Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Long-term progestin contraceptives (LTPOC) induce aberrant angiogenesis, oxidative stress and apoptosis in the guinea pig uterus: A model for abnormal uterine bleeding in humans

Long-term progestin contraceptives (LTPOC) induce aberrant angiogenesis, oxidative stress and... Background: Irregular uterine bleeding is the major side effect of, and cause for, discontinuation of long-term progestin-only contraceptives (LTPOCs). The endometria of LTPOC-treated women display abnormally enlarged, fragile blood vessels (BV ), decreased endometrial blood flow and oxidative stress. However, obtaining sufficient, good quality tissues have precluded elucidation of the mechanisms underlying these morphological and functional vascular changes. Methods: The current study assessed the suitability of the guinea pig (GP) as a model for evaluating the uterine effects of LTPOC administration. Thus GPs were treated with a transdermal pellet for 21 days and examined for endometrial histology, angiogenic markers as well as markers of oxidative stress and apoptosis. Results and Discussion: We now demonstrate that GP uteri were enlarged by both estradiol (E2) and medroxyprogesterone acetate (MPA) (p < 0.001). Effects of MPA on uterine weight differed significantly depending on E2 levels (p < 0.001), where MPA opposed the E2 effect in combined treatments. Angiogenesis parameters were similarly impacted upon: MPA alone increased BV density (p = 0.036) and BV average area (p = 0.002). The presence of E2 significantly decreased these parameters. These changes were associated with highly elevated of the lipid peroxidation product, 8-isoprostane (8-isoP) content in E2+MPA-treated and by nuclear 8-OH-deoxyguanosine (8oxoG) staining compared to all other groups (p < 0.001). Abnormalities in the E2+MPA group were consistent with chromatin redistribution, nuclear pyknosis, karyolysis and increased apoptosis as observed by a marked increase in TUNEL labeling. Conclusions: LTPOC exposure alters endometrial vascular and tissue morphology consistent with oxidative stress and apoptosis in a complex interplay with endogenous estrogens. These findings are remarkably similar to in vivo change observed in the human uterus following LTPOC administration. Hence, the GP is an excellent model for the study of LTPOC effects on the uterus and will be extremely useful in determining the mechanistic pathways involved in this process which cannot be conducted on humans. Introduction ine bleeding in the majority of users [1,2]. Such bleeding Because of their safety and efficacy, long-term progestin- disturbances are the primary indication for discontinua- only contraceptives (LTPOCs) are well-suited for women tion of therapy[1,2]. with restricted access to health care or in whom estrogen Endometria from LTPOC-treated patients display containing contraceptives are contraindicated. Unfortu- dilated, thin walled, fragile vessels that are irregularly dis- nately, administration of LTPOCs leads to irregular uter- tributed across the endometrial surface [3-5]. Previous studies from our laboratory [6,7] as well as others [8-14] * Correspondence: graciela.krikun@yale.edu demonstrated that LTPOC therapy produced a statisti- Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University School of Medicine, New Haven, CT, 06510, USA cally significant increase in mean lumen diameter of Full list of author information is available at the end of the article © 2010 Krikun et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in BioMed Central any medium, provided the original work is properly cited. Krikun et al. Journal of Angiogenesis Research 2010, 2:8 Page 2 of 7 http://www.jangiogenesis.com/content/2/1/8 microvessels at bleeding versus non-bleeding sites [7] and Immunohistochemistry that the key regulators of endometrial angiogenesis, vas- Sections were stained for von Willebrand factor (vWF) cular endothelial growth factor (VEGF) and angiopoie- with the AB6994 primary antibody (Abcam, Cambridge, tin-2 (Ang-2) were up-regulated in endometria treated MA) at 1-10,000 dilution or 8-oxoG with the x 24326 pri- with LTPOC[6,9]. Moreover, we demonstrated that mary antibody at 1-100 (Oxis International, Foster City, hypoxia and reactive oxygen species (ROS) induced aber- CA). For negative controls, normal IgG isotypes which rant angiogenesis by reducing endometrial blood flow, were derived from the animals from which the antibodies inducing hypoxia, decreasing the ratio of the angiostatic were prepared and used at the same concentrations as the agent, angiopoietin-1 to the angiogenic factor, angiopoie- primary antibody. The sections were washed and the tin-2 [3,5,6]. appropriate secondary biotinylated antibody (Vector While past studies produced descriptive information Laboratories, Inc., Burlingame, CA, USA) was added per regarding the possible causes of abnormal uterine bleed- the manufacturer's instructions. ing following LTPOC treatment, considerations of diffi- The antigen-antibody complex was detected with 3,3'- culty in attaining good quality tissues from humans diaminobenzidine with or without nickel sulfate as the preclude functional studies of the mechanistic pathways chromogen solution (Vector Laboratories). When nickel involved in this process. Poor understanding of the mech- sulfate was used, no hematoxylin counterstaining was anisms underlying bleeding has limited the development conducted. For each condition, 3 different slides were of effective therapies for abnormal bleeding with LTPOC. assessed and at least three independent areas of each To further understand these mechanisms, we determined slide photographed. For vWF, 6 randomly selected fields whether the guinea pig (GP) was a relevant model to were digitally captured at 200× magnification using an study the uterine effects of LTPOC administration. The Olympus microscope with digital camera. Vessel size, GP was chosen because its endometria display functional density and heterogeneity were measured in each field by estrogen and progesterone receptors [15] as well as other computerized selection of the stained vessels with the properties closely related to the humans including spon- public domain image analysis software Image J as previ- taneous estrous cycling and hemochorial placentation ously described by others [21,22]. All steps including field [16-19]. In order to elucidate mechanisms underlying acquisition and vessel morphology measurement were LTPOC-induced abnormal uterine bleeding, we evalu- performed blinded. For the other endpoints immunos- ated the separate and interactive effects of estrogen and taining intensities were ascertained with Image-J. progestin on GP-endometrial weight, vascular morphol- Lipid peroxidation profile ogy, oxidative stress and apoptosis. The isoprostanes are a family of eicosanoids of non-enzy- matic origin produced by the random oxidation of tissue Materials and methods phospholipids by oxygen radicals [5]. The levels of 8-IsoP Guinea pigs were measured in all 4 treatment groups on the frozen Eighteen nulliparous female GPs, aged 2-6 months, were uterine horn obtained as described above. The samples subjected to bilateral oopherectomy and then given sub- were sonicated in 0.1 M Tris (pH 7.4) and diluted 1:5 in cutaneous implants of 50 mg medroxyprogesterone ace- eicosanoid affinity buffer (Cayman Chemical Company, tate (MPA)-cholesterol-based 21 day time-release pellets Ann Arbor, MI) and 8-isoP was detected by ELISA as we or 5 mg estradiol (E2) cholesterol based 21 day time- previously described [5]. The sensitivity of this ELISA is release pellets (Innovative Research of America; Sarasota, 10 pg/ml. Limit of detection: 80% B/B0: 2.7 pg/ml FL) or both. Thus, animals received the treatment as fol- lows: MPA (n = 6), E2 (n = 6), E2+MPA (n = 3) or placebo Apoptosis (n = 3). After three weeks, hysterectomy was performed Assessment of apoptosis was conducted on formalin- and the right uterine horn was formalin fixed whereas the fixed, paraffin-embedded tissues with ApoTag peroxidase left horn was snap frozen for subsequent studies. These labeling kit (Chemicon International, Temecula, CA) as studies were approved by both Charles River (Wilming- per the manufacturer's instructions. The procedure is ton, MA) and Yale University IACUC offices. based on detection of free 3'OH DNA termini in situ. To assess differences in apoptotic levels, photographs were Histology taken from 3 representative areas of each slide at x200 The specimens were weighed and then formalin fixed, magnitude under identical camera settings. The slides and paraffin embedded. Five micron sections were cut were analyzed with Image J by setting the minimum and stained with Hematoxylin-Eosin or Trichrome threshold that allowed for the visualization of the stained Mason (Sigma-Aldrich, St. Louis, MO) by conventional nuclei only. This threshold was maintained throughout histological procedures as described [20] or used for the analysis of all subsequent slides. All values were sub- immunohistochemistry as illustrated below. Krikun et al. Journal of Angiogenesis Research 2010, 2:8 Page 3 of 7 http://www.jangiogenesis.com/content/2/1/8 jected to analysis with Sigma Stat (Systat Software Inc, els, where MPA opposed the E2 effect in combined treat- Chicago, ILL) utilizing the recommended ANOVA pro- ments. vided by the program based on sample distribution. The Histological analysis of the samples was conducted mean particle stained average +/- standard error of the after fixation and tissues were cut at 5 μ. Samples were mean were then represented by bar graphs. stained with H&E or Mason Trichrome (Figure 2). Sub- endometrial edema is observed in MPA-treated animals. Statistical Analysis Statistical analysis was conducted by ANOVA using the Angiogenic parameters Sigma Stat Program (SPSS Inc., Chicago, IL). Figure 3 displays staining for the endothelial marker, vWF in endometria treated with placebo, E2, MPA or E2+MPA. Based on that staining, the following endpoints Results Morphology were analyzed as follows: Blood vessel density (BVD) = Figure 1 demonstrates that E2 (p < 0.001), MPA (p < 0.02) area occupied by blood vessel lumen/area analyzed × 100. and E2+MPA (p < 0.05) all increase uterine wet weight Blood vessel size (BVS) = average blood vessel diameter compared with controls. The greatest effects were for the field analyzed (microns) and Blood vessel hetero- observed with E2 treatment, consistent with the prolifer- geneity (BVH) = relative SD of blood vessel area for the ative effects seen in human endometrial following estro- field analyzed (SD/average area × 100). gen-only treatment. As expected, the effects of MPA on Thus, angiogenic parameters were impacted upon as uterine weight differed significantly depending on E2 lev- follows: MPA alone increased BV density (p = 0.036) and Figure 1 Gross morphological analysis. A) Transverse and longitudinal sections of GP uteri following treatment with placebo, E2, MPA or E2+MPA as described in Methods. B) Bar graph represents the mean +/- standard error of the mean for 6 experiments as determined by ANOVA followed by Student-Newman-Keuls test. * p < 0.01, ** p = 0.25 Krikun et al. Journal of Angiogenesis Research 2010, 2:8 Page 4 of 7 http://www.jangiogenesis.com/content/2/1/8 BV average area (p = 0.002). The presence of E2 signifi- cantly decreased these parameters (BV density mean SEM: CRL: 9.4 1.0%, E2: 10.3 1.6%, MPA: 13.6 1.1%, E2+MPA: 6.0 0.7%, p = 0.002). Oxidative stress Levels of 8-isoprostane (8-IsoP) production were evalu- ated in endometrial extracts obtained from uteri treated with the various steroids. Figure 4 demonstrates that an eight-fold elevation in 8-IsoP levels occurred in uteri derived from E2+MPA-treated animals compared to all other groups (p < 0.001). Figure 5 displays immunohis- Figure 2 Histological analysis. Samples were stained with H&E or Ma- tochemical staining for 7,8-dihydro-8-oxoguanine (8- son Trichrome as described in Methods. Controls (CRL: Panels a-c). Sub- oxoG) which reflects ROS damage to DNA [23]. It is endometrial edema is observed in MPA-treated animals (Panels d-f). important to note however, that this endpoint alone is not conclusive of oxidative damage but also necrosis. That is Figure 3 Vascular morphology: (Top) vWF staining was conducted on formalin fixed, paraffin embedded tissues for placebo. Bar graphs rep- resents the mean +/- standard error of the mean for placebo control (CTL), E2, MPA or E2+MPA. A) Average blood vessel density, B) blood vessel size and C) blood vessel area heterogeneity. Statistical analysis were conducted by a two way ANOVA followed by Student-Newman-Keuls test for the 4 treatment groups. (Bottom) From left to right, representative IHC of vWF for CTL, E2, MPA and E2+MPA (20×). Krikun et al. Journal of Angiogenesis Research 2010, 2:8 Page 5 of 7 http://www.jangiogenesis.com/content/2/1/8 Apoptosis In humans, LTPOC treatment results in enhanced apop- tosis of the endometrial glands and stroma [24]. Figure 6 demonstrates a similar apoptotic profile of E2+MPA group as reflected by increased TUNEL labeling. By con- trast, MPA did not displayed statistical differences in TUNEL labeling compared to E2 or placebo. Discussion Endometria from LTPOC-treated patients display dilated vessels that are irregularly distributed across the endome- Figure 4 Lipid peroxidation profile. The levels of 8-IsoP were mea- trial surface [3-5]. These dilated vessels bleed on minimal sured in all 4 treatment groups as described in Methods. Bar graph rep- pressure and have deficient vascular basement membrane resents the mean ELISA values +/- standard error of the mean. Statistical analysis were conducted by one way ANOVA with Student- components [25,26] In the present studies we examined Newman-Keuls post hoc test (n = 6, *p < 0.001) the effects of hormone treatment on a guinea pig LTPOC model. Because they undergo estrous cycling, the guinea pig more closely emulates the reproductive system of why the study includes one of the best endpoints for oxi- humans [27,28] compared to other rodents. dative stress which is 8-isoP (see above) These studies demonstrate that treatment of guinea Expression of 8-oxoG was significantly higher in pigs with progestin and in particular with E2+MPA E2+MPA treated groups compared to E2 alone. A signifi- resulted in changes in endometrial vascular morphology, cant, though diminished effect was observed with MPA as well as increased markers of apoptosis and oxidative alone. However, no statistical differences were observed stress similar to that observed in human LTPOC-treated between E2 and the placebo control. endometria. In humans, LTPOC treatment occurs in the Figure 5 DNA oxidation. The levels of 8-oxoG were measured in all 4 treatment groups as described in Methods. (Top) Bar graph represents the av- erage staining intensity/total field studied +/- standard error of the mean. Staining intensity was analyzed by Image J as previously described [21,22]. Statistical analysis were conducted by one way ANOVA with Student-Newman-Keuls post hoc test (n = 6, *p < 0.001 compared to E2). (Bottom) From left to right, representative IHC of 8oxoG for E2, MPA and E2+MPA (20×). Krikun et al. Journal of Angiogenesis Research 2010, 2:8 Page 6 of 7 http://www.jangiogenesis.com/content/2/1/8 Figure 6 Apoptosis: The apoptotic index was established for all 4 treatment groups after staining nuclei with Apotag as described in Meth- ods. The bar graph represents the average staining intensity/total field studied +/- standard error of the mean following analysis with Image J. Statis- tical analysis were conducted by one way ANOVA with Student-Newman-Keuls post hoc test (n = 3,*p < 0.05). setting of continuous low-level ovarian-derived estrogen gleaned from the experimental results presented in this production. Since the GPs used in the current study are study are expected to ultimately improve the formula- ovariectomized, the E2+MPA treated animals are likely to tions and acceptability of LTPOC therapies by reducing most closely resemble LTPOC-treated humans. aberrant angiogenesis and related irregular, unpredict- In prior studies, we have demonstrated that LTPOC able bleeding. results in both reduced endometrial blood flow and Competing interests increased oxidative stress [5,6]. These findings are associ- The authors declare that they have no competing interests. ated with immunohistochemical evidence of increased Authors' contributions angiogenic factor production. Further, hypoxia and oxi- GK and CL conceived and designed the experiments and wrote the manu- dative stress induce increased production of vascular script. IB, FS and LB carried out the ELISAs and immunohistochemical proce- endothelial growth factor (VEGF) and reduced produc- dures and statistical analysis. MH contributed in the critical analysis of the paper. tion of the angiostatic agent, angiopoietin-1 in cultured human endometrial stromal cells [4,6,9]. Hypoxia and Acknowledgements oxidative stress also greatly enhance production of the This work was supported by an NIH grant RO1HD33937 (CJL) highly angiogenic molecule, angiopoietin-2, in cultured Author Details endometrial endothelial cells [29]. However, it is unclear Department of Obstetrics, Gynecology and Reproductive Sciences, Yale how LTPOCs exert the initial vasoconstrictive effects on 2 University School of Medicine, New Haven, CT, 06510, USA and School of Women's and Infants' Health, University of Western Australia, Subiaco, WA human endometrium. The availability of this animal 6008, Australia model should allow dissection of the underlying mecha- nism driving this vascular phenomenon. Information Received: 12 March 2010 Accepted: 27 April 2010 Published: 27 April 2010 Krikun et al. Journal of Angiogenesis Research 2010, 2:8 Page 7 of 7 http://www.jangiogenesis.com/content/2/1/8 T © T Jo h h 2 u i is s 0 r a na i1 sr 0 an t l o K icl r f O e i A ikp u n se n a gn i et v o a A g a icce e la n l;b l es i ls c e f s is e arti R n ro s em e scle e e:a B h r d c ito t h istri p Med 2010 ://bu w C w te , e 2 w d n :8 . u t jr aa n n ld Lt ge io r th d g . en e te esirm s.cs o om f/ th co e n C te re na t/ ti 2ve /1 C /8ommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 22. Noursadeghi M, Tsang J, Haustein T, Miller RF, Chain BM, Katz DR: Quantitative imaging assay for NF-kappaB nuclear translocation in References primary human macrophages. J Immunol Methods 2008, 329:194-200. 1. Affandi B: An integrated analysis of vaginal bleeding patterns in clinical 23. Thorslund T, Sunesen M, Bohr VA, Stevnsner T: Repair of 8-oxoG is slower trials of Implanon. Contraception 1998, 58:99S-107S. in endogenous nuclear genes than in mitochondrial DNA and is 2. Collins J, Crosignani PG: Hormonal contraception without estrogens. without strand bias. DNA Repair (Amst) 2002, 1:261-273. Hum Reprod Update 2003, 9:373-386. 24. Maruo T, Laoag-Fernandez JB, Pakarinen P, Murakoshi H, Spitz IM, 3. Krikun G, Critchley H, Schatz F, Wan L, Caze R, Baergen RN, Lockwood CJ: Johansson E: Effects of the levonorgestrel-releasing intrauterine system Abnormal uterine bleeding during progestin-only contraception may on proliferation and apoptosis in the endometrium. Hum Reprod 2001, result from free radical-induced alterations in angiopoietin expression. 16:2103-2108. Am J Pathol 2002, 161:979-986. 25. Hickey M, Dwarte D, Fraser IS: Superficial endometrial vascular fragility 4. Hague S, MacKenzie IZ, Bicknell R, Rees MC: In-vivo angiogenesis and in Norplant users and in women with ovulatory dysfunctional uterine progestogens. Hum Reprod 2002, 17:786-793. bleeding. Human Reproduction 2000, 15:1509-1514. 5. Hickey M, Krikun G, Kodaman P, Schatz F, Carati C, Lockwood CJ: Long- 26. Hickey M, Simbar M, Markham R, Young L, Manconi F, Russell P, Fraser IS: term progestin-only contraceptives result in reduced endometrial Changes in vascular basement membrane in the endometrium of blood flow and oxidative stress. J Clin Endocrinol Metab 2006, Norplant users. Hum Reprod 1999, 14:716-721. 91:3633-3638. 27. Quandt LM, Hutz RJ: Induction by estradiol-17 beta of polycystic ovaries 6. Lockwood CJ, Schatz F, Krikun G: Angiogenic factors and the in the guinea pig. Biol Reprod 1993, 48:1088-1094. endometrium following long term progestin only contraception. 28. Mularoni A, Mahfoudi A, Beck L, Coosemans V, Bride J, Nicollier M, Adessi Histol Histopathol 2004, 19:167-172. GL: Progesterone control of fibronectin secretion in guinea pig 7. Runic R, Schatz F, Krey L, Demopoulos R, Thung S, Wan L, Lockwood CJ: endometrium. Endocrinology 1992, 131:2127-2132. Alterations in endometrial stromal cell tissue factor protein and 29. Krikun G, Schatz F, Finlay T, Kadner S, Mesia A, Gerrets R, Lockwood CJ: messenger ribonucleic acid expression in patients experiencing Expression of angiopoietin-2 by human endometrial endothelial cells: abnormal uterine bleeding while using Norplant-2 contraception. J regulation by hypoxia and inflammation. Biochem Biophys Res Commun Clin Endocrinol Metab 1997, 82:1983-1988. 2000, 275:159-163. 8. Hickey M, Simbar M, Young L, Markham R, Russell P, Fraser IS: A longitudinal study of changes in endometrial microvascular density in Norplant implant users. Contraception 1999, 59:123-129. doi: 10.1186/2040-2384-2-8 9. Charnock-Jones DS, Macpherson AM, Archer DF, Leslie S, Makkink WK, Cite this article as: Krikun et al., Long-term progestin contraceptives Sharkey AM, Smith SK: The effect of progestins on vascular endothelial (LTPOC) induce aberrant angiogenesis, oxidative stress and apoptosis in the guinea pig uterus: A model for abnormal uterine bleeding in humans Journal growth factor, oestrogen receptor and progesterone receptor of Angiogenesis Research 2010, 2:8 immunoreactivity and endothelial cell density in human endometrium. Hum Reprod 2000, 15(Suppl 3):85-95. 10. Shaw ST Jr, Macaulay LK, Aznar R, Gonzalez-Angulo A, Roy S: Effects of a progesterone-releasing intrauterine contraceptive device on endometrial blood vessels: a morphometric study. Am J Obstet Gynecol 1981, 141:821-827. 11. Guttinger A, Critchley HOD: Endometrial effects of intrauterine levonorgestrel. Contraception 2007, 75(6 Suppl):S93-98. 12. Hickey M, Fraser IS: The structure of endometrial microvessels. Hum Reprod 2000, 15(Suppl 3):57-66. 13. Palmer JA, Lau TM, Hickey M, Simbah M, Rogers PA: Immunohistochemical study of endometrial microvascular basement membrane components in women using Norplant. Hum Reprod 1996, 11:2142-2150. 14. Subakir SB, Hadisaputra W, Siregar B, Irawati D, Santoso DI, Cornain S, Affandi B: Reduced endothelial cell migratory signal production by endometrial explants from women using Norplant contraception. Hum Reprod 1995, 10:2579-2583. 15. Alkhalaf M, Propper AY, Chaminadas G, Adessi GL: Ultrastructural changes in guinea pig endometrial cells during the estrous cycle. J Morphol 1992, 214:83-96. 16. Makker A, Bansode FW, Srivastava VM, Singh MM: Antioxidant defense system during endometrial receptivity in the guinea pig: effect of ormeloxifene, a selective estrogen receptor modulator. J Endocrinol 2006, 188:121-134. 17. Lee KY, DeMayo FJ: Animal models of implantation. Reproduction 2004, 128:679-695. 18. Hutz RJ, Bejvan SM, Durning M, Dierschke DJ: Changes in follicular populations, in serum estrogen and progesterone, and in ovarian steroid secretion in vitro during the guinea pig estrous cycle. Biol Reprod 1990, 42:266-272. 19. Carter AM: Animal models of human placentation--a review. Placenta 2007, 28(Suppl A):S41-47. 20. Noci I, Borri P, Chieffi O, Scarselli G, Biagiotti R, Moncini D, Paglierani M, Taddei G: I. Aging of the human endometrium: a basic morphological and immunohistochemical study. Eur J Obstet Gynecol Reprod Biol 1995, 63:181-185. 21. Marcilhac A, Raynaud F, Clerc I, Benyamin Y: Detection and localization of calpain 3-like protease in a neuronal cell line: possible regulation of apoptotic cell death through degradation of nuclear IkappaBalpha. Int J Biochem Cell Biol 2006, 38:2128-2140. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Angiogenesis Research Springer Journals

Long-term progestin contraceptives (LTPOC) induce aberrant angiogenesis, oxidative stress and apoptosis in the guinea pig uterus: A model for abnormal uterine bleeding in humans

Download PDF
7 pages

Loading next page...
 
/lp/springer-journals/long-term-progestin-contraceptives-ltpoc-induce-aberrant-angiogenesis-iSFkQVjq0m

References (58)

Publisher
Springer Journals
Copyright
Copyright © 2010 by Krikun et al; licensee BioMed Central Ltd.
Subject
Medicine & Public Health; Angiology; Cardiology; Cancer Research; Cell Biology; Developmental Biology
ISSN
2040-2384
eISSN
2040-2384
DOI
10.1186/2040-2384-2-8
pmid
20423489
Publisher site
See Article on Publisher Site

Abstract

Background: Irregular uterine bleeding is the major side effect of, and cause for, discontinuation of long-term progestin-only contraceptives (LTPOCs). The endometria of LTPOC-treated women display abnormally enlarged, fragile blood vessels (BV ), decreased endometrial blood flow and oxidative stress. However, obtaining sufficient, good quality tissues have precluded elucidation of the mechanisms underlying these morphological and functional vascular changes. Methods: The current study assessed the suitability of the guinea pig (GP) as a model for evaluating the uterine effects of LTPOC administration. Thus GPs were treated with a transdermal pellet for 21 days and examined for endometrial histology, angiogenic markers as well as markers of oxidative stress and apoptosis. Results and Discussion: We now demonstrate that GP uteri were enlarged by both estradiol (E2) and medroxyprogesterone acetate (MPA) (p < 0.001). Effects of MPA on uterine weight differed significantly depending on E2 levels (p < 0.001), where MPA opposed the E2 effect in combined treatments. Angiogenesis parameters were similarly impacted upon: MPA alone increased BV density (p = 0.036) and BV average area (p = 0.002). The presence of E2 significantly decreased these parameters. These changes were associated with highly elevated of the lipid peroxidation product, 8-isoprostane (8-isoP) content in E2+MPA-treated and by nuclear 8-OH-deoxyguanosine (8oxoG) staining compared to all other groups (p < 0.001). Abnormalities in the E2+MPA group were consistent with chromatin redistribution, nuclear pyknosis, karyolysis and increased apoptosis as observed by a marked increase in TUNEL labeling. Conclusions: LTPOC exposure alters endometrial vascular and tissue morphology consistent with oxidative stress and apoptosis in a complex interplay with endogenous estrogens. These findings are remarkably similar to in vivo change observed in the human uterus following LTPOC administration. Hence, the GP is an excellent model for the study of LTPOC effects on the uterus and will be extremely useful in determining the mechanistic pathways involved in this process which cannot be conducted on humans. Introduction ine bleeding in the majority of users [1,2]. Such bleeding Because of their safety and efficacy, long-term progestin- disturbances are the primary indication for discontinua- only contraceptives (LTPOCs) are well-suited for women tion of therapy[1,2]. with restricted access to health care or in whom estrogen Endometria from LTPOC-treated patients display containing contraceptives are contraindicated. Unfortu- dilated, thin walled, fragile vessels that are irregularly dis- nately, administration of LTPOCs leads to irregular uter- tributed across the endometrial surface [3-5]. Previous studies from our laboratory [6,7] as well as others [8-14] * Correspondence: graciela.krikun@yale.edu demonstrated that LTPOC therapy produced a statisti- Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University School of Medicine, New Haven, CT, 06510, USA cally significant increase in mean lumen diameter of Full list of author information is available at the end of the article © 2010 Krikun et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in BioMed Central any medium, provided the original work is properly cited. Krikun et al. Journal of Angiogenesis Research 2010, 2:8 Page 2 of 7 http://www.jangiogenesis.com/content/2/1/8 microvessels at bleeding versus non-bleeding sites [7] and Immunohistochemistry that the key regulators of endometrial angiogenesis, vas- Sections were stained for von Willebrand factor (vWF) cular endothelial growth factor (VEGF) and angiopoie- with the AB6994 primary antibody (Abcam, Cambridge, tin-2 (Ang-2) were up-regulated in endometria treated MA) at 1-10,000 dilution or 8-oxoG with the x 24326 pri- with LTPOC[6,9]. Moreover, we demonstrated that mary antibody at 1-100 (Oxis International, Foster City, hypoxia and reactive oxygen species (ROS) induced aber- CA). For negative controls, normal IgG isotypes which rant angiogenesis by reducing endometrial blood flow, were derived from the animals from which the antibodies inducing hypoxia, decreasing the ratio of the angiostatic were prepared and used at the same concentrations as the agent, angiopoietin-1 to the angiogenic factor, angiopoie- primary antibody. The sections were washed and the tin-2 [3,5,6]. appropriate secondary biotinylated antibody (Vector While past studies produced descriptive information Laboratories, Inc., Burlingame, CA, USA) was added per regarding the possible causes of abnormal uterine bleed- the manufacturer's instructions. ing following LTPOC treatment, considerations of diffi- The antigen-antibody complex was detected with 3,3'- culty in attaining good quality tissues from humans diaminobenzidine with or without nickel sulfate as the preclude functional studies of the mechanistic pathways chromogen solution (Vector Laboratories). When nickel involved in this process. Poor understanding of the mech- sulfate was used, no hematoxylin counterstaining was anisms underlying bleeding has limited the development conducted. For each condition, 3 different slides were of effective therapies for abnormal bleeding with LTPOC. assessed and at least three independent areas of each To further understand these mechanisms, we determined slide photographed. For vWF, 6 randomly selected fields whether the guinea pig (GP) was a relevant model to were digitally captured at 200× magnification using an study the uterine effects of LTPOC administration. The Olympus microscope with digital camera. Vessel size, GP was chosen because its endometria display functional density and heterogeneity were measured in each field by estrogen and progesterone receptors [15] as well as other computerized selection of the stained vessels with the properties closely related to the humans including spon- public domain image analysis software Image J as previ- taneous estrous cycling and hemochorial placentation ously described by others [21,22]. All steps including field [16-19]. In order to elucidate mechanisms underlying acquisition and vessel morphology measurement were LTPOC-induced abnormal uterine bleeding, we evalu- performed blinded. For the other endpoints immunos- ated the separate and interactive effects of estrogen and taining intensities were ascertained with Image-J. progestin on GP-endometrial weight, vascular morphol- Lipid peroxidation profile ogy, oxidative stress and apoptosis. The isoprostanes are a family of eicosanoids of non-enzy- matic origin produced by the random oxidation of tissue Materials and methods phospholipids by oxygen radicals [5]. The levels of 8-IsoP Guinea pigs were measured in all 4 treatment groups on the frozen Eighteen nulliparous female GPs, aged 2-6 months, were uterine horn obtained as described above. The samples subjected to bilateral oopherectomy and then given sub- were sonicated in 0.1 M Tris (pH 7.4) and diluted 1:5 in cutaneous implants of 50 mg medroxyprogesterone ace- eicosanoid affinity buffer (Cayman Chemical Company, tate (MPA)-cholesterol-based 21 day time-release pellets Ann Arbor, MI) and 8-isoP was detected by ELISA as we or 5 mg estradiol (E2) cholesterol based 21 day time- previously described [5]. The sensitivity of this ELISA is release pellets (Innovative Research of America; Sarasota, 10 pg/ml. Limit of detection: 80% B/B0: 2.7 pg/ml FL) or both. Thus, animals received the treatment as fol- lows: MPA (n = 6), E2 (n = 6), E2+MPA (n = 3) or placebo Apoptosis (n = 3). After three weeks, hysterectomy was performed Assessment of apoptosis was conducted on formalin- and the right uterine horn was formalin fixed whereas the fixed, paraffin-embedded tissues with ApoTag peroxidase left horn was snap frozen for subsequent studies. These labeling kit (Chemicon International, Temecula, CA) as studies were approved by both Charles River (Wilming- per the manufacturer's instructions. The procedure is ton, MA) and Yale University IACUC offices. based on detection of free 3'OH DNA termini in situ. To assess differences in apoptotic levels, photographs were Histology taken from 3 representative areas of each slide at x200 The specimens were weighed and then formalin fixed, magnitude under identical camera settings. The slides and paraffin embedded. Five micron sections were cut were analyzed with Image J by setting the minimum and stained with Hematoxylin-Eosin or Trichrome threshold that allowed for the visualization of the stained Mason (Sigma-Aldrich, St. Louis, MO) by conventional nuclei only. This threshold was maintained throughout histological procedures as described [20] or used for the analysis of all subsequent slides. All values were sub- immunohistochemistry as illustrated below. Krikun et al. Journal of Angiogenesis Research 2010, 2:8 Page 3 of 7 http://www.jangiogenesis.com/content/2/1/8 jected to analysis with Sigma Stat (Systat Software Inc, els, where MPA opposed the E2 effect in combined treat- Chicago, ILL) utilizing the recommended ANOVA pro- ments. vided by the program based on sample distribution. The Histological analysis of the samples was conducted mean particle stained average +/- standard error of the after fixation and tissues were cut at 5 μ. Samples were mean were then represented by bar graphs. stained with H&E or Mason Trichrome (Figure 2). Sub- endometrial edema is observed in MPA-treated animals. Statistical Analysis Statistical analysis was conducted by ANOVA using the Angiogenic parameters Sigma Stat Program (SPSS Inc., Chicago, IL). Figure 3 displays staining for the endothelial marker, vWF in endometria treated with placebo, E2, MPA or E2+MPA. Based on that staining, the following endpoints Results Morphology were analyzed as follows: Blood vessel density (BVD) = Figure 1 demonstrates that E2 (p < 0.001), MPA (p < 0.02) area occupied by blood vessel lumen/area analyzed × 100. and E2+MPA (p < 0.05) all increase uterine wet weight Blood vessel size (BVS) = average blood vessel diameter compared with controls. The greatest effects were for the field analyzed (microns) and Blood vessel hetero- observed with E2 treatment, consistent with the prolifer- geneity (BVH) = relative SD of blood vessel area for the ative effects seen in human endometrial following estro- field analyzed (SD/average area × 100). gen-only treatment. As expected, the effects of MPA on Thus, angiogenic parameters were impacted upon as uterine weight differed significantly depending on E2 lev- follows: MPA alone increased BV density (p = 0.036) and Figure 1 Gross morphological analysis. A) Transverse and longitudinal sections of GP uteri following treatment with placebo, E2, MPA or E2+MPA as described in Methods. B) Bar graph represents the mean +/- standard error of the mean for 6 experiments as determined by ANOVA followed by Student-Newman-Keuls test. * p < 0.01, ** p = 0.25 Krikun et al. Journal of Angiogenesis Research 2010, 2:8 Page 4 of 7 http://www.jangiogenesis.com/content/2/1/8 BV average area (p = 0.002). The presence of E2 signifi- cantly decreased these parameters (BV density mean SEM: CRL: 9.4 1.0%, E2: 10.3 1.6%, MPA: 13.6 1.1%, E2+MPA: 6.0 0.7%, p = 0.002). Oxidative stress Levels of 8-isoprostane (8-IsoP) production were evalu- ated in endometrial extracts obtained from uteri treated with the various steroids. Figure 4 demonstrates that an eight-fold elevation in 8-IsoP levels occurred in uteri derived from E2+MPA-treated animals compared to all other groups (p < 0.001). Figure 5 displays immunohis- Figure 2 Histological analysis. Samples were stained with H&E or Ma- tochemical staining for 7,8-dihydro-8-oxoguanine (8- son Trichrome as described in Methods. Controls (CRL: Panels a-c). Sub- oxoG) which reflects ROS damage to DNA [23]. It is endometrial edema is observed in MPA-treated animals (Panels d-f). important to note however, that this endpoint alone is not conclusive of oxidative damage but also necrosis. That is Figure 3 Vascular morphology: (Top) vWF staining was conducted on formalin fixed, paraffin embedded tissues for placebo. Bar graphs rep- resents the mean +/- standard error of the mean for placebo control (CTL), E2, MPA or E2+MPA. A) Average blood vessel density, B) blood vessel size and C) blood vessel area heterogeneity. Statistical analysis were conducted by a two way ANOVA followed by Student-Newman-Keuls test for the 4 treatment groups. (Bottom) From left to right, representative IHC of vWF for CTL, E2, MPA and E2+MPA (20×). Krikun et al. Journal of Angiogenesis Research 2010, 2:8 Page 5 of 7 http://www.jangiogenesis.com/content/2/1/8 Apoptosis In humans, LTPOC treatment results in enhanced apop- tosis of the endometrial glands and stroma [24]. Figure 6 demonstrates a similar apoptotic profile of E2+MPA group as reflected by increased TUNEL labeling. By con- trast, MPA did not displayed statistical differences in TUNEL labeling compared to E2 or placebo. Discussion Endometria from LTPOC-treated patients display dilated vessels that are irregularly distributed across the endome- Figure 4 Lipid peroxidation profile. The levels of 8-IsoP were mea- trial surface [3-5]. These dilated vessels bleed on minimal sured in all 4 treatment groups as described in Methods. Bar graph rep- pressure and have deficient vascular basement membrane resents the mean ELISA values +/- standard error of the mean. Statistical analysis were conducted by one way ANOVA with Student- components [25,26] In the present studies we examined Newman-Keuls post hoc test (n = 6, *p < 0.001) the effects of hormone treatment on a guinea pig LTPOC model. Because they undergo estrous cycling, the guinea pig more closely emulates the reproductive system of why the study includes one of the best endpoints for oxi- humans [27,28] compared to other rodents. dative stress which is 8-isoP (see above) These studies demonstrate that treatment of guinea Expression of 8-oxoG was significantly higher in pigs with progestin and in particular with E2+MPA E2+MPA treated groups compared to E2 alone. A signifi- resulted in changes in endometrial vascular morphology, cant, though diminished effect was observed with MPA as well as increased markers of apoptosis and oxidative alone. However, no statistical differences were observed stress similar to that observed in human LTPOC-treated between E2 and the placebo control. endometria. In humans, LTPOC treatment occurs in the Figure 5 DNA oxidation. The levels of 8-oxoG were measured in all 4 treatment groups as described in Methods. (Top) Bar graph represents the av- erage staining intensity/total field studied +/- standard error of the mean. Staining intensity was analyzed by Image J as previously described [21,22]. Statistical analysis were conducted by one way ANOVA with Student-Newman-Keuls post hoc test (n = 6, *p < 0.001 compared to E2). (Bottom) From left to right, representative IHC of 8oxoG for E2, MPA and E2+MPA (20×). Krikun et al. Journal of Angiogenesis Research 2010, 2:8 Page 6 of 7 http://www.jangiogenesis.com/content/2/1/8 Figure 6 Apoptosis: The apoptotic index was established for all 4 treatment groups after staining nuclei with Apotag as described in Meth- ods. The bar graph represents the average staining intensity/total field studied +/- standard error of the mean following analysis with Image J. Statis- tical analysis were conducted by one way ANOVA with Student-Newman-Keuls post hoc test (n = 3,*p < 0.05). setting of continuous low-level ovarian-derived estrogen gleaned from the experimental results presented in this production. Since the GPs used in the current study are study are expected to ultimately improve the formula- ovariectomized, the E2+MPA treated animals are likely to tions and acceptability of LTPOC therapies by reducing most closely resemble LTPOC-treated humans. aberrant angiogenesis and related irregular, unpredict- In prior studies, we have demonstrated that LTPOC able bleeding. results in both reduced endometrial blood flow and Competing interests increased oxidative stress [5,6]. These findings are associ- The authors declare that they have no competing interests. ated with immunohistochemical evidence of increased Authors' contributions angiogenic factor production. Further, hypoxia and oxi- GK and CL conceived and designed the experiments and wrote the manu- dative stress induce increased production of vascular script. IB, FS and LB carried out the ELISAs and immunohistochemical proce- endothelial growth factor (VEGF) and reduced produc- dures and statistical analysis. MH contributed in the critical analysis of the paper. tion of the angiostatic agent, angiopoietin-1 in cultured human endometrial stromal cells [4,6,9]. Hypoxia and Acknowledgements oxidative stress also greatly enhance production of the This work was supported by an NIH grant RO1HD33937 (CJL) highly angiogenic molecule, angiopoietin-2, in cultured Author Details endometrial endothelial cells [29]. However, it is unclear Department of Obstetrics, Gynecology and Reproductive Sciences, Yale how LTPOCs exert the initial vasoconstrictive effects on 2 University School of Medicine, New Haven, CT, 06510, USA and School of Women's and Infants' Health, University of Western Australia, Subiaco, WA human endometrium. The availability of this animal 6008, Australia model should allow dissection of the underlying mecha- nism driving this vascular phenomenon. Information Received: 12 March 2010 Accepted: 27 April 2010 Published: 27 April 2010 Krikun et al. Journal of Angiogenesis Research 2010, 2:8 Page 7 of 7 http://www.jangiogenesis.com/content/2/1/8 T © T Jo h h 2 u i is s 0 r a na i1 sr 0 an t l o K icl r f O e i A ikp u n se n a gn i et v o a A g a icce e la n l;b l es i ls c e f s is e arti R n ro s em e scle e e:a B h r d c ito t h istri p Med 2010 ://bu w C w te , e 2 w d n :8 . u t jr aa n n ld Lt ge io r th d g . en e te esirm s.cs o om f/ th co e n C te re na t/ ti 2ve /1 C /8ommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 22. Noursadeghi M, Tsang J, Haustein T, Miller RF, Chain BM, Katz DR: Quantitative imaging assay for NF-kappaB nuclear translocation in References primary human macrophages. J Immunol Methods 2008, 329:194-200. 1. Affandi B: An integrated analysis of vaginal bleeding patterns in clinical 23. Thorslund T, Sunesen M, Bohr VA, Stevnsner T: Repair of 8-oxoG is slower trials of Implanon. Contraception 1998, 58:99S-107S. in endogenous nuclear genes than in mitochondrial DNA and is 2. Collins J, Crosignani PG: Hormonal contraception without estrogens. without strand bias. DNA Repair (Amst) 2002, 1:261-273. Hum Reprod Update 2003, 9:373-386. 24. Maruo T, Laoag-Fernandez JB, Pakarinen P, Murakoshi H, Spitz IM, 3. Krikun G, Critchley H, Schatz F, Wan L, Caze R, Baergen RN, Lockwood CJ: Johansson E: Effects of the levonorgestrel-releasing intrauterine system Abnormal uterine bleeding during progestin-only contraception may on proliferation and apoptosis in the endometrium. Hum Reprod 2001, result from free radical-induced alterations in angiopoietin expression. 16:2103-2108. Am J Pathol 2002, 161:979-986. 25. Hickey M, Dwarte D, Fraser IS: Superficial endometrial vascular fragility 4. Hague S, MacKenzie IZ, Bicknell R, Rees MC: In-vivo angiogenesis and in Norplant users and in women with ovulatory dysfunctional uterine progestogens. Hum Reprod 2002, 17:786-793. bleeding. Human Reproduction 2000, 15:1509-1514. 5. Hickey M, Krikun G, Kodaman P, Schatz F, Carati C, Lockwood CJ: Long- 26. Hickey M, Simbar M, Markham R, Young L, Manconi F, Russell P, Fraser IS: term progestin-only contraceptives result in reduced endometrial Changes in vascular basement membrane in the endometrium of blood flow and oxidative stress. J Clin Endocrinol Metab 2006, Norplant users. Hum Reprod 1999, 14:716-721. 91:3633-3638. 27. Quandt LM, Hutz RJ: Induction by estradiol-17 beta of polycystic ovaries 6. Lockwood CJ, Schatz F, Krikun G: Angiogenic factors and the in the guinea pig. Biol Reprod 1993, 48:1088-1094. endometrium following long term progestin only contraception. 28. Mularoni A, Mahfoudi A, Beck L, Coosemans V, Bride J, Nicollier M, Adessi Histol Histopathol 2004, 19:167-172. GL: Progesterone control of fibronectin secretion in guinea pig 7. Runic R, Schatz F, Krey L, Demopoulos R, Thung S, Wan L, Lockwood CJ: endometrium. Endocrinology 1992, 131:2127-2132. Alterations in endometrial stromal cell tissue factor protein and 29. Krikun G, Schatz F, Finlay T, Kadner S, Mesia A, Gerrets R, Lockwood CJ: messenger ribonucleic acid expression in patients experiencing Expression of angiopoietin-2 by human endometrial endothelial cells: abnormal uterine bleeding while using Norplant-2 contraception. J regulation by hypoxia and inflammation. Biochem Biophys Res Commun Clin Endocrinol Metab 1997, 82:1983-1988. 2000, 275:159-163. 8. Hickey M, Simbar M, Young L, Markham R, Russell P, Fraser IS: A longitudinal study of changes in endometrial microvascular density in Norplant implant users. Contraception 1999, 59:123-129. doi: 10.1186/2040-2384-2-8 9. Charnock-Jones DS, Macpherson AM, Archer DF, Leslie S, Makkink WK, Cite this article as: Krikun et al., Long-term progestin contraceptives Sharkey AM, Smith SK: The effect of progestins on vascular endothelial (LTPOC) induce aberrant angiogenesis, oxidative stress and apoptosis in the guinea pig uterus: A model for abnormal uterine bleeding in humans Journal growth factor, oestrogen receptor and progesterone receptor of Angiogenesis Research 2010, 2:8 immunoreactivity and endothelial cell density in human endometrium. Hum Reprod 2000, 15(Suppl 3):85-95. 10. Shaw ST Jr, Macaulay LK, Aznar R, Gonzalez-Angulo A, Roy S: Effects of a progesterone-releasing intrauterine contraceptive device on endometrial blood vessels: a morphometric study. Am J Obstet Gynecol 1981, 141:821-827. 11. Guttinger A, Critchley HOD: Endometrial effects of intrauterine levonorgestrel. Contraception 2007, 75(6 Suppl):S93-98. 12. Hickey M, Fraser IS: The structure of endometrial microvessels. Hum Reprod 2000, 15(Suppl 3):57-66. 13. Palmer JA, Lau TM, Hickey M, Simbah M, Rogers PA: Immunohistochemical study of endometrial microvascular basement membrane components in women using Norplant. Hum Reprod 1996, 11:2142-2150. 14. Subakir SB, Hadisaputra W, Siregar B, Irawati D, Santoso DI, Cornain S, Affandi B: Reduced endothelial cell migratory signal production by endometrial explants from women using Norplant contraception. Hum Reprod 1995, 10:2579-2583. 15. Alkhalaf M, Propper AY, Chaminadas G, Adessi GL: Ultrastructural changes in guinea pig endometrial cells during the estrous cycle. J Morphol 1992, 214:83-96. 16. Makker A, Bansode FW, Srivastava VM, Singh MM: Antioxidant defense system during endometrial receptivity in the guinea pig: effect of ormeloxifene, a selective estrogen receptor modulator. J Endocrinol 2006, 188:121-134. 17. Lee KY, DeMayo FJ: Animal models of implantation. Reproduction 2004, 128:679-695. 18. Hutz RJ, Bejvan SM, Durning M, Dierschke DJ: Changes in follicular populations, in serum estrogen and progesterone, and in ovarian steroid secretion in vitro during the guinea pig estrous cycle. Biol Reprod 1990, 42:266-272. 19. Carter AM: Animal models of human placentation--a review. Placenta 2007, 28(Suppl A):S41-47. 20. Noci I, Borri P, Chieffi O, Scarselli G, Biagiotti R, Moncini D, Paglierani M, Taddei G: I. Aging of the human endometrium: a basic morphological and immunohistochemical study. Eur J Obstet Gynecol Reprod Biol 1995, 63:181-185. 21. Marcilhac A, Raynaud F, Clerc I, Benyamin Y: Detection and localization of calpain 3-like protease in a neuronal cell line: possible regulation of apoptotic cell death through degradation of nuclear IkappaBalpha. Int J Biochem Cell Biol 2006, 38:2128-2140.

Journal

Journal of Angiogenesis Research – Springer Journals

Published: Apr 27, 2010

There are no references for this article.