Divergent regulation of angiopoietin-1 and -2, Tie-2, and thrombospondin-1 expression by estrogen in the baboon endometrium

Abstract

Estrogen has an important role in the reconstruction of a new vascular network in the endometrium during each menstrual cycle; however, the underlying mechanisms are incompletely understood. Angiopoietin-1 (Ang-1) promotes vessel assembly, whereas Ang-2 and thrombospondin-1 (TSP-1) cause vessel breakdown. To determine the potential effect of estrogen on the expression of these angioregulatory factors in the endometrium, Ang-1, Ang-2, TSP-1, and Tie-2 receptor mRNA levels were assessed by real-time reverse transcriptase polymerase chain reaction in glandular epithelial and stromal cells isolated from the endometrium of ovariectomized baboons treated acutely with estradiol. Corresponding protein expression was assessed by immunocytochemistry and the proximity ligation assay (PLA) during advancing stages of the baboon menstrual cycle. Serum estradiol levels in ovariectomized baboons were 400 pg/ml within 4-6 hr of estradiol treatment. Ang-1 mRNA levels in glandular epithelial cells increased threefold (P < 0.01) within 4 hr of estradiol administration. In contrast, TSP-1 mRNA levels decreased four- to fivefold (P < 0.01) in endometrial glandular epithelial and stromal cells 4-6 hr after estradiol, whereas Ang-2 and Tie-2 expression was unaltered. Immunostaining for Ang-1 increased, TSP-1 decreased, and Ang-2 and Tie-2 were unaltered in the endometrium during the secretory compared with the proliferative phase of the cycle. Endometrial Ang-1 protein expression, quantified by PLA, increased 10-fold (P < 0.05) between the early proliferative and late proliferative/mid-secretory phases of the menstrual cycle in association with the rise in estrogen. In summary, estrogen induced a rapid, divergent, and cell-specific change in expression of angiostimulatory and angioinhibitory growth factors in the endometrium of the nonhuman primate.

Figure 1

Serum estradiol levels (means ± SE) in ovariectomized baboons after administration of estradiol (bolus intravenous injection at 1 µg/kg body weight and subcutaneous insertion of three SILASTIC capsules containing estradiol) at time 0 hr (n = 11 baboons).

Figure 2

Endometrial angiopoietin-1 (Ang-1; A)andthrombospondin-1 (TSP-1; B) mRNA levels (means ± SE, corrected for 18S rRNA) determined by real-time reverse transcriptase-polymerase chain reaction in glandular epithelial and stromal cells isolated by laser capture microdissection from ovariectomized baboons (n = 11) treated at time 0 hr with estradiol as detailed in the legend of Figure 1. *Ang-1 mRNA levels different (P < 0.01) from values at time 0hr (ANOVA and Newman–Keul’s multiple comparison test). *TSP-1 mRNA levels collectively at 4 and 6hr different (P < 0.03, glands; P < 0.01, stroma) from values at 0 or 2 hr (Mann–Whitney test).

Figure 3

Angiopoietin-2 (Ang-2; A) and Tie-2 (B) mRNA levels (ratio to 18S rRNA) in glandular epithelial and stromal cells of the estradiol-treated baboons in which Ang-1 and thrombospondin-1 mRNA levels are shown in Figure 2.

Figure 4

Representative immunocytochemical localization (brown precipitate) of angiopoietin-1 (Ang-1; A, B), Ang-2 (C, D), Tie-2 receptor (E, F), and thrombospondin-1 (G, H) in the endometrium during the early (A, G) and late (C, E) proliferative and mid-late secretory (B,D,F,H) phases of the baboon menstrual cycle; (I) replacement of primary antibody with goat immunoglobulin G; (J) replacement of secondary anti-goat immunoglobulin in the Tie-2 reaction with secondary anti-mouse immunoglobulin. G, gland; M, microvessel. Final approximate magnification 200× in each panel, 640× in the inserts of panels A and B, and 400× in the remaining inserts.

Figure 5

Angiopoietin-1 protein expression assessed by the proximity ligation assay (PLA) in the endometrium during the early proliferative (A), late proliferative (B), and mid-secretory (C) phases of the baboon menstrual cycle. (D) Negative control with omission of primary Ang-1 antibody. Each red PLA signal/dot represents a single molecule of Ang-1 protein detected by primary Ang-1 antibody tagged with secondary antibody conjugated to fluorescently labeled oligonucleotide. Nuclei are labeled blue and red blood cells autofluoresce as yellow. G, glandular epithelium.

Figure 6

Angiopoietin-1 protein expression quantified by the proximity ligation assay (PLA) and Image J software (mean ± SE; PLA signals/nuclear area × 10⁴, panel A; or per 32,350 µm² endometrial area, panel B) in the endometrium during the early proliferative (EP, n = 3 baboons), late proliferative (LP, n = 3), and mid-secretory (MS, n = 3) phases of the baboon menstrual cycle. Values are different (*P < 0.01 or **P< 0.05) from EP (ANOVA and Neuman–Keul’s multiple comparison test).