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, 7 (271), 271ra9

Dual Suppression of Estrogenic and Inflammatory Activities for Targeting of Endometriosis

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Dual Suppression of Estrogenic and Inflammatory Activities for Targeting of Endometriosis

Yuechao Zhao et al. Sci Transl Med.

Abstract

Estrogenic and inflammatory components play key roles in a broad range of diseases including endometriosis, a common estrogen-dependent gynecological disorder in which endometrial tissue creates inflammatory lesions at extrauterine sites, causing pelvic pain and reduced fertility. Current medical therapies focus primarily on reducing systemic levels of estrogens, but these are of limited effectiveness and have considerable side effects. We developed estrogen receptor (ER) ligands, chloroindazole (CLI) and oxabicycloheptene sulfonate (OBHS), which showed strong ER-dependent anti-inflammatory activity in a preclinical model of endometriosis that recapitulates the estrogen dependence and inflammatory responses of the disease in immunocompetent mice and in primary human endometriotic stromal cells in culture. Estrogen-dependent phenomena, including cell proliferation, cyst formation, vascularization, and lesion growth, were all arrested by CLI or OBHS, which prevented lesion expansion and also elicited regression of established lesions, suppressed inflammation, angiogenesis, and neurogenesis in the lesions, and interrupted crosstalk between lesion cells and infiltrating macrophages. Studies in ERα or ERβ knockout mice indicated that ERα is the major mediator of OBHS effectiveness and ERβ is dominant in CLI actions, implying involvement of both ERs in endometriosis. Neither ligand altered estrous cycling or fertility at doses that were effective for suppression of endometriosis. Hence, CLI and OBHS are able to restrain endometriosis by dual suppression of the estrogen-inflammatory axis. Our findings suggest that these compounds have the desired characteristics of preventive and therapeutic agents for clinical endometriosis and possibly other estrogen-driven and inflammation-promoted disorders.

Figures

Fig. 1
Fig. 1. Effects of OBHS and CLI in the endometriosis prevention model
(A) Uterine fragments from intact donor mice were engrafted to the peritoneal wall of ovariectomized syngeneic recipient mice, which were then treated with ligands or control vehicle (Veh; n = 6 mice per group) for up to 14 days. (B) Structures of compounds. (C) Ovariectomized recipient mice were treated with control vehicle, E2 (0.125 mg per pellet), E2 + OBHS, or E2 + CLI (0.125 mg of E2 and 0.25 mg of OBHS or CLI per pellet), and ectopic lesion growth was measured over the 14 days of treatment. *P < 0.05 [n = 6 per group, two-way analysis of variance (ANOVA) with Bonferroni's multiple comparison test]. Original data for each animal are given in table S4, and exact P values in table S5. (D) H&E staining of ectopic tissues from recipients treated with different ligands or control vehicle for 14 days. Scale bar, 200 μm. (E) Immunohistochemistry of Ki67 in ectopic tissue after 14 days of treatment. Scale bar, 100 μm. (F) Quantification of Ki67-positive cells as a percent of the number of total cells in lesions (n = 6 per group). (G) PECAM immunofluorescence was analyzed to observe the vasculature in ectopic lesions after 14 days of ligand treatment (n = 6 per group). Scale bar, 100 μm. DAPI, diamidino-2-phenylindole. (H) Quantification of microvessel density in ectopic lesions at day 14 (n = 6 per group). (I) Level of Cyr61 and Vegfa profiled by quantitative polymerase chain reaction (PCR) (n = 6 per group). (J) Immunohistochemistry for CYR61 and VEGFA protein in ectopic tissues after 3 days of vehicle or ligand treatment (n = 6 per group). Scale bars, 100 μm. BV, blood vessel; C, cyst; E, epithelial cells; S, stromal cells; P, peritoneal wall. Lowercase letters indicate P < 0.05 by one-way ANOVA with Bonferroni's multiple comparison test.
Fig. 2
Fig. 2. Effects of OBHS and CLI in the therapeutic model
(A) Uterine donor tissue was transplanted to the peritoneal wall of ovary-intact recipient (8-week-old) female mice. After 2 weeks of lesion establishment, recipient animals received OBHS or CLI or control vehicle for up to 6 weeks. Both ectopic and eutopic tissues were collected from recipients at diestrus. (B) Effect of ligand dosage (0, 0.1, and 0.25 mg per pellet) on lesion growth and eutopic recipient uterine weight at 6 weeks of ligand or vehicle treatment (n = 6 per group). Lowercase letters indicate P < 0.05 by two-way ANOVA with Bonferroni's multiple comparison test. (C) Lesion volume was monitored over the 6 weeks of ligand treatment (0.25 mg per pellet, n = 6 per group). *P < 0.05. Original data for each animal are given in table S4, and exact P values in table S5. (D) Immunohistochemistry of Ki67 in ectopic tissue after 6 weeks of treatment. Scale bar, 50 μm. (E) Quantification of Ki67-positive cells as a percentage of the number of total cells in lesions (n = 6 per group). (F) PECAM immunofluorescence to observe the vasculature in ectopic lesions after 6 weeks of treatment. Scale bar, 50 μm. (G) Quantification of microvessel density in ectopic lesions at 6 weeks (n = 6 per group). *P < 0.05 versus vehicle. Lowercase letters indicate P < 0.05 by one-way ANOVA with Bonferroni's multiple comparison test.
Fig. 3
Fig. 3. Anti-inflammatory effects of OBHS and CLI
(A) Expression of cytokines in ectopic tissues was analyzed by quantitative reverse transcription PCR (RT-PCR) over 6 weeks of treatment. mRNA levels are expressed relative to transcript level in eutopic uterine donor tissue, set at 1.0 (n = 6 per group). *P < 0.05 by two-way ANOVA with Bonferroni's multiple comparison test. Exact P values are given in table S5. (B) IL-6 immunohistochemistry in ectopic lesions after 2 weeks of treatment. immunoglobulin G (IgG) served as negative control for the anti–IL-6 antibody. Scale bar, 50 μm. (C) Quantification of IL-6 staining signal in ectopic lesions (n = 6 per group). (D) NFκB activity monitored by immunostaining of p65 in ectopic lesions after 2 weeks. Scale bar, 50 μm. (E) Quantification of p65 nuclear staining in ectopic lesions (n = 6 per group). (F) COX2 immunofluorescence in ectopic lesions after 2 weeks of treatment. Scale bar, 200 μm. (G) Fluorescence images of TUNEL staining in ectopic lesions. Scale bar, 50 μm. (H) CD3 and F4/80 immunofluorescence to monitor T cells and macrophages, respectively, in ectopic lesions, and quantification of CD3- and F4/80-positive cells (n = 6 per group). Scale bar, 50 μm. E, epithelial compartment; S, stromal compartment. Lowercase letters indicate P < 0.05 by one-way ANOVA with Bonferroni's multiple comparison test.
Fig. 4
Fig. 4. Effects of ligands on innervation and neuron activation in ectopic endometriotic lesions
(A and B) Dual immunofluorescence of PGP 9.5 and SP (A), or PGP 9.5 and CGRP (B), in ectopic lesions after 2 weeks of treatment with vehicle or ligand. Costaining signals are highlighted by white arrows. (C) Double immunofluorescence of NGF and NGFR-p75 in ectopic lesions. Costaining signals are highlighted by white arrows. Scale bars, 50 μm.
Fig. 5
Fig. 5. Cotreatment with letrozole in combination with ligands in the therapeutic model
(A and B) Ectopic lesion volume (A) and eutopic uterine weight (B) after 2 weeks of letrozole (0.75 mg per pellet) with or without OBHS or CLI ligand (0.25 mg per pellet), or with OBHS or CLI alone (n = 6 per group). Original data for all animals are given in table S4. (C) Proliferation monitored by Ki67 staining (n = 6 per group). (D) Quantitative RT-PCR analysis of cytokine mRNA expression in ectopic tissues, with transcript level in vehicle-treated tissue set at 1.0 (n = 6 per group). (E) Angiogenesis and innervation were assessed by immunofluorescence of PECAM and PGP 9.5 after 2 weeks. Scale bars, 50 μm. Let, letrozole. Lowercase letters indicate P < 0.05 by one-way ANOVA with Bonferroni's multiple comparison test. Data for Ki67 staining, cytokine expression, angiogenesis, and innervation for lesions after OBHS or CLI treatment alone were shown in Figs. 2 to 4.
Fig. 6
Fig. 6. Effects of OBHS and CLI on hESCs in vitro
(A) Primary cultured hESCs were treated with ligands for 6 days, and cell proliferation was monitored (n = 6 per group). (B) Immunofluorescence of phosphorylated Ser10 of phospho–histone 3 (p-H3) was performed to monitor the mitotic activity of endometriotic stromal cells after 24 hours. p-H3–positive nuclei are highlighted by white arrows (n = 6 per group). Scale bar, 50 μm. (C) Quantification of p-H3–positive cells (n = 6 per group). (D) Apoptotic DAPI-stained nuclei are red by TUNEL assay after 72 hours. White arrows indicate apoptotic foci. Scale bar, 50 μm. (E) Percentage of apoptotic cells (n = 6 per group). (F) Cytokine mRNA levels in hESCs after treatment for 24 hours (n = 6 per group). E2, 10 nM; TNFα, 20 ng/ml; OBHS, 1 μM; CLI, 1 μM; ICI, 1 μM. Lowercase letters indicate P < 0.05 by one-way ANOVA with Bonferroni's multiple comparison test.
Fig. 7
Fig. 7. Role of macrophages in the prevention of lesion establishment by OBHS or CLI
(A) Serial injections of clodronate-containing liposomes (Clod; 1 mg per injection) were used to deplete macrophages from ovariectomized recipient mice. Donor tissues were transplanted followed by 2 weeks of OBHS or CLI treatment. (B) Lack of F4/80 staining, quantified in stroma, confirmed the absence of macrophages in ectopic tissue in Clod-treated hosts. Scale bar, 50 μm. S, stromal compartment. (C) Lesion volume with and without clodronate and treatment with vehicle, E2, E2 + OBHS, or E2 + CLI for 2 weeks (n = 6 per group). (D) Quantitative RT-PCR analysis of cytokine transcript levels in ectopic lesions, with transcript level in vehicle-treated lesions without clodronate set at 1.0 (n = 6 per group). (E) Eutopic uterine weight with and without clodronate and treatment with vehicle, E2, E2 + OBHS, or E2 + CLI for 2 weeks (n = 6 per group). Original data for all animals are given in table S4. Lowercase letters indicate P < 0.05 by one-way ANOVA with Bonferroni's multiple comparison test.
Fig. 8
Fig. 8. Effectiveness of ligand treatment against lesion progression in ER transgenic mice
Intact wild-type (WT), αKO, or βKO mice were used as recipient animals (n = 6 per group). (A) Lesion volume was quantified after 2 weeks of lesion establishment from WT donor uterine tissue and 2 weeks of vehicle, OBHS, or CLI treatment (0.25 mg per pellet). P values determined by one-way ANOVA with Bonferroni's multiple comparison test. (B and C) Quantitative RT-PCR analysis of relative mRNA expression levels of cytokines in ectopic tissue. Transcript level in vehicle-treated WT ectopic tissue transplanted in WT recipients was set at 1.0 (n = 6 per group). Note different y-axis scales in (B) and (C). (D) Schematic model depicting the mechanisms by which OBHS and CLI exert their antiestrogenic and anti-inflammatory effects via ERs to block events contributing to establishment and progression of endometriosis.

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