2-methoxyestradiol inhibits hypoxia-inducible factor-1{alpha} and suppresses growth of lesions in a mouse model of endometriosis - PubMed (original) (raw)

2-methoxyestradiol inhibits hypoxia-inducible factor-1{alpha} and suppresses growth of lesions in a mouse model of endometriosis

Christian M Becker et al. Am J Pathol. 2008 Feb.

Abstract

Endometriosis, the presence of ectopic endometrial tissue outside the uterine cavity, is a common disease affecting women during their reproductive years. Current therapeutic success is often unsatisfactory because of limited insight into disease mechanisms. Nevertheless, angiogenesis plays an essential role in the pathogenesis of the disease, making it a potential novel target for therapy. In the current study, we demonstrate in an established mouse model of endometriosis that transient hypoxia in transplanted endometriosis-like lesions results in the up-regulation of hypoxia-inducible factor-1alpha (HIF-1alpha), leading to the expression of vascular endothelial growth factor (VEGF), a key player in endometriosis-associated angiogenesis. Systemic treatment with the angiogenesis inhibitor 2-methoxyestradiol suppressed HIF-1alpha expression in vivo, resulting in a decreased downstream expression of HIF-1alpha target genes, such as for VEGF, phosphoglycerate kinase, and glucose transporter-1. 2-Methoxyestradiol also suppressed VEGF-induced vascular permeability, as demonstrated in a modified Miles assay. Finally, systemic treatment with 2-methoxyestradiol significantly inhibited the growth of endometriosis-like lesions in a dose-dependent manner. In conclusion, hypoxia appears to play an important role in the pathogenesis of endometriosis and endometriosis-associated angiogenesis, and the angiogenesis inhibitor 2-methoxyestradiol may be a potential candidate for systemic treatment in the future.

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Figures

Figure 1

Figure 1

A: Hypoxyprobe staining. Representative murine endometriotic tissue removed at different time points after transplantation. D: 1 hour. E: 4 hours. F: 24 hours. G: 48 hours. H: 1 week. I: 6 months. Each mouse was injected with pimonidazole hydrochloride (hypoxyprobe-1) 30 minutes before removal. Eutopic uterine lesions from the same mice were used as negative controls (A–C). Staining with hypoxyprobe antibody and counterstaining with hematoxylin. Control endometriotic tissue (4 hours; J) stained with an isotype-matched irrelevant murine IgG1 monoclonal antibody directed against Shigela toxin.

Figure 2

Figure 2

A: Immunohistochemistry staining of endometriosis-like lesions with anti-HIF-1α antibody. Counterstaining with hematoxylin. Increasing immunoreactivity (brown color) in glandular epithelial and stromal cells (arrows) over time (0, 1, 3, and 6 hours). Control (6 hours) stained with nonspecific rabbit IgG antibody. B: Western blot of endometriotic tissue stained with ant-HIF-1α antibody from different time points after transplantation. Loading control of same Western blot stained with anti-β-actin antibody. C: Real-time RT PCR of endometriosis-like lesions at different time points using primers for HIF-1α-dependent genes (VEGF, PGK, and Glut-1). Bars indicate SEM. D: Semiquantitative measurement of Western blot for HIF-1α as seen in B. Error bars indicate SEM. E: Endometriosis-like lesion from a green fluorescent protein+/+ mouse on the peritoneal wall of a wild-type recipient. Typical growth of blood vessels into the endometriosis-like lesion.

Figure 3

Figure 3

Systemic 2-methoxyestradiol inhibits growth of endometriosis-like lesions. Endometriosis-like lesions were measured in two perpendicular diameters (_D_1 and _D_2) with a caliper, and CSA was calculated using the formula of an ellipse (_D_1 × _D_2 × π/4) as previously described.,, A: Mean CSA of endometriosis-like lesions (n = 7 lesions) of mice treated with vehicle (▪) or 2-methoxyestradiol (□) (n = 6/group). B: Mean CSA after 4 weeks of oral treatment with different doses of 2-methoxyestradiol. Bars indicate the SEM.

Figure 4

Figure 4

Systemic 2-methoxyestradiol treatment reduces expression of HIF-1α target genes. Results of a representative experiment using real-time RT-PCR of endometriosis-like lesions removed after different time intervals after autotransplantation. Mice had been treated systemically with 2-methoxyestradiol before the induction of endometriosis. Lesions were removed at different time points (baseline, 1 hour, 3 hours, 24 hours, 72 hours, and 1 week) and tested for mRNA expression of VEGF (A), PGK (B), and Glut-1 (C). Bars indicate SEM.

Figure 5

Figure 5

A: Western blot of endometriotic tissue samples at baseline (0 hours) and 6 hours. Lewis lung carcinoma is used as positive control in equivalent concentration. Staining with anti-HIF-1α antibody and anti-β actin as loading control. B: A modified Miles assay shows that systemic 2-methoxyestradiol treatment inhibits vascular permeability. Mice were pretreated orally with tap water (control), vehicle, and 2-methoxyestradiol for 5 days and then anesthetized and injected with Evans blue (i.v.). After 10 minutes, mice were given intradermal injections of PBS (50 μl; top two spots) and VEGF/vascular permeability factor (50 ng in 50 μl; bottom two spots), and mice were euthanized after 20 minutes. Reduced extravasation of dye in the skin of mice treated with 2-methoxyestradiol. C: Dye from excised skin was extracted with formamide for 5 days, and dye contents were measured at 620 nm. Data are expressed as mean ± SEM.

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