Catecholestrogens are biologically active metabolites of 17β-estradiol (E2) synthesized by cytochrome P450 enzymes forming 2-Hydroxyestradiol (OHE2) and 4-OHE2. Although catecholestrogens bind estrogen receptors (ERs) with low affinity, their high affinity for β2-adrenergic receptors (β2-AR) regulates uterine endothelial cell proliferation in gestation1. Women with endometriosis have aberrant E2 metabolizing enzymes potentially inducing catecholestrogen production2. We hypothesize that ectopic and eutopic endometrial microvascular endothelial cells express β2-AR and that, under stimulation by 2-OHE2, endometrial endothelial cells have increased cell viability and an effect on endothelial tube stabilization. Paired eutopic/ectopic endometrial sections from women with endometriosis in the proliferative (n=5) or secretory (n=4) phases were immunostained for β2-AR and evaluated semi-quantitatively by HSCORE. Confluent human endometrial endothelial cells (HEEC) grown in 96-well plates were pretreated with control or with β-AR antagonist propranolol (2x10-6 M) followed by treatment with vehicle (control) or with 10-8 M E2 or 2-OHE2. Then, vascular tube formation (15x103 cells/well, n=3) and endothelial proliferation (5x103 cells/well, n=3) were evaluated. Tube formation was analyzed at 2, 4, 8, 16, and 24h post-treatment. Images were utilized to score endothelial tube formation and degradation based on the following scores: initial tube coalescence= 1, initiation of tube networks= 2, completed tube networks= 3, vascular destabilization= 4, and complete loss of vascular network= 5. XTT assays measured cell viability as an indicator of proliferation at 24h post-treatment. Statistical analysis used a t-test or One-way ANOVA and post hoc Tukey test. Immunohistochemistry analysis revealed increased β2-AR staining in vascular endothelial cells in ectopic compared to eutopic tissue during the proliferative phase (P<0.05) and showed a trend toward increased β2-AR staining in eutopic compared to ectopic tissue in the secretory phase; however, it did not achieve significance (P=0.055). In vitro endothelial tube formation analyses revealed no difference in scores at 2, 4, and 8h post-treatment with both E2 and 2-OHE2 compared with control. However, a difference at 16 and 24h post-treatment existed in that treatment with E2 and 2-OHE2 led to lower scores (mean = 3) compared to control (mean = 5) indicating a stabilization of endothelial tube structure in the treatment groups. XTT analyses revealed that compared to control, there were no significant differences in proliferative index in the treatment groups compared to control (P>0.05).Figure 2Endothelial Tube Formation Assay showing control compared to E2 10-8 M or 2-OHE2 10-8 M at hours 2, 4, 8, 16, and 24h post-treatment.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The increased β2-AR expression in endothelial cells in ectopic endometriotic tissue during the proliferative phase suggests it plays a role in angiogenesis during implantation of ectopic endometrial tissue. Our in vitro data, that stabilization of newly formed endothelial tube network by 2-OHE2, suggest that the effect of 2-OHE2 may be mediated by β2-AR on endometriotic endothelial cells to maturate newly formed vasculatures during angiogenesis, thereby contributing to endometriosis pathogenesis.