Estrogen-Mediated Endothelial Progenitor Cell Biology and Kinetics For Physiological Postnatal Vasculogenesis
Estrogen has been demonstrated to promote therapeutic reendothelialization after vascular injury by bone marrow (BM)–derived endothelial progenitor cell (EPC) mobilization and phenotypic modulation. We investigated the primary hypothesis that estrogen regulates physiological postnatal vasculogenesis by modulating bioactivity of BM-derived EPCs through the estrogen receptor (ER), in cyclic hormonally regulated endometrial neovascularization. Cultured human EPCs from peripheral blood mononuclear cells (PB-MNCs) disclosed consistent gene expression of ER α as well as downregulated gene expressions of ER β. Under the physiological concentrations of estrogen (17β-estradiol, E2), proliferation and migration were stimulated, whereas apoptosis was inhibited on day 7 cultured EPCs. These estrogen-induced activities were blocked by the receptor antagonist, ICI182,780 (ICI). In BM transplanted (BMT) mice with ovariectomy (OVX) from transgenic mice overexpressing β-galactosidase (lacZ) regulated by an endothelial specific Tie-2 promoter (Tie-2/lacZ/BM), the uterus demonstrated a significant increase in BM-derived EPCs (lacZ expressing cells) incorporated into neovasculatures detected by CD31 immunohistochemistry after E2 administration. The BM-derived EPCs that were incorporated into the uterus dominantly expressed ER α, rather than ER β in BMT mice from BM of transgenic mice overexpressing EGFP regulated by Tie-2 promoter with OVX (Tie-2/EGFP/BMT/OVX) by ERs fluorescence immunohistochemistry. An in vitro assay for colony forming activity as well as flow cytometry for CD133, CD34, KDR, and VE-cadherin, using human PB-MNCs at 5 stages of the female menstrual-cycle (early-proliferative, pre-ovulatory, post-ovulatory, mid-luteal, late-luteal), revealed cycle-specific regulation of EPC kinetics. These findings demonstrate that physiological postnatal vasculogenesis involves cyclic, E2-regulated bioactivity of BM-derived EPCs, predominantly through the ERα.
In the female reproductive system, neovascularization is a recurring phenomemnon controlled by cyclic development of transient structure and cyclical repair of damaged tissue.1 The ovarian sex steroid hormones, estrogen and progesterone, are primarily uterotropic and control the cyclical patterns of uterine cell proliferation and vascular growth that occur throughout the nonpregnant menstrual cycle. Given the synchronized nature of neovascularization in this cyclical mannter, it is assumed that angiogenic growth factor expression is induced by steroid hormones and regulates blood vessel formation in reproductive organogenesis.2–5
Despite clinical evidence for the significant role of steroid hormones in endometrial neovascularization, further investigation using in vitro and in vivo experiments have yielded inconclusive results regarding pathophysiological mechanisms in angiogenesis.6–10 Moreover, estrogen has been shown to exhibit an inhibitory effect on certain hematopoietic kinetics, including lymphocytes and monocytes, both in terms of number and function.11–14 Endometrial vascularization has formerly been considered to develop via “angiogenesis”, ie, proliferation and migration of fully differentiated endothelial cells (ECs) from preexisting “parent” vessels.15 However, circulating EPCs have been shown to incorporate into foci of neovascularization in adult species,16 consistent with the notion of postnatal “vasculogenesis”.17 EPCs comprise of undifferentiated blood MNCs which are mobilized from BM by ischemic stimuli and angiogenic/hematopoietic factors,17 and subsequently home to, differentiate, and proliferate in foci of neovascularization.18
Given this new understanding of adult neovascularization, it is also possible that vasculogenesis could also be responsible for ovarian hormonal regulation of endometrial neovascularization. Recently, 2 groups have demonstrated promotive effect of estrogen on reendothelialization after vascular injury via EPC incorporation. Iwakura et al disclosed an NO-dependent estrogen effect on EPCs using eNOS KO mice,19,20 and Strehlow et al indicated an estrogen dependent antiapoptotic effect on EPC biology. Hamada et al demonstrated the functional importance of ER expression by EPCs.21 Together, these reports suggest a therapeutic application of altering estrogen levels directly, or its receptor agonists, for vascular repair.
Therefore, in the current study, we investigated the hypothesis that E2 regulates physiological neovascularization of the endometrium by modulating the biology and kinetics of BM-derived EPCs.
EPC Culture of Human and Mouse EPCs
Human and mouse EPCs were cultured by using a modified protocol that has previously been reported.18,22,23 Phenol-red free (PRF) endothelial basal media (EBM, Clonetics) was used to delete estrogenic effect, as described in Supplementary Method (SM)-I, available online at http://circres.ahajournals.org.24,25
Effects of Estrogen on EPCs: Differentiation, Proliferation, Migration, and Antiapoptosis In Vitro
The assays for EPC bioactivity effected by E2 were performed, according to the detailed description in SM-II, as previously reported.19,26
RT-PCR for Endothelial Gene Expression in Cultured Human EPCs
The protocol for RT-PCR assay was described in SM-III.
Real Time PCR Assay for Gene Expression of Estrogen Receptors in Cultured Human EPCs
The protocol of real time PCR assay was described in SM-IV.
Mouse Cultured EPC Assay
The protocol of mouse cultured EPC assay was described in SM-V.18,22,23
Mouse Cornea Neovascularization Assay
The effect of E2-induced EPC kinetics on neovascularization was studied by E2 pretreated OVX mice for 4 days as described in SM-6.18,22,27
Study Design of BMT Animal Experiments
BMT animal models with endogenous sex hormone depletion were developed as follows: female nude SCID mice (NIHS-bg-nu-xid, Taconic, Albany, NY; 4 weeks) were lethally irradiated and received BM cells from age-matched female Tie-2 transgenic mice overexpressing β-galactosidase by Tie-2 promoter (FVB/N-TgN[TIE2LacZ]182Sato, Jackson Laboratory, Bar Harbor, Me).17,22,28 The protocol of BMT animal experiments was depicted in SM-VII.
Cellular Identification of LacZ Expressing Cells in Uterus or Cornea of Tie-2/LacZ/BMT/OVX Mice
The uterus of mice euthanized at day 2, day 4, and day 7 after subcutaneous E2 pellet implantation was processed for CD31 immunohistochemistry as well as LacZ staining,17 as described in SM-VIII. LacZ positive cells in whole area, or localized in vascular wall or stroma per uterus tissue section were counted. The percentage of LacZ positive cells localized in each part versus whole area was assayed.
Six days after making the cornea model, the cornea was observed, after staining eye balls with LacZ solution. LacZ stained tissues embedded in paraffin were processed to CD31 immunohistochemistry.
Investigation of ER α and ER β Expression by BM-Derived EPCs Incorporated Into the Uterus of Tie-2/EGFP/BMT/OVX Mice
The protocol was described in SM-IX.
EPC Culture Assay and Flow Cytometry in Menstrual Cycle of Premenopausal Women
EPC culture assay and flow cytometrical analysis were performed, using PB-MNCs of 6 healthy premenopausal females (aged 20 to 40) at 5 separate stages of the menstrual cycle: T1=early proliferative, T2=preovulatory, T3=postovulatory, T4=mid luteal, and T5=late luteal, as previously described.29 Flow cytometrical analysis (FACS) was performed on a FACStar flow cytometer (Becton Dickinson) and a Cell Quest software (Becton Dickinson), as described in SM-X.
EPC Colony Forming Activity of PB in Menstrual Cycle of Premenopausal Women
EPC colony forming activity was also assessed by SM-XI and XII.
Notice on Experiments in Animal and Human Subjects
Notice on experiments in animal and human subjects was described in SM-XIII.
All results are expressed as mean±SE. Statistical significance was evaluated using unpaired Student t test for the comparison between 2 groups and ANOVA followed by Fisher post hoc test for the comparison among multiple groups. A probability value less than 0.05 was interpreted to denote statistical significance.
Quantitative Real Time PCR Assay of ER α and ER β Gene Expressions in Cultured Human EPCs
In day 7 cultured human EPCs, endothelial gene expressions of von Willbrand Factor (vWF) and CD31 were detected (Figure 1A). To establish the potential for a direct effect of E2 on EPCs, mRNA expression of ER α and ER β was assessed by quantitative real time PCR assay. The gene expressions of ER α and ER β in cultured EPCs varied during the culture period. The expression of ER α did not change between day 4 and day 7, whereas the expression of ER β was remarkably downregulated at day 7 to the level of 0.12 fold relative to day 4 (Figure 1B).
Receptor Mediated E2 Effects on EPC Activity
EPC bioactivities upregulated by E2 were deleted in the presence of ICI, suggesting the ER mediated bioactivities, as described in supplemental Figure (SF)-I.
Upregulation of EPC Kinetics After Ε2 Administration
To explore the systemic effects of E2 on EPC kinetics, we administered E2 to OVX female and CST male mice. The EPC culture assay22 revealed a significant increase in endothelial lineage cells in cultures of PB-MNCs isolated at 2 to 4 days after subcutaneous implantation of E2 pellet in both OVX and CST mice. EPCs, identified by acLDL-DiI uptake and BS-1 lectin- FITC reactivity, consisted principally of spindle-shaped cells, often forming colonies. The number of cultured EPCs decreased to or below the level of pre-implantation by day 7 (Figure 2A and 2B). In controls, pellet implant did not increase EPC numbers significantly. These results thus provide quantitative evidence that E2 mobilizes EPCs from BM into the peripheral circulation.
Enhanced Cornea Neovascularization After E2 Administration
Examination of the cornea in E2- or P-treated mice established the extent of vascular development induced by implantation of a VEGF-containing pellet in the mouse cornea, presented in SF-II.
Identification of BM-Derived EPCs Within the Endometrium of Tie-2/LacZ/BMT/OVX Mice After E2 Administration
Before pellet implantation, macroscopic examination of the uterus of Tie-2/LacZ/BMT/OVX mice from both groups disclosed occasional blue LacZ-stained cells located mainly in the mesometrium. In mice with P pellet implants, the uterus remained atrophic, and the location and frequency of LacZ-stained BM-derived EPCs did not change during observation. In contrast, Tie-2/LacZ/BMT/OVX mice with implanted E2 pellet revealed an evolving pattern of BM-derived, LacZ-positive cells within the uterus. Two days after E2 pellet implant, the frequency of LacZ-positive cells increased throughout the uterus but remained concentrated in the outer layer of myometrium. By day 4 after implantation, EPCs continued to increase in number and were now identified in the outer and inner layers of myometrium and endometrium. EPCs finally accumulated in large numbers within the endometrium 7 days after E2 implantation. Immunohistochemical staining for CD31 in the P group demonstrated LacZ-stained CD31-positive EPCs appearing as round cells localized in the stroma adjacent to established vessels and incorporated as spindle-shaped cells into vascular walls. After E2 pellet implantation, EPCs were more frequently found to be incorporated within the vascular walls of the endometrium (Figure 3A). Counterstaining with antibody F4/80, as a tissue macrophage marker, and LacZ staining, revealed that the macrophages did not express β-gal; thereby indicating that they do not express Tie-2 and are therefore a completely separate population from the EPCs identified by Tie-2 promoter driven LacZ expression (Figure 3B). EPCs incorporation into foci of uterine neovascularization increased significantly by approximately 4.5-, 5.5-, or 7.8-fold at day 2, day 4, or day 7 after E2 pellet implantation versus endometrial tissues harvested from P pellet implants examined at identical time points. The number of incorporated EPCs per uterine section were as follows: for E2 group, day 2=9.0±1.8, day 4=11.0±1.7, day 7=14.0±3.6; in contrast, for P group, day 2=2.1±0.4, day 4=2.0±0.3, and day 7=1.8±0.5 (Figure 3C). Also, after E2 pellet implantation, the percentage of BM-derived EPCs incorporated into the neovasculature (of the total BM-derived EPCs per uterine section) increased significantly (day 2=10.44±2.7%, day 4=33.01±3.7%, day 7=36.73±6.9%); in contrast, the percentage for P group remained low (day 2=3.85±3.8%, day 4=8.56±4.1%, and day 7=4.44±2.9%; Figure 3D). On the other hand, the percentage of BM-derived EPCs in the stroma of the E2 group decreased inversely (day 2=89.57±2.7%, day 4=66.99±3.7%, day 7=63.27±6.9%), whereas the percentage for P group remained high (day 2=96.15±3.8%, day 4=91.44±4.1%, and day 7=95.56±2.9%; Figure 3D).
These findings indicate that BM-derived EPCs incorporate into foci of neovascularization during E2-induced endometrial maturation. This effect was restricted to E2-responsive organs: incorporated EPCs in other organs such as lung, liver, or skin,17 could not be enhanced by E2 (data not shown). The sequence of histologic patterns observed suggests that E2 mobilizes BM-derived EPCs via the circulation (vide infra) into the myometrium from mesometrium, which precedes accumulation and incorporation into the neovasculature of the endometrium. The representative feature of BM-derived EPC incorporation into endothelial layer of vessel wall in uterine endometrium was recognized at day 7 after E2 pellet implantation by fluorescence immunohistochemistry of EGFP cellular positivity in CD31 positive endothelial layer (SF-III).
BM-derived EPCs (Tie-2/EGFP positive cells) incorporated into uterine tissues in Tie-2/EGFP/BMT/OVX mice, expressing ER α by stimulation of E2 pellet implantation for 4 days, but not ER β by fluorescence immunohistochemistry (Figure 3E). In this context, it is intriguing to note that the pattern of EPC recruitment and incorporation is identical to the previously established pattern of in situ VEGF expression in the hormone-regulated cycle of endometrial development and regression.3
Recruitment of BM-Derived EPCs into Cornea Neovascularization of Tie-2/LacZ/BMT/OVX Mice Following E2 Administration
Macroscopically, BM-derived EPCs stained with LacZ were observed more frequently in cornea of E2 pellet implanted mice, as compared with P pellet implanted, as presented in SF-4.
EPC Kinetics Through Human Menstrual Cycle
The morphology of cultured EPCs varied at different phases of the menstrual cycle (Figure 4A). At the preovulatory phase (T2), cultured EPCs, identified by double staining with acLDL-DiI and UEA-1-FITC, were recognized as isolated round adhesive cells, seldom forming colonies. After ovulation through the luteal phase (T3 to T5), EPCs appeared spindle-shaped, frequently exhibiting colony formation. Frequent colony formation was noted during the early proliferative phase (T1). The frequency of EPCs in culture decreased to the lowest level at the preovulatory phase (T2), increased gradually through ovulation, and remained high even during the early proliferative phase (Figure 4B).
As shown in Figure 4C, ovulation was identified between T2 and T3 by a surge of luteinizing hormone; the associated expression patterns of E2 and progesterone conform to the typical pattern of the menstrual cycle. VEGF levels were lowest at T1, then increased rapidly, reaching a peak at the T2 before slowly decreasing through the luteal phase. The pattern of VEGF expression was thus synchronized with E2. The numeric values of hormones and EPC numbers during menstrual cycle are shown in supplemental Table I.
Flow cytometrical analysis of PB-MNCs was used to determine the frequency of endothelial-specific antigen expressing cells as well as circulating immature EPCs according to the phase of the menstrual cycle. KDR was more frequently expressed by circulating cells from the preovulatory through luteal phases (Figure 4D-b). VE-cadherin antigen positive cells also increased, but the peak expression was until the later luteal phase. Differentiated ECs, positive for P1H12 antigen,30 were identified at T1, immediately after menstruation, and did not augment, whereas KDR or VE-cadherin–positive endothelial lineage cells increased, after steroid hormone peaks (Figure 4D-b). Of note, the cell populations of CD133-positive or CD34-positive cells involving circulating immature EPCs in PB during the menstrual cycle disclosed a fluctuating pattern with significant amelioration at T3 following E2 peak (Figure 4D-c and 4D-d).
The female reproductive system constitutes a unique exception to the quiescent vasculature of the normal healthy adult as the requirement for neovascularization recurring on a cyclic basis. Specifically, in every estrous cycle, the sequential maturation of the endometrium, as well as ovarian follicles and corpora lutea, is accompanied by concomitant development of elaborate capillary networks. Given the extent of newly forming vascular volume in endometrial development, it is possible that vasculogenic mechanisms may play a significant role in this cyclic organization. In this regard, we hypothesized that one of the main gender hormones, estrogen, controls EPC biology for cyclic neovascularization. The present findings provide evidence that the physiologic cycle of estrogen regulates EPC kinetics, ie, differentiation, proliferation, migration, apoptosis, mobilization, and ultimately incorporation into foci of neovascularization in the developing endometrium. Although the therapeutic potential of E2 for enhancing the contributions of EPCs for reendothelialization after vascular injury has been suggested, the physiological role of estrogen for EPC mediated vascular development has not been well established.19,20,31
Our EPC culture assay experiments demonstrated variations in EPC number and morphology throughout the phases of the menstrual cycle. These morphological changes are indicative of enhanced differentiation potential of circulating EPCs corresponding to cyclic hormonal changes. The peak increase in EPC number followed the peak serum concentrations of estrogen, as well as VEGF. This interval may potentially reflect a combination of estrogen effect on EPC proliferation, differentiation, and estrogen-induced mobilization from BM that has been suggested previously.19,20,31
The increase in the number of circulating EPCs expressing KDR or VE-cadherin antigen after peak estrogen levels and the decrease after downregulation of sex hormones were demonstrated by flow cytometrical analysis. Given their essential function in embryonic vasculogenesis,32–35 KDR and VE-cadherin were used to detect EPCs in PB-MNC population. Similarly, Strehlow et al have shown that estrogen mobilizes BM-derived EPCs (CD34 positive/KDR positive cells) into circulation of human subjects.31 A temporal discrepancy in expression between KDR and VE-cadherin antigen in preovulatory and postovulatory phases was observed, which may suggest a differential effect of ovulatory estrogen on EPC biology. The basis for this differential expression may be related to the former findings that the expression of Flk-1 (homologue of mouse KDR) has been considered to represent a very early endothelial lineage marker during embryogenesis, whereas VE-cadherin–positive EPCs are considered differentiated from Flk-1–positive/VE-cadherin–negative cells.35 The initial increase in KDR-positive cells seen during pre- to post-ovulatory phases may result from mobilization of immature EPCs into circulation, followed by an increase in committing EPCs during the luteal phase.35 In contrast to KDR and VE-cadherin, P1H12 was used as a marker for differentiated ECs and was present at lesser frequency during preovulatory to luteal phases than other markers. Thus, differentiated ECs may circulate in PB only when dislodged by physiological blood vessel regression during menstruation.
The animal experiments provide potential insights into the significance of cyclic changes in EPC kinetics observed in human subjects. Systemic E2 pretreatment enhanced EPC mobilization detected in EPC culture assay and promoted enhanced neovascularization in the mouse cornea micropocket model. These results indicate that systemic estrogen stimulates EPC kinetics in the circulation, subsequently contributing to neovascularization via vasculogenesis. BMT experiments have demonstrated recruitment and incorporation of BM-derived Tie-2 receptor expressing cells, putative EPCs, during estrogen-induced endometrial development. It is intriguing to note that this pattern of EPC recruitment and incorporation is identical to the previously established pattern of in situ VEGF expression in the hormone-regulated cycle of endometrial development and regression.3 In addition to EPCs found have incorporated into vascular structures, other round and at times even spindle-shaped cells were frequently identified in the uterine stroma. Regarding the finding, the cells could represent tissue macrophages derived from BM incorporating into uterine stroma during estrous cycle. The existence of BM-derived cells defined as macrophages (stained by F4/80) is also important to discuss because this may identify a significant role of blood bone cells in endometrial formation, especially regarding neovascularization. The similar findings were pointed out by several publications.36,37 Tie-2 expressing BM-derived EPCs need vasculogenic environments introduced by angiogenic cytokines secreted from BM-derived macrophages. Therefore, this balance of EPCs and macrophages might play a pivotal role in neovascularization in endometrial formation.
Although the fate of the EPCs is currently uncertain, such cells may comprise EC reservoirs for the next round of endometrial development. The concept of BM-derived progenitor cell reservoirs in normal tissues is consistent with the notion of BM-derived satellite myoblasts and mesenchymal stem cells in muscle or other normal organs.5,38 Using the same BMT models, we have previously demonstrated similar stroma-localized EPCs in growing neoplasms, wound healing, severe ischemia, and even though more sparsely—in normal organs.17 Flk-1–positive cells previously demonstrated in the uterine myometrium39 may represent similar cells. We have considered that the effect of estrogen may be direct or indirect (eg, mediated via VEGF). Evidence for a direct effect was given by the fact that EPC kinetics in CST male mice, lacking reproductive organs to respond to estrogen, responded equivalently in the case of OVX female mice, leaving a potentially estrogen-responsive (ie, VEGF-producing) uterus. This finding suggests that estrogen enhances EPC kinetics by direct interaction with EPCs or associated cells, such as in the BM microenvironment.
Our in vitro assay presented E2 promotion on EPC differentiation, migration, proliferation, and apoptosis inhibition, as partly indicated previously.19,31 The findings that enhanced biological activities, such as proliferation, migration, antiapoptosis, and differentiation stimulated by E2 were blocked by an nonselective ERs antagonist (ICI), supported the fact that these effects of estrogen on EPCs were via functional ERs which were detected by mRNA in EPCs.
The importance of ERs on EPC bioactivity, using a myocardial infarcted model of ER α and ER β knockout mice has been recently documented by Hamada et al.21 The authors described that ER α expressed in EPCs plays a more potent role in pathological vasculogenesis, rather than ER β. The present study disclosed the higher significance of ER α versus ER β in physiological vasculogenesis as well. During the culture period of human EPCs for 7 days through 4 days, the ER α expression was remained at the high level with the downregulation of ER β expression by real time PCR assay, as shown in Figure 1B. Accordingly, the EPC bioactivities disclosed in vitro study of day 7 cultured EPCs are considered to be brought through ER α. Furthermore, in vivo study of Tie-2/EGFP/BMT/OVX mice, the incorporated EPCs in uterus via E2 stimulation disclosed the expression of ER α, but not ER β, as shown in Figure 3E.
These findings may suggest that each ER shares the roles on EPC differentiation cascade, ie, the predominant function of ER β for EPC immature stage at provasculogenic state or ER α for EPC maturing stage at vasculogenic state, although the ER β function especially has yet to be elucidated.
Given the consideration, even in pathological vasculogenesis, ie, coronary vessel formation in infracted hearts, as shown by Hamada et al21 as well as physiological vasculogenesis, each ER may have a unique role on EPC differentiation.
The basis for organogenesis has become a seminal issue for organ transplantation or therapeutic regeneration of damaged organs. Embryogenesis as well as physiological organogenesis in adult species reveal essential elements of organogenesis, devoid of pathological stimuli, including inflammation. The physiological regenerative processes constitute natural models that indicate how organs are established and survive. Blood vessel development is clearly one of the essential processes for organogenesis. The present study demonstrates that the unique system of cyclical blood vessel development and regression during the menstrual cycle, which occurs more than 300 times in a female life span, involves hormone-mediated in situ proliferation, incorporation, differentiation, and survival of BM-derived progenitor cells. Above all, the unique EPC kinetics during menstrual cycle provides “dogma” for EPC biology as shown in supplemental Table I. Also, these findings have important implications for the impact of estrogen on vessel formation in disease states. Further insights regarding the precise mechanisms responsible for such physiological vasculogenesis will likely contribute to advanced methods and concepts for organ development in vivo as well as ex vivo.
This paper is dedicated to Dr. Jeffrey M. Isner. We would like to gratefully acknowledge him for his inspirational leadership, friendship and encouraging support. EPC colony assay was performed following the pretrial using cord blood under the approval of the ethical committees of Cord Blood Bank and Clinical Investigation in Tokai University School of Medicine. We also thank Dr. Kiyoshi Ando in Research Center for Regenerative Medicine for the management of the research facility; Dr. Yoshinori Okada and Dr. Jobu Itoh in Teaching and Research Support Center for the technical advices and supports; members of the animal facility in Tokai University School of Medicine as well as Miss. Sachie Ota as the secretary assistant. Especially, we greatfully thank Dr. Oren Tepper in Institute of Reconstructive Plastic Surgery, New York University Medical Center to finally check English grammar and style in the revised text.
Sources of Funding
This work was supported by grants from the National Institutes of Health (HL53354, and HL57516), Bethesda, MD; the ministry of Health, Labor and Welfare (H14-trans-001, H14-trans-002, H17- 014), Japan; the ministry of Education, Culture, Sports, Science and Technology (Academic Frontier Promotion Program), Japan. H.M. is supported in part by Uehara Memorial Foundation and Kanagawa Nanbyo Foundation in Japan, and C.K. by a Cologne Fortune Grant in Germany. Estrogen receptor antagonist (ICI182,780) was kindly gifted by Astra Zeneca Pharmaceutical company.
Original received November 3, 2006; revision received June 11, 2007; accepted July 16, 2007.
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