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Effects of progesterone, levonorgestrel and medroxyprogesterone acetate on apoptosis in human endometrial endothelial cells
Contraception 79 (2009) 139 – 145
Original research article
Effects of progesterone, levonorgestrel and medroxyprogesterone acetate on apoptosis in human endometrial endothelial cells☆,☆☆ Chainarong Choksuchata,b , Shumei Zhaoa , Todd D. Deutcha , Thomas D. Kimblea , David F. Archera,⁎ a
CONRAD Clinical Research Center, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, VA 23507, USA b Faculty of Medicine, Department of Obstetrics and Gynecology, Prince of Songkla University, Hat-Yai, Songkhla 90110, Thailand Received 7 August 2008; revised 27 August 2008; accepted 28 August 2008
Abstract Background: We evaluated apoptosis in human endometrial endothelial cells (HEECs) incubated with progesterone, levonorgestrel (LNG) and medroxyprogesterone acetate (MPA). Study Design: HEECs were cultured to near confluence, and the progestogens were added. Setting: Academic Department of Obstetrics and Gynecology. Patients: No patients were involved. Interventions: Progestogens at 5-, 250- and 500-ng/mL concentrations were added to incubations of HEECs for 12, 24 and 48 h. Main Outcome Measure: Apoptosis based on terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick-end labeling (TUNEL), and semiquantification of Bax and Bcl-2. Results: No apoptosis was found by TUNEL, Bax and Bcl-2 after 12 h incubation with any progestogen. TUNEL increased after incubation for 24 and 48 h with progesterone 500 ng/mL; LNG 250, 500 ng/mL and all concentrations of MPA (pb.001), Bax increased and Bcl-2 decreased at all concentrations of MPA and the two highest concentrations of LNG at 48 h (pb.05). Conclusion: MPA results in apoptosis of HEECs. © 2009 Elsevier Inc. All rights reserved. Keywords: Apoptosis; Endometrial endothelial cells; Progesterone; Levonorgestrel; Medroxyprogesterone acetate
1. Introduction Endometrial angiogenic factors, metalloproteinases and tissue factor have been hypothesized to alter capillary vessel integrity and result in the abnormal endometrial spotting and
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The research was supported in part by National Institutes of Health Grant R01 HD43175-01 and CONRAD Clinical Research Center Internal Funds. ☆☆ Conflict of interest: C.C. has nothing to disclose. S.Z. has nothing to disclose. T.D.D. has nothing to disclose. T.D.K. has nothing to disclose. D.F. A. has received research funding from Wyeth, Novo Nordisk, Bayer Healthcare, Warner Chilcott and Glaxo SmithKline; has served as a consultant to Wyeth, Bayer Healthcare, Novo Nordisk, Schering Plough, Merck, Warner Chilcott, Abbott Laboratories, and Agile Therapeutics and has received honoraria from Organon, Wyeth, Novo Nordisk and Ascend Therapeutics. ⁎ Corresponding author. Tel.: +1 757 446 7444; fax: +1 757 446 8998. E-mail address: [email protected] (D.F. Archer). 0010-7824/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.contraception.2008.08.008
bleeding and spotting/bleeding (S/B) in women using progestin-only contraceptives [1–3]. Vascular dysfunction has been manifest by changes in endometrial capillary vessel density, and altered endometrial levels of metalloproteinases, free radicals and tissue factor [4–8]. Vascular fragility in levonorgestrel subcutaneous implant users (LNG-SI), is suggested by the hysteroscopic findings of local bleeding following changes in intrauterine pressure [4,5]. Endothelial cells play a critical role in the maintenance of vessel integrity and vascular homeostasis. Spotting and bleeding associated with progestin-only contraceptives appear to be associated with a change in vessel wall integrity [3–5]. We hypothesized that apoptosis of the endometrial endothelial cells could be one of the mechanisms that could result in increased vascular fragility and loss of vascular integrity resulting in endometrial S/B. The aim of this study was to evaluate the effect of progesterone, levonorgestrel
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(LNG) and medroxyprogesterone acetate (MPA) on indices of apoptosis using immortalized human endometrial endothelial cells (HEECs), in vitro. 2. Materials and methods The experiments were carried out on immortalized endothelial cells, in vitro, in a basic research laboratory. No patients were involved, and institutional review board approval was not required. 2.1. Cell culture and hormonal treatment The immortalized HEECs were provided by Dr. Charles J. Lockwood (Yale University, New Haven, CT, USA). The cells have previously been characterized and confirmed as endothelial cells [9]. We have demonstrated von Willebrand factor mRNA and protein expression in these HEECs by real-time polymerase chain reaction and Western blot analysis, respectively (data not shown). The cells were maintained in endothelial cell growth medium-2 (Cambrex, Walkersville, MD. USA) at 37°C in a humidified atmosphere with 5% CO2 and grown as monolayer cultured in 100-mm tissue culture dishes (Corning, Acton, MA, USA) coated with 2% gelatin (Sigma-Aldrich, St. Louis, MO, USA). The cells were harvested by trypsin-EDTA (0.05% trypsin with EDTA 4Na) (Invitrogen, Grand Island, NY, USA). The cells were then plated in 25-cm2 tissue culture flasks (Corning) coated with 2% gelatin in 7 mL of culture medium containing phenol red and 2% serum. After reaching 80% confluence, the incubations were continued using serum-free medium containing phenol red at a concentration of 1.24 mg/L. Each progestogen was added at concentrations of 5, 250 and 500 ng/mL. Control incubations contained only serum-free media with phenol red, and ethanol at 0.01%. Progesterone, LNG and MPA (Sigma–Aldrich, St. Louis, MO, USA) were dissolved in absolute ethanol; the final ethanol concentration in each culture medium was 0.01%. 2.2. Apoptosis indices Apoptosis in HEECs was assessed after 12, 24 and 48 h of incubation by terminal deoxynucleotidyl transferasemediated deoxy-UTP nick-end labeling (TUNEL), and Bcl-2 and Bax were evaluated by Western blot and semiquantitated comparing their concentrations to actin. Each experiment was performed in triplicate. 2.3. Detection of DNA strand break by TUNEL assays Apoptosis was assessed using TUNEL kits (In Situ Cell Death Detection Kit, Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer's instruction. The HEECs were fixed in 40% paraformaldehyde solution in phosphate-buffered saline (PBS) (United States Biochemical, Cleveland, OH, USA), pH 7.4, for 60 min at 25°C. After
washing twice in PBS, cells were incubated with permeabilization solution (0.1% Trixon X-100 in 0.1% sodium citrate) for 10 min at room temperature. Following two washes with PBS, cells were covered with TUNEL reaction mixture [terminal deoxynucleotidyl transferase from calf thymus (EC 2.7.7.31)], recombinant in Escherichia coli, in storage buffer 5 μL and nucleotide mixture in reaction buffer 45 μL and incubated in a humidified incubator for 60 min at 37°C. Staurosporine (Sigma–Aldrich) 1.5 mM and DNase I recombinant (Roche Diagnostics) 10 mcg/mL were used as positive controls [10]. The slides were washed twice with PBS. The cells were then double-stained with propidium iodide (Sigma–Aldrich) 1:10,000 for 30 s to detect morphological changes. The slides were covered with mounting medium for fluorescence (H-1000, Vector Laboratories, Burlingame, CA, USA) to prevent rapid loss of fluorescence during microscopic examination and analyzed under a fluorescence microscope. Five fields were randomly analyzed and averaged for cells exhibiting apoptosis. 2.4. Protein extraction and Bax and Bcl-protein expression by Western blot analysis Proteins were extracted from HEECs by using RadioImmuno Precipitation Assay buffer (Technova, Hollister, CA, USA) with protease inhibitor cocktail (Pierce Biotechnology, Rockford, IL, USA) according to the manufacturer's instruction. A Micro BCA protein assay kit (Pierce Biotechnology) measured protein concentration. Total proteins 20 mcg were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. Nonspecific binding sites were blocked for 1 h in Tris-buffered saline (4 mM Tris-Cl, 100 mM NaCl) containing fat-free milk (5%) and Tween 20 (0.5%) at room temperature. The membranes were incubated overnight at 4°C with anti-Bax (N-20) (sc-493; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and antiBcl-2 (N-19) (sc-492) (Santa Cruz Biotechnology) in a concentration of 1:100 and 1:200, respectively. The PVDF membranes were then incubated for 1 h with secondary antirabbit IgG (1:1000, sc-2004, Santa Cruz Biotechnology). Actin was evaluated to assess consistency of protein loading using anti-actin antibody (1:5000) (BD Biosciences, Franklin Lakes, NJ, USA). The membranes were developed with Western Lighting Chemiluminescence Reagent Plus (NEL 104) (Perkin-Elmer Life Sciences, Boston, MA, USA) for 1 min. Images were registered using Kodak imaging station, and Kodak image analysis software (Carestream Health, Rochester, NY, USA) was used to determine net intensities of identified bands. 2.5. Statistical analysis Normal distribution of all data was found after a log 10 transformation. An analysis of variance with post hoc Dunnett t test was performed to compare the outcomes between the various treatments compared to control and the
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effect of incubation time using SPSS 13.0 for MS Windows (SPSS, Chicago, IL, USA). Significance was based on a 95% confidence interval and a p value of b.05. 3. Results 3.1. TUNEL TUNEL images from representative experiments with HEECs are shown in Fig. 1. The green fluorescence indicates apoptosis. DNase I recombinant 10 mcg/mL and 1.5 mM Staurosporine were used as positive controls and resulted in apoptosis (Fig. 1B), compared with the negative control (Fig. 1A). A difference in apoptosis by TUNEL was found between control (Fig. 1C) and MPA treatment (Fig. 1D) after 24 h. The incidence of TUNEL-positive cells was between 4% and 9% after 12 h incubation with all treatments, and not different from control (data not shown). TUNEL incidence was 7.5% in the control group after 24-h incubation (Fig. 2) with the range of TUNEL-positive cells in the progesterone and LNG groups of 6.3%-11.5% and 7.6%-12.4%, respectively, not different from control. TUNEL-positivity was significantly increased only with MPA 500 ng/mL (25.8%) treatment for 24 h when compared with control (Fig. 2).
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Significant increases in TUNEL-positive cells were found with progesterone 500 ng/mL (27.5%), LNG 250 and 500 ng/mL (29.7% and 40%, respectively) and all concentrations of MPA (range 24.2–49.9%) after 48 h compared to control (12.1%) (Fig. 2). 3.2. Bax expression Bax expression (proapoptotic) was similar between groups and comparable to control after 12 h of incubation (data not shown) and was increased significantly with LNG 500 ng/mL and all concentrations of MPA at 24 h (Fig. 3). Progesterone and the two lowest concentrations of LNG had no effect on Bax expression after 24 h (Fig. 3). Increased Bax expression at 48 h was found with LNG 500 ng/mL and all concentrations of MPA, but not with progesterone and the two lower concentrations of LNG (Fig. 3). 3.3. Bcl-2 expression Bcl-2 expression (antiapoptotic) after 12 h was not different between treatments (data not shown). Bcl-2 expression increased with progesterone 5 ng/mL at 24 h but was not different from control. Bcl-2 decreased significantly compared to control values after 24 h with LNG 250 ng/mL, 500 ng/mL and all concentrations of MPA but not with progesterone and the lowest concentration of
Fig. 1. TUNEL assays images from representative experiments. The green fluorescence indicates apoptosis (arrows). The red fluorescence demonstrated cell morphology. (A) The negative control, which was not stained with TUNEL solution. (B) Apoptosis induced by DNase I recombinant/staurosporine. HEECs cultured in serum free medium and incubated for 24 h are: control (C) and MPA 250 ng/mL (D), respectively.
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Fig. 2. TUNEL results after 24 h incubation found a significant increase in the incidence of apoptotic cells after treatment with MPA 500 ng/mL only. TUNEL at 48 h detected significant increases in the incidence of apoptotic cells with progesterone 500 ng/mL, LNG 250 and 500 ng/mL and all concentrations of MPA. *, +pb.001 when compared between control and treatment at 24 h, respectively.
Fig. 3. Bax expression by Western blot at 24 h was significantly increased after treatment with 500 ng/mL LNG and all concentrations of MPA. Bax expression at 48 h was significantly increased by 500 ng/mL LNG and all concentrations of MPA. Progesterone at all concentrations had no effect on Bax expression. *,+pb.05 when compared between control and treatment at 12 and 24 h, respectively.
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Fig. 4. Bcl-2 expression at 24 h was significantly decreased after treatment with LNG 250, 500 ng/mL and all concentrations of MPA using Western blot. Bcl-2 expression at 48 h decreased significantly when incubated with 250 and 500 ng/mL LNG and 250 and 500 ng/mL MPA. Progesterone had no significant effect on Bcl-2 expression. *,+pb.05 when compared between control and treatment at 12 and 24 h, respectively.
LNG (Fig. 4). Bcl-2 decreased significantly with LNG and MPA concentrations of 250 and 500 ng/ml after 48 h (Fig. 4).
4. Discussion Women using progestogen-only contraception such as LNG-SI and MPA injections [DepoProvera (DMPA); Pfizer, New York, NY, USA) experience irregular, unpredictable S/B [11,12]. A dysfunction in endometrial capillary integrity has been postulated as a cause of the S/B and has been termed “vessel fragility” based on visualizing overt spurts of bleeding associated with changes in intrauterine pressure [4,13,14]. Our hypothesis was that progestational agents could have a direct effect on the endometrial endothelial cell, altering its integrity. There is no compelling evidence for the progesterone receptor (PR) in human or nonhuman primate endometrial endothelial cells [15–17]. Both the glucocorticoid receptor (GR) and androgen receptor are present in endothelial cells [18–21]. One article reports finding PR using real time polymerase chain reaction that increased the mRNA levels [15]. This report did not find any significant PR after five passages of the HEECs, and the authors were unable to detect PR in the endometrial endothelial cells in tissue sections [15].
Despite this, we have indirect evidence that LNG did induce apoptotic changes using TUNEL after 24 and 48 h and that the ratio of Bax to Bcl-2 was altered only with LNG 500 ng/mL after 24 and 48 h. Serum concentrations of LNG after LNG implants, and the LNG intrauterine system (LNG-IUS) are 0.4–1.6 and 0.2 ng/mL, respectively [22–24]. The LNGIUS has concentrations of LNG in myometrial, fallopian tube and fat tissue of between 1 and 5 ng/gm of wet tissue weight [25]. Thus, the serum and tissue levels in contraceptors using LNG implants and IUS are significantly lower that the concentrations used in our current experiments. The relative ratio of Bcl-2 to Bax may be important in determining the result of the multiple stimuli that initiate apoptosis but do not provide an mechanism for apoptosis [26]. A previous report found no evidence of endometrial epithelial, glandular or stromal cell immunostaining for Bcl2, Fas and Caspase-3 at 3 and 12 months following insertion of a LNG-SI [27]. This investigation did not identify any apoptotic markers in the endothelial cell although they were specifically sought [27]. The possible mechanism resulting in apoptosis in the presence of high concentrations of LNG in vitro is unknown. Our current data suggest that the effect if any of LNG on vascular permeability and fragility in progestinonly contraceptives is probably secondary to mechanisms other than endothelial cell apoptosis [6,9,14,27,28].
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MPA had a significant apoptotic effect in HEECs in this study in both a concentration and time dependent manner. MPA increased TUNEL and the expression of Bax, a proapoptotic protein, and decreased Bcl-2, an anti-apoptosic protein. MPA peak plasma concentrations of 1–7 ng/mL are found at 3 weeks after an injection of DMPA [29–31]. We feel that the lowest MPA concentration used in our experiments is equivalent to the peripheral plasma concentrations found following injection of DMPA. MPA has been shown to exert its effect on apoptosis via the GR in human umbilical vein endothelial cells (HUVEC) in vitro [32,33]. We have evidence that GR is present in the HEECs used in these experiments (unpublished observations). MPA is both angiostatic and apoptotic in a variety of cancer cells [34–37]. MPA alters both Bcl-2 and Bax in the decidualized endometrium of rodents leading to tissue remodeling [38]. We believe that MPA could cause endothelial cell apoptosis via the GR. Progesterone 500 ng/mL after 48 h incubation resulted in increased TUNEL positivity. Progesterone had no effect on Bax and Bcl-2 at any concentration or time. Progesterone at concentrations of 0.05–1 μM inhibited caspase-3, an apoptosis-related cysteine peptidase in HUVEC in vitro, but this could have been mediated through a non-genomic pathway [39], since there is no evidence for PR in endometrial endothelial cells in either the human or nonhuman primate [15,16]. Progesterone was not found to induce consistent and significant apoptotic changes in HEECs in our study. Progesterone given as a 200 mg intravaginal suppository reduced TUNEL, Bax protein and increased Bcl-2 in the endometrial epithelium of normal women, without evidence of apoptosis in endometrial blood vessels [40]. Our data do not support progesterone as affecting endothelial cell apoptosis. 5. Conclusion Our current findings demonstrate changes in apoptotic markers in HEECs following incubation with MPA, which we believe is mediated via the GR. This finding may have clinical relevance by contributing to an increased vascular fragility in users of DMPA. We believe that LNG-related S/B is due to mechanism(s) other than apoptosis of endothelial cells, and we have no evidence that progesterone induces apoptosis in endometrial endothelial cells. References [1] Fraser IS, Hickey M. Endometrial vascular changes and bleeding disturbances with long-acting progestins. Steroids 2000;65:665–70. [2] Hickey M, Crewe J, Mahoney LA, Doherty DA, Fraser IS, Salamonsen LA. Mechanisms of irregular bleeding with hormone therapy: the role of matrix metalloproteinases and their tissue inhibitors. J Clin Endocrinol Metab 2006;91:3189–98. [3] Hickey M. Bleeding with menopausal hormone therapy: physiological or pathological? Menopause Int 2007;13:188–90.
[4] Hickey M, Dwarte D, Fraser IS. Superficial endometrial vascular fragility in Norplant users and in women with ovulatory dysfunctional uterine bleeding. Hum Reprod 2000;15:1509–14. [5] Hickey M, Fraser IS. Human uterine vascular structures in normal and diseased states. Microsc Res Tech 2003;60:377–89. [6] Krikun G, Critchley H, Schatz F, et al. Abnormal uterine bleeding during progestin-only contraception may result from free radicalinduced alterations in angiopoietin expression. Am J Pathol 2002;161: 979–86. [7] Vincent AJ, Zhang J, Ostor A, et al. Decreased tissue inhibitor of metalloproteinase in the endometrium of women using depot medroxyprogesterone acetate: a role for altered endometrial matrix metalloproteinase/tissue inhibitor of metalloproteinase balance in the pathogenesis of abnormal uterine bleeding? Hum Reprod 2002;17:1189–98. [8] Freitas S, Meduri G, Le Nestour E, Bausero P, Perrot-Applanat M. Expression of metalloproteinases and their inhibitors in blood vessels in human endometrium. Biol Reprod 1999;61:1070–82. [9] Krikun G, Mor G, Huang J, Schatz F, Lockwood CJ. Metalloproteinase expression by control and telomerase immortalized human endometrial endothelial cells. Histol Histopathol 2005;20:719–24. [10] Kabir J, Lobo M, Zachary I. Staurosporine induces endothelial cell apoptosis via focal adhesion kinase dephosphorylation and focal adhesion disassembly independent of focal adhesion kinase proteolysis. Biochem J 2002;367(Pt 1):145–55. [11] Abdel-Aleem H, d'Arcangue C, Vogelsong K, Gulmezoglu AM. Treatment of vaginal bleeding irregularities induced by progestin only contraceptives. Cochrane Database Syst Rev 2007(2):CD003449. [12] Livingstone M, Fraser IS. Mechanisms of abnormal uterine bleeding. Hum Reprod Update 2002;8:60–7. [13] Hickey M, Fraser IS. The structure of endometrial microvessels. Hum Reprod 2000;15(Suppl 3):57–66. [14] Hickey M, Simbar M, Markham R, et al. Changes in vascular basement membrane in the endometrium of Norplant users. Hum Reprod 1999; 14:716–21. [15] Krikun G, Schatz F, Taylor R, et al. Endometrial endothelial cell steroid receptor expression and steroid effects on gene expression. J Clin Endocrinol Metab 2005;90:1812–8. [16] Brenner RM, Slayden OD. Steroid receptors in blood vessels of the rhesus macaque endometrium: a review. Arch Histol Cytol 2004;67: 411–6. [17] Critchley HO, Brenner RM, Henderson TA, et al. Estrogen receptor beta, but not estrogen receptor alpha, is present in the vascular endothelium of the human and nonhuman primate endometrium. J Clin Endocrinol Metab 2001;86:1370–8. [18] Vani S, Critchley HO, Fraser IS, Hickey M. Endometrial expression of steroid receptors in postmenopausal hormone replacement therapy users: relationship to bleeding patterns. J Fam Plann Reprod Health Care 2008;34:27–34. [19] Bamberger AM, Milde-Langosch K, Loning T, Bamberger CM. The glucocorticoid receptor is specifically expressed in the stromal compartment of the human endometrium. J Clin Endocrinol Metab 2001;86:5071–4. [20] Narvekar N, Critchley HO, Cheng L, Baird DT. Mifepristone-induced amenorrhoea is associated with an increase in microvessel density and glucocorticoid receptor and a decrease in stromal vascular endothelial growth factor. Hum Reprod 2006;21:2312–8. [21] Liang T, Hoyer S, Yu R, et al. Immunocytochemical localization of androgen receptors in human skin using monoclonal antibodies against the androgen receptor. J Invest Dermatol 1993;100:663–6. [22] Brache V, Alvarez-Sanchez F, Faundes A, Tejada AS, Cochon L. Ovarian endocrine function through five years of continuous treatment with NORPLANT subdermal contraceptive implants. Contraception 1990;41:169–77. [23] Ganong WF. Physiology of reproduction in women. In: DeCherney AH, et al, editor. Current Diagnosis & Treatment Obstetrics & Gynecology. New York: Lange Medical Books/McGraw-Hill; 2007. p. 126–48.
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[33] Zerr-Fouineau M, Chataigneau M, Blot C, Schini-Kerth VB. Progestins overcome inhibition of platelet aggregation by endothelial cells by down-regulating endothelial NO synthase via glucocorticoid receptors. FASEB J 2007;21:265–73. [34] Yamamoto T, Terada N, Nishizawa Y, Petrow V. Angiostatic activities of medroxyprogesterone acetate and its analogues. Int J Cancer 1994; 56:393–9. [35] Ozdemir F, Ovali E, Aydin F, et al. The in-vitro effects of medroxyprogesterone acetate on acidic pH induced apoptosis of peripheral blood mononuclear cells. J Exp Clin Cancer Res 2004;23: 93–5. [36] Aydin F, Ozdemir F, Kavgaci H, Yilmaz M, Karti S. The effects of medroxyprogesterone acetate on apoptosis of CD34+ cells. Chemotherapy 2003;49:66–70. [37] Abe M, Yamashita J, Ogawa M. Medroxyprogesterone acetate inhibits human pancreatic carcinoma cell growth by inducing apoptosis in association with Bcl-2 phosphorylation. Cancer 2000; 88:2000–9. [38] Akcali KC, Khan SA, Moulton BC. Effect of decidualization on the expression of Bax and Bcl-2 in the rat uterine endometrium. Endocrinology 1996;137:3123–31. [39] Williams TA, Verhovez A, Milan A, Veglio F, Mulatero P. Protective effect of spironolactone on endothelial cell apoptosis. Endocrinology 2006;147:2496–505. [40] Lovely LP, Fazleabas AT, Fritz MA, McAdams DG, Lessey BA. Prevention of endometrial apoptosis: randomized prospective comparison of human chorionic gonadotropin versus progesterone treatment in the luteal phase. J Clin Endocrinol Metab 2005;90: 2351–6.
Original research article
Effects of progesterone, levonorgestrel and medroxyprogesterone acetate on apoptosis in human endometrial endothelial cells☆,☆☆ Chainarong Choksuchata,b , Shumei Zhaoa , Todd D. Deutcha , Thomas D. Kimblea , David F. Archera,⁎ a
CONRAD Clinical Research Center, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, VA 23507, USA b Faculty of Medicine, Department of Obstetrics and Gynecology, Prince of Songkla University, Hat-Yai, Songkhla 90110, Thailand Received 7 August 2008; revised 27 August 2008; accepted 28 August 2008
Abstract Background: We evaluated apoptosis in human endometrial endothelial cells (HEECs) incubated with progesterone, levonorgestrel (LNG) and medroxyprogesterone acetate (MPA). Study Design: HEECs were cultured to near confluence, and the progestogens were added. Setting: Academic Department of Obstetrics and Gynecology. Patients: No patients were involved. Interventions: Progestogens at 5-, 250- and 500-ng/mL concentrations were added to incubations of HEECs for 12, 24 and 48 h. Main Outcome Measure: Apoptosis based on terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick-end labeling (TUNEL), and semiquantification of Bax and Bcl-2. Results: No apoptosis was found by TUNEL, Bax and Bcl-2 after 12 h incubation with any progestogen. TUNEL increased after incubation for 24 and 48 h with progesterone 500 ng/mL; LNG 250, 500 ng/mL and all concentrations of MPA (pb.001), Bax increased and Bcl-2 decreased at all concentrations of MPA and the two highest concentrations of LNG at 48 h (pb.05). Conclusion: MPA results in apoptosis of HEECs. © 2009 Elsevier Inc. All rights reserved. Keywords: Apoptosis; Endometrial endothelial cells; Progesterone; Levonorgestrel; Medroxyprogesterone acetate
1. Introduction Endometrial angiogenic factors, metalloproteinases and tissue factor have been hypothesized to alter capillary vessel integrity and result in the abnormal endometrial spotting and
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The research was supported in part by National Institutes of Health Grant R01 HD43175-01 and CONRAD Clinical Research Center Internal Funds. ☆☆ Conflict of interest: C.C. has nothing to disclose. S.Z. has nothing to disclose. T.D.D. has nothing to disclose. T.D.K. has nothing to disclose. D.F. A. has received research funding from Wyeth, Novo Nordisk, Bayer Healthcare, Warner Chilcott and Glaxo SmithKline; has served as a consultant to Wyeth, Bayer Healthcare, Novo Nordisk, Schering Plough, Merck, Warner Chilcott, Abbott Laboratories, and Agile Therapeutics and has received honoraria from Organon, Wyeth, Novo Nordisk and Ascend Therapeutics. ⁎ Corresponding author. Tel.: +1 757 446 7444; fax: +1 757 446 8998. E-mail address: [email protected] (D.F. Archer). 0010-7824/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.contraception.2008.08.008
bleeding and spotting/bleeding (S/B) in women using progestin-only contraceptives [1–3]. Vascular dysfunction has been manifest by changes in endometrial capillary vessel density, and altered endometrial levels of metalloproteinases, free radicals and tissue factor [4–8]. Vascular fragility in levonorgestrel subcutaneous implant users (LNG-SI), is suggested by the hysteroscopic findings of local bleeding following changes in intrauterine pressure [4,5]. Endothelial cells play a critical role in the maintenance of vessel integrity and vascular homeostasis. Spotting and bleeding associated with progestin-only contraceptives appear to be associated with a change in vessel wall integrity [3–5]. We hypothesized that apoptosis of the endometrial endothelial cells could be one of the mechanisms that could result in increased vascular fragility and loss of vascular integrity resulting in endometrial S/B. The aim of this study was to evaluate the effect of progesterone, levonorgestrel
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(LNG) and medroxyprogesterone acetate (MPA) on indices of apoptosis using immortalized human endometrial endothelial cells (HEECs), in vitro. 2. Materials and methods The experiments were carried out on immortalized endothelial cells, in vitro, in a basic research laboratory. No patients were involved, and institutional review board approval was not required. 2.1. Cell culture and hormonal treatment The immortalized HEECs were provided by Dr. Charles J. Lockwood (Yale University, New Haven, CT, USA). The cells have previously been characterized and confirmed as endothelial cells [9]. We have demonstrated von Willebrand factor mRNA and protein expression in these HEECs by real-time polymerase chain reaction and Western blot analysis, respectively (data not shown). The cells were maintained in endothelial cell growth medium-2 (Cambrex, Walkersville, MD. USA) at 37°C in a humidified atmosphere with 5% CO2 and grown as monolayer cultured in 100-mm tissue culture dishes (Corning, Acton, MA, USA) coated with 2% gelatin (Sigma-Aldrich, St. Louis, MO, USA). The cells were harvested by trypsin-EDTA (0.05% trypsin with EDTA 4Na) (Invitrogen, Grand Island, NY, USA). The cells were then plated in 25-cm2 tissue culture flasks (Corning) coated with 2% gelatin in 7 mL of culture medium containing phenol red and 2% serum. After reaching 80% confluence, the incubations were continued using serum-free medium containing phenol red at a concentration of 1.24 mg/L. Each progestogen was added at concentrations of 5, 250 and 500 ng/mL. Control incubations contained only serum-free media with phenol red, and ethanol at 0.01%. Progesterone, LNG and MPA (Sigma–Aldrich, St. Louis, MO, USA) were dissolved in absolute ethanol; the final ethanol concentration in each culture medium was 0.01%. 2.2. Apoptosis indices Apoptosis in HEECs was assessed after 12, 24 and 48 h of incubation by terminal deoxynucleotidyl transferasemediated deoxy-UTP nick-end labeling (TUNEL), and Bcl-2 and Bax were evaluated by Western blot and semiquantitated comparing their concentrations to actin. Each experiment was performed in triplicate. 2.3. Detection of DNA strand break by TUNEL assays Apoptosis was assessed using TUNEL kits (In Situ Cell Death Detection Kit, Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer's instruction. The HEECs were fixed in 40% paraformaldehyde solution in phosphate-buffered saline (PBS) (United States Biochemical, Cleveland, OH, USA), pH 7.4, for 60 min at 25°C. After
washing twice in PBS, cells were incubated with permeabilization solution (0.1% Trixon X-100 in 0.1% sodium citrate) for 10 min at room temperature. Following two washes with PBS, cells were covered with TUNEL reaction mixture [terminal deoxynucleotidyl transferase from calf thymus (EC 2.7.7.31)], recombinant in Escherichia coli, in storage buffer 5 μL and nucleotide mixture in reaction buffer 45 μL and incubated in a humidified incubator for 60 min at 37°C. Staurosporine (Sigma–Aldrich) 1.5 mM and DNase I recombinant (Roche Diagnostics) 10 mcg/mL were used as positive controls [10]. The slides were washed twice with PBS. The cells were then double-stained with propidium iodide (Sigma–Aldrich) 1:10,000 for 30 s to detect morphological changes. The slides were covered with mounting medium for fluorescence (H-1000, Vector Laboratories, Burlingame, CA, USA) to prevent rapid loss of fluorescence during microscopic examination and analyzed under a fluorescence microscope. Five fields were randomly analyzed and averaged for cells exhibiting apoptosis. 2.4. Protein extraction and Bax and Bcl-protein expression by Western blot analysis Proteins were extracted from HEECs by using RadioImmuno Precipitation Assay buffer (Technova, Hollister, CA, USA) with protease inhibitor cocktail (Pierce Biotechnology, Rockford, IL, USA) according to the manufacturer's instruction. A Micro BCA protein assay kit (Pierce Biotechnology) measured protein concentration. Total proteins 20 mcg were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. Nonspecific binding sites were blocked for 1 h in Tris-buffered saline (4 mM Tris-Cl, 100 mM NaCl) containing fat-free milk (5%) and Tween 20 (0.5%) at room temperature. The membranes were incubated overnight at 4°C with anti-Bax (N-20) (sc-493; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and antiBcl-2 (N-19) (sc-492) (Santa Cruz Biotechnology) in a concentration of 1:100 and 1:200, respectively. The PVDF membranes were then incubated for 1 h with secondary antirabbit IgG (1:1000, sc-2004, Santa Cruz Biotechnology). Actin was evaluated to assess consistency of protein loading using anti-actin antibody (1:5000) (BD Biosciences, Franklin Lakes, NJ, USA). The membranes were developed with Western Lighting Chemiluminescence Reagent Plus (NEL 104) (Perkin-Elmer Life Sciences, Boston, MA, USA) for 1 min. Images were registered using Kodak imaging station, and Kodak image analysis software (Carestream Health, Rochester, NY, USA) was used to determine net intensities of identified bands. 2.5. Statistical analysis Normal distribution of all data was found after a log 10 transformation. An analysis of variance with post hoc Dunnett t test was performed to compare the outcomes between the various treatments compared to control and the
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effect of incubation time using SPSS 13.0 for MS Windows (SPSS, Chicago, IL, USA). Significance was based on a 95% confidence interval and a p value of b.05. 3. Results 3.1. TUNEL TUNEL images from representative experiments with HEECs are shown in Fig. 1. The green fluorescence indicates apoptosis. DNase I recombinant 10 mcg/mL and 1.5 mM Staurosporine were used as positive controls and resulted in apoptosis (Fig. 1B), compared with the negative control (Fig. 1A). A difference in apoptosis by TUNEL was found between control (Fig. 1C) and MPA treatment (Fig. 1D) after 24 h. The incidence of TUNEL-positive cells was between 4% and 9% after 12 h incubation with all treatments, and not different from control (data not shown). TUNEL incidence was 7.5% in the control group after 24-h incubation (Fig. 2) with the range of TUNEL-positive cells in the progesterone and LNG groups of 6.3%-11.5% and 7.6%-12.4%, respectively, not different from control. TUNEL-positivity was significantly increased only with MPA 500 ng/mL (25.8%) treatment for 24 h when compared with control (Fig. 2).
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Significant increases in TUNEL-positive cells were found with progesterone 500 ng/mL (27.5%), LNG 250 and 500 ng/mL (29.7% and 40%, respectively) and all concentrations of MPA (range 24.2–49.9%) after 48 h compared to control (12.1%) (Fig. 2). 3.2. Bax expression Bax expression (proapoptotic) was similar between groups and comparable to control after 12 h of incubation (data not shown) and was increased significantly with LNG 500 ng/mL and all concentrations of MPA at 24 h (Fig. 3). Progesterone and the two lowest concentrations of LNG had no effect on Bax expression after 24 h (Fig. 3). Increased Bax expression at 48 h was found with LNG 500 ng/mL and all concentrations of MPA, but not with progesterone and the two lower concentrations of LNG (Fig. 3). 3.3. Bcl-2 expression Bcl-2 expression (antiapoptotic) after 12 h was not different between treatments (data not shown). Bcl-2 expression increased with progesterone 5 ng/mL at 24 h but was not different from control. Bcl-2 decreased significantly compared to control values after 24 h with LNG 250 ng/mL, 500 ng/mL and all concentrations of MPA but not with progesterone and the lowest concentration of
Fig. 1. TUNEL assays images from representative experiments. The green fluorescence indicates apoptosis (arrows). The red fluorescence demonstrated cell morphology. (A) The negative control, which was not stained with TUNEL solution. (B) Apoptosis induced by DNase I recombinant/staurosporine. HEECs cultured in serum free medium and incubated for 24 h are: control (C) and MPA 250 ng/mL (D), respectively.
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Fig. 2. TUNEL results after 24 h incubation found a significant increase in the incidence of apoptotic cells after treatment with MPA 500 ng/mL only. TUNEL at 48 h detected significant increases in the incidence of apoptotic cells with progesterone 500 ng/mL, LNG 250 and 500 ng/mL and all concentrations of MPA. *, +pb.001 when compared between control and treatment at 24 h, respectively.
Fig. 3. Bax expression by Western blot at 24 h was significantly increased after treatment with 500 ng/mL LNG and all concentrations of MPA. Bax expression at 48 h was significantly increased by 500 ng/mL LNG and all concentrations of MPA. Progesterone at all concentrations had no effect on Bax expression. *,+pb.05 when compared between control and treatment at 12 and 24 h, respectively.
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Fig. 4. Bcl-2 expression at 24 h was significantly decreased after treatment with LNG 250, 500 ng/mL and all concentrations of MPA using Western blot. Bcl-2 expression at 48 h decreased significantly when incubated with 250 and 500 ng/mL LNG and 250 and 500 ng/mL MPA. Progesterone had no significant effect on Bcl-2 expression. *,+pb.05 when compared between control and treatment at 12 and 24 h, respectively.
LNG (Fig. 4). Bcl-2 decreased significantly with LNG and MPA concentrations of 250 and 500 ng/ml after 48 h (Fig. 4).
4. Discussion Women using progestogen-only contraception such as LNG-SI and MPA injections [DepoProvera (DMPA); Pfizer, New York, NY, USA) experience irregular, unpredictable S/B [11,12]. A dysfunction in endometrial capillary integrity has been postulated as a cause of the S/B and has been termed “vessel fragility” based on visualizing overt spurts of bleeding associated with changes in intrauterine pressure [4,13,14]. Our hypothesis was that progestational agents could have a direct effect on the endometrial endothelial cell, altering its integrity. There is no compelling evidence for the progesterone receptor (PR) in human or nonhuman primate endometrial endothelial cells [15–17]. Both the glucocorticoid receptor (GR) and androgen receptor are present in endothelial cells [18–21]. One article reports finding PR using real time polymerase chain reaction that increased the mRNA levels [15]. This report did not find any significant PR after five passages of the HEECs, and the authors were unable to detect PR in the endometrial endothelial cells in tissue sections [15].
Despite this, we have indirect evidence that LNG did induce apoptotic changes using TUNEL after 24 and 48 h and that the ratio of Bax to Bcl-2 was altered only with LNG 500 ng/mL after 24 and 48 h. Serum concentrations of LNG after LNG implants, and the LNG intrauterine system (LNG-IUS) are 0.4–1.6 and 0.2 ng/mL, respectively [22–24]. The LNGIUS has concentrations of LNG in myometrial, fallopian tube and fat tissue of between 1 and 5 ng/gm of wet tissue weight [25]. Thus, the serum and tissue levels in contraceptors using LNG implants and IUS are significantly lower that the concentrations used in our current experiments. The relative ratio of Bcl-2 to Bax may be important in determining the result of the multiple stimuli that initiate apoptosis but do not provide an mechanism for apoptosis [26]. A previous report found no evidence of endometrial epithelial, glandular or stromal cell immunostaining for Bcl2, Fas and Caspase-3 at 3 and 12 months following insertion of a LNG-SI [27]. This investigation did not identify any apoptotic markers in the endothelial cell although they were specifically sought [27]. The possible mechanism resulting in apoptosis in the presence of high concentrations of LNG in vitro is unknown. Our current data suggest that the effect if any of LNG on vascular permeability and fragility in progestinonly contraceptives is probably secondary to mechanisms other than endothelial cell apoptosis [6,9,14,27,28].
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MPA had a significant apoptotic effect in HEECs in this study in both a concentration and time dependent manner. MPA increased TUNEL and the expression of Bax, a proapoptotic protein, and decreased Bcl-2, an anti-apoptosic protein. MPA peak plasma concentrations of 1–7 ng/mL are found at 3 weeks after an injection of DMPA [29–31]. We feel that the lowest MPA concentration used in our experiments is equivalent to the peripheral plasma concentrations found following injection of DMPA. MPA has been shown to exert its effect on apoptosis via the GR in human umbilical vein endothelial cells (HUVEC) in vitro [32,33]. We have evidence that GR is present in the HEECs used in these experiments (unpublished observations). MPA is both angiostatic and apoptotic in a variety of cancer cells [34–37]. MPA alters both Bcl-2 and Bax in the decidualized endometrium of rodents leading to tissue remodeling [38]. We believe that MPA could cause endothelial cell apoptosis via the GR. Progesterone 500 ng/mL after 48 h incubation resulted in increased TUNEL positivity. Progesterone had no effect on Bax and Bcl-2 at any concentration or time. Progesterone at concentrations of 0.05–1 μM inhibited caspase-3, an apoptosis-related cysteine peptidase in HUVEC in vitro, but this could have been mediated through a non-genomic pathway [39], since there is no evidence for PR in endometrial endothelial cells in either the human or nonhuman primate [15,16]. Progesterone was not found to induce consistent and significant apoptotic changes in HEECs in our study. Progesterone given as a 200 mg intravaginal suppository reduced TUNEL, Bax protein and increased Bcl-2 in the endometrial epithelium of normal women, without evidence of apoptosis in endometrial blood vessels [40]. Our data do not support progesterone as affecting endothelial cell apoptosis. 5. Conclusion Our current findings demonstrate changes in apoptotic markers in HEECs following incubation with MPA, which we believe is mediated via the GR. This finding may have clinical relevance by contributing to an increased vascular fragility in users of DMPA. We believe that LNG-related S/B is due to mechanism(s) other than apoptosis of endothelial cells, and we have no evidence that progesterone induces apoptosis in endometrial endothelial cells. References [1] Fraser IS, Hickey M. Endometrial vascular changes and bleeding disturbances with long-acting progestins. Steroids 2000;65:665–70. [2] Hickey M, Crewe J, Mahoney LA, Doherty DA, Fraser IS, Salamonsen LA. Mechanisms of irregular bleeding with hormone therapy: the role of matrix metalloproteinases and their tissue inhibitors. J Clin Endocrinol Metab 2006;91:3189–98. [3] Hickey M. Bleeding with menopausal hormone therapy: physiological or pathological? Menopause Int 2007;13:188–90.
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