after 24 and 72 h of treatment. Four hours before the end of each experiment, MTS solution was added to all wells (10 µl/100 µl medium/well), and plates were incubated at 37 C. At the end of the incubation period, plates were read with a multiwell plate reader (Thermomax, Molecular Devices Corp., Menlo Park, CA). Data were expressed in OD units. Experiments were conducted with replicates of 12 wells/treatment condition. Similar experiments were conducted on at least three different occasions with cells prepared from four different endometrial tissues.
In vitro assay of steroid-driven angiogenic capacity and vascular tube formation
Third passage HEEC (1 x 104) was cultured on ECMatrix (a 96-well plate format of angiogenesis assay kit, Chemicon International) and collagen I (collagen-coated 12-well culture dishes). Short-term (4–48 h) and long-term (3–8 d) angiogenesis assays were performed in the presence or absence of sex steroids. Tube formation is a multistep time-dependent process involving cell adhesion, migration, differentiation, and growth. This in vitro angiogenesis assay kit provides a convenient system for evaluation of tube formation by endothelial cells (21, 22). ECMatrix consists of laminin, collagen type IV, heparan sulfate proteoglycans, entactin, and nidogen. It also contains various growth factors (TGF-ß and fibroblast growth factor) and proteolytic enzymes (plasminogen, tissue plasminogen activator, and matrix metalloproteinases). The short-term angiogenesis assay was performed as described in the manufacturer’s instructions (Chemicon International). A long-term angiogenesis assay was performed on collagen I-coated, 12-well plates. HEEC (0.5 x 105/well) were seeded in 12-well culture plates. Overall, in both short- and long-term angiogenesis assays, the vascular tube formation process was numerically scored as: 1, cells beginning to align with each other; 2, visible capillary tubes; 3, sprouting of secondary capillary tubes; 4, closed polygons of capillaries beginning to form; and 5, complex mesh-like capillary structures.
Statistical analysis
Cell proliferation, Western blot, and angiogenic scores were normally distributed as assessed by Kolmogorov-Smirnov test. ANOVA and post hoc Tukey test for pairwise comparisons were used in statistical analysis. P < 0.05 was considered significant. Statistical calculations were performed using SigmaStat for Windows (version 2.0, Jandel Scientific Corp., San Rafael, CA).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
HEEC were isolated by selection of CD105-positive cells using petri dishes coated with magnetic bead-conjugated anti-CD105 as described above. Morphological analysis pointed out that the HEEC formed a monolayer on noncoated culture plates (Fig. 1
, A and B). Morphologically, subconfluent monolayer stage of endothelial cells had many cytoplasmic extensions that extend to adjacent cells (Fig. 1). Moreover, the culture on gelatin-coated plates resulted in formation of networks and tube-like structures in some areas, which were observed by phase-contrast microscopy (Fig. 1, C and D).
|
). CD31 was localized mainly in the plasma membrane. In contrast, CD146 was observed mostly membranous, but also weakly cytoplasmic. Interestingly, CD146 immunocytochemistry also revealed a strong expression in cell-cell contact areas (Fig. 2D, inset). Furthermore, cells showed no immunoreactivity when stained with CD45 antibody, a common marker for leukocytes (data not shown). As an epithelial cell marker, cytokeratin antibody did not reveal any immunoreactivity in cultured HEEC. Only few cells were negative for endothelial markers (1–5%). On the other hand, after six or seven passages, most of the cells lost the morphological appearance of endothelial cells.
|
By immunocytochemistry, HEEC in culture showed a strong immunoreactivity for ERß (Fig. 3A), but a very weak immunoreactivity for ER (Fig. 3B). Mostly nuclear, but also cytoplasmic, immunoreactivity was observed for ERß. HEEC also revealed a weak immunoreactivity for PR (Fig. 3C). Similarly, in vivo analysis of HEEC demonstrated a clear immunoreactivity for ERß (Fig. 3D). However, ER immunoreactivity was absent (Fig. 3E). No PR immunoreactivity was detected in endothelial cells (Fig. 3F), which was confirmed by double immunostaining for CD31 and PR (Fig. 3F, inset). Confirming the immunocytochemistry and immunohistochemistry results, Western blot analysis revealed that HEEC have a clear 56-kDa band for ERß, whereas no band for ER was detected (Fig. 4). At the same time, although no band was detected around the expected 94 and 116 kDa sizes for PR A and B (PRA and PRB, respectively), a nonspecific band was observed around 50 kDa (Fig. 4)
|
|
Concentration-dependent proliferative effects of E2 (10–12–10–8 M) and P4 (10–12–10–8 M) on HEEC were assessed using the MTS colorimetric cell proliferation assay. HEEC were treated with ovarian steroids for 24–48 h. The proliferative effect of E2 was significant at 10–10–10–8 m concentration after 48 h of treatment (P < 0.05; Fig. 5A). The proliferative effect of progesterone was significant at 10–12–10–8 M after 48 h of treatment compared with control (P < 0.05; Fig. 5A). When combined with E2, P4 induced additional HEEC proliferation (P < 0.05; Fig. 5A). Moreover, when the cells treated with E2 combined with ICI 182,780 and progesterone combined with RU486, they did not reveal a significant increase in cell proliferation compared with control (Fig. 5B).
|
We assessed the effect of E2 and P4 on HEEC’s angiogenic capacity and formation of capillary-like tubular structures in vitro. Angiogenic pattern and vascular tube formation was numerically scored as: 1, cells beginning to align with each other; 2, differentiation to visible capillary-like structures; 3, sprouting of secondary capillary tubes; 4, closed polygons of capillaries beginning to form; and 5, complex mesh-like capillary structures. HEECs were cultured on ECMatrix for 4–48 h (short-term) and were scored every 8 h. In control cells, angiogenic pattern was generally scored 1 or 2, and 3. At 48 h there was no significant difference in P4-treated (10–10 M) cells compared with control cells. In contrast, E2 (10–8 M) treatment alone or combined with P4, induced angiogenic scores of 2–3, a trend for a higher scores than vehicle (control) and P4-treated cells (P < 0.12; Fig. 6).
|
).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Endometrial endothelial cells are the least studied cells of the human uterus. Most of the previous studies have focused on endometrial stromal, epithelial, myometrial cells and/or leukocytes. In the present study we evaluated the direct and functional effects of ovarian steroids on human endometrial vascular endothelial cells.
Using a novel isolation technique of HEEC by anti-CD105-microbead technique that we are presenting, we have obtained a quite pure population of endothelial cells confirmed by markers of endothelial cells such as CD31, CD146, and vWF. Additionally, morphological appearance (confluent monolayer with cobblestone-like tightly packed endothelial cells) and emergence of capillary-like tube structures on gelatin-coated plates confirmed the endothelial aspect of isolated cells.
The antibody P1H12 used in this study binds specifically to CD146. In the blood vessel and bone marrow it reacts only with endothelial cells and has been used to detect endothelial cells. It positively stains normal, primary endothelial cells in culture. Anti-CD146 positively stains endothelial cells of all blood vessels in frozen sections of human tissues, such as skin, intestine, ovary, tonsil, and uterus. CD146 is a membrane glycoprotein that functions as an adhesion molecule involved in heterophilic cell-cell interactions. Its expression has been demonstrated in a very limited number of normal cell types and some malignant neoplasms (23). Although the biological role of CD146 in normal tissue and malignant tumors remains unclear, CD146 has been suggested to play an important role in tumor progression or suppression in a tissue-specific manner. In human endometrium, only vascular endothelial cells are immunoreactive for CD146 antibody (24, 25). Positive expression of CD146 in HEEC supports the high purity of the endothelial cell isolation. Moreover, increased CD146 immunoreactivity in cell-cell contact areas of HEEC supports a role for CD146 in endometrial angiogenesis.
Although the isolation of HEEC was previously achieved (10, 14, 15, 16), none of these reports assessed the direct and functional effect of sex steroids on HEEC’s tube formation. One study described the presence of steroid receptors and proliferative and antiproliferative effects of E2 and P4 on HEEC, respectively (10). Thus, to our knowledge, the present study is the first using HEEC to evaluate vascular tube formation in the presence of ovarian steroids.
Previously, several in vivo studies were carried out in endometrial tissues to evaluate the expression of PR in human vascular endothelium (11, 26, 27, 28, 29). Although some of these studies were able to show PR expression, others did not observe PR expression in HEEC. Our in vitro and in vivo results showing the absence of PR in the endothelium support later studies. Moreover, immunocytochemistry analysis revealing a weak immunoreactivity for PR in our study is most likely due to a nonspecific binding of the antibody, because no specific band for either PRA or PRB was detected in the Western blot analysis, except for a nonspecific band around 50 kDa. However, this band may also be an alternative form of PRA or PRB (30).
Similar to PR expression, the results of previous in vivo studies reporting the expression of ER and ERß in HEEC are conflicting (11, 12, 13, 31). Although some studies reported the presence of ER and ERß (13), many other studies reported the absence of ER, but the presence of ERß, in endometrial vascular endothelium (11, 12, 31). Supporting these later studies, the present study confirmed the absence of or very weak ER signal and a strong ERß signal in HEEC. Therefore, we conclude that HEEC express ER in vitro, and ERß is the main ER in these cells, suggesting the possibility for a direct receptor-dependent response to estrogen.
Iruela-Arispe et al. (10) previously reported that although estrogen stimulates HEEC proliferation, P4 inhibits it. Although we have similar results for E2, in our study P4 has a proliferative effect, rather than an antiproliferative effect, in HEEC in culture. It is possible that culture conditions may cause these contradictory results, because Iruela-Arispe et al. (10) carried out their experiments in the presence of fibroblast growth factor-2 and vascular endothelial growth factor (VEGF), whereas we performed our experiments in the presence of 2% FBS. Interestingly, HEEC seem to be highly responsive to P4 even at low concentrations (10–12 M) compared with E2. Although our study has demonstrated a proliferative effect of P4 on HEEC, this effect might not be specific only for these cells, but may also be observed in other endothelial cells. Treatment with E2 and with E2 combined with P4 was shown to have proliferative effects, whereas treatment with P4 alone had an antiproliferative effect in an in vivo model in mice (32). In contrast, chronic treatment of cycling rhesus monkeys with ZK 137 316, a P4 antagonist, resulted in a clear dose-dependent reduction in the thickness of the endometrium, the abundance of glands, and endometrial atrophy. In the same study, after treatment with 0.1 mg ZK 137 316, spiral arteries and small veins were observed as more atrophied and with degenerative hyalinization compared with the control (33). Moreover, Johannisson et al. (34) reported that after administration of the P4 antagonist RU486 in secretory phase, necrosis was observed in endometrial capillary structures. However, the mechanism of contraceptive progestin-associated breakthrough bleeding and blood vessel rupture is not well understood (35). None of these studies reported whether the effect of P4 is due to a direct effect on vasculature or because of a decreased production of VEGF by stromal and/or epithelial cells. However, our results suggest the possibility of a direct effect of P4 and its antagonist RU486 on HEEC.
Numerous angiogenic and angiostatic factors have been identified in the human endometrium. Most of these studies have focused on VEGF (3, 4, 5). Although other researchers have performed HEEC isolation successfully, to our knowledge this is the first study assessing the effects of sex steroids on vascularization of HEEC in culture. Koolwijk et al. (16) reported that HEEC begin forming capillary-like tubes within 3 d when cultured over a fibrin matrix in the presence of 20% human serum. Moreover, increased angiogenic capacity was observed when HEEC were cultured on rat collagen type I. Similarly, we observed a better capillary-like structure formation on collagen I compared with Matrigel. Because the highest proliferative responses to E2 and P4 in cell proliferation assay were obtained at 10–8 and 10–10 M, respectively, we used these concentrations for the analysis of vascularization in vitro.
In the presence of sex steroids, HEEC exhibit an enhanced capacity to form tube-like structures and sprouts on gelatin and collagen I. The higher angiogenic score of HEEC probably reflects the direct effect of E2, because HEEC express ER in culture. However, we do not yet know how P4 may achieve its proliferative and angiogenic effects because of the absence of PR in HEEC. One possibility is that a direct, but nongenomic, effect of P4 in HEEC may explain these results (30). Previous studies reported several examples of P4-induced, non-PR-dependent, nongenomic effects, including the initiation of meiosis in amphibian oocytes, the inhibition of cholesterol synthesis, the stimulation of human acrosome reaction, a rapid increase in the intracellular calcium concentration, tyrosine phosphorylation of proteins, the activation of protein kinase C and of extracellular signal-regulated kinases, and the binding to oxytocin receptor (36, 37, 38, 39, 40, 41, 42). In contrast, it seems that a long incubation period (5–8 d) is needed to reveal the effect of sex steroids on vascular-like tube formation assay. We also observed spontaneous tube formation in the control group, which may be a response of HEEC to growth factors in FBS.
In conclusion, HEEC display increased proliferative and angiogenic activities in response to ovarian steroids. Our results suggest direct effects of E2 and P4 on HEEC function, providing additional understanding of physiological role of the endothelium in endometrial function. Additional studies are needed to investigate the roles of epithelium, stroma, trophoblast, and leukocytes on endometrial angiogenesis in the presence and absence of sex steroids in the physiological and pathological events of the endometrium.
|
Footnotes |
|---|
Abbreviations: E2, Estradiol; ER, estrogen receptor; FBS, fetal bovine serum; HEEC, human endometrial endothelial cell; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; P4, progesterone; PBS-T, 0.05% Tween 20 in PBS, pH 7.4; PR, progesterone receptor; PRA, progesterone receptor A; PRB, progesterone receptor B; VEGF, vascular endothelial growth factor; vWF, von Willebrand factor.
Received May 12, 2004.
Accepted July 21, 2004.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, is present in the vascular endothelium of the human and nonhuman primate endometrium. J Clin Endocrinol Metab 86:1370–1378This article has been cited by other articles:
 |
|
 |
|
  N. C. Douglas, H. Tang, R. Gomez, B. Pytowski, D. J. Hicklin, C. M. Sauer, J. Kitajewski, M. V. Sauer, and R. C. Zimmermann Vascular Endothelial Growth Factor Receptor 2 (VEGFR-2) Functions to Promote Uterine Decidual Angiogenesis during Early Pregnancy in the Mouse Endocrinology, August 1, 2009; 150(8): 3845 - 3854. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. W. Aberdeen, S. J. Wiegand, T. W. Bonagura Jr., G. J. Pepe, and E. D. Albrecht Vascular Endothelial Growth Factor Mediates the Estrogen-Induced Breakdown of Tight Junctions between and Increase in Proliferation of Microvessel Endothelial Cells in the Baboon Endometrium Endocrinology, December 1, 2008; 149(12): 6076 - 6083. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Hirota, S. Tranguch, T. Daikoku, A. Hasegawa, Y. Osuga, Y. Taketani, and S. K. Dey Deficiency of Immunophilin FKBP52 Promotes Endometriosis Am. J. Pathol., December 1, 2008; 173(6): 1747 - 1757. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Murk, C. S. Atabekoglu, H. Cakmak, A. Heper, A. Ensari, U. A. Kayisli, and A. Arici Extracellularly Signal-Regulated Kinase Activity in the Human Endometrium: Possible Roles in the Pathogenesis of Endometriosis J. Clin. Endocrinol. Metab., September 1, 2008; 93(9): 3532 - 3540. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Sun, J. Wu, H. Fujii, J. Wu, S.-H. Li, S. Porozov, A. Belleli, V. Fulga, Y. Porat, and R.-K. Li Human angiogenic cell precursors restore function in the infarcted rat heart: A comparison of cell delivery routes Eur J Heart Fail, June 1, 2008; 10(6): 525 - 533. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X.-D. Fu, M. Flamini, A. M. Sanchez, L. Goglia, M. S. Giretti, A. R. Genazzani, and T. Simoncini Progestogens regulate endothelial actin cytoskeleton and cell movement via the actin-binding protein moesin Mol. Hum. Reprod., April 1, 2008; 14(4): 225 - 234. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Sha, D. Wu, L. Zhang, X. Chen, M. Lei, H. Sun, S. Lin, and J. Lang Differentially expressed genes in human endometrial endothelial cells derived from eutopic endometrium of patients with endometriosis compared with those from patients without endometriosis Hum. Reprod., December 1, 2007; 22(12): 3159 - 3169. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. B. Gupta, D. Proia, O. Cingoz, J. Weremowicz, S. P. Naber, R. A. Weinberg, and C. Kuperwasser Systemic Stromal Effects of Estrogen Promote the Growth of Estrogen Receptor-Negative Cancers Cancer Res., March 1, 2007; 67(5): 2062 - 2071. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Seli, O. Guzeloglu-Kayisli, H. Cakmak, U. A. Kayisli, B. Selam, and A. Arici Estradiol Increases Apoptosis in Human Coronary Artery Endothelial Cells by Up-Regulating Fas and Fas Ligand Expression J. Clin. Endocrinol. Metab., December 1, 2006; 91(12): 4995 - 5001. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M.-L. Huang, H. Tian, J. Wu, K. Matsubayashi, R. D. Weisel, and R.-K. Li Myometrial cells induce angiogenesis and salvage damaged myocardium Am J Physiol Heart Circ Physiol, November 1, 2006; 291(5): H2057 - H2066. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. M Walter, P. A W Rogers, and J. E Girling The role of progesterone in endometrial angiogenesis in pregnant and ovariectomised mice Reproduction, June 1, 2005; 129(6): 765 - 777. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Luk, Y. Seval, U. A. Kayisli, M. Ulukus, C. E. Ulukus, and A. Arici Regulation of Interleukin-8 Expression in Human Endometrial Endothelial Cells: A Potential Mechanism for the Pathogenesis of Endometriosis J. Clin. Endocrinol. Metab., March 1, 2005; 90(3): 1805 - 1811. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
