Vascular endothelial growth factor mediates the estrogen-induced breakdown of tight junctions between and increase in proliferation of microvessel endothelial cells in the baboon endometrium

Abstract

To assess whether there is a link between estrogen, vascular endothelial growth factor (VEGF), and early aspects of uterine angiogenesis, an acute temporal study was conducted in which ovariectomized baboons were pretreated with VEGF Trap, which sequesters endogenous VEGF, and administered estradiol at time 0 h. Serum estradiol levels approximated 500 pg/ml 4-6 h after estradiol administration. VEGF mRNA levels in endometrial glandular epithelial and stromal cells were increased to values 6 h after estradiol that were 3.74 +/- 0.99-fold (mean +/- se) and 5.70 +/- 1.60-fold greater (P < 0.05), respectively, than at 0 h. Microvessel interendothelial cell tight junctions, which control paracellular permeability, were present in the endometrium at time 0 h, but not evident 6 h after estradiol administration. Thus, microvessel paracellular cleft width increased (P < 0.01, ANOVA) from 5.03 +/- 0.22 nm at 0 h to 7.27 +/- 0.48 nm 6 h after estrogen. In contrast, tight junctions remained intact, and paracellular cleft widths were unaltered in estradiol/VEGF Trap and vehicle-treated animals. Endometrial microvessel endothelial cell mitosis, i.e. percent Ki67+/Ki67- immunolabeled endothelial cells, increased (P < 0.05) from 2.9 +/- 0.3% at 0 h to 21.4 +/- 7.0% 6 h after estrogen treatment but was unchanged in estradiol/VEGF Trap and vehicle-treated animals. In summary, the estrogen-induced disruption of endometrial microvessel endothelial tight junctions and increase in endothelial cell proliferation were prevented by VEGF Trap. Therefore, we propose that VEGF mediates the estrogen-induced increase in microvessel permeability and endothelial cell proliferation as early steps in angiogenesis in the primate endometrium.

Figure 1

Serum estradiol levels (means ± se) in ovariectomized baboons after administration of estradiol (bolus iv injection at 1 μg/kg body weight and sc insertion of three SILASTIC brand capsules containing estradiol) at time 0 h (n = 10), VEGF Trap (bolus iv injection at 1 mg/kg body weight) at −24 h plus estradiol at 0 h (n = 4), or saline/hFc vehicle at 0 h (n = 8).

Figure 2

Endometrial VEGF mRNA levels (fold increase vs. 0 h, means ± se) determined by real-time RT-PCR in glandular epithelial and stromal cells isolated by LCM from ovariectomized baboons treated with estradiol as detailed in the legend of Fig. 1. *, Significantly different (P < 0.05, ANOVA and Dunnett test) vs. value at 0 h.

Figure 3

Electron photomicrographs illustrating endometrial tight junctions (arrowheads) and paracellular clefts between adjacent microvessel endothelial cells of baboons 0 h (A and C) and 6 h (B and D) after administration of estradiol (A and B) or estradiol plus VEGF Trap (C and D). Scale bar, 20 nm.

Figure 4

Net gain (means ± se) in paracellular cleft width (nm) between adjacent endometrial microvessel endothelial cells of ovariectomized baboons before (0 h) and 6 h after acute administration of estradiol (E₂) (n = 10), estradiol plus VEGF Trap (n = 4), or saline/hFc vehicle (n = 8). *, Different at P < 0.001 (ANOVA and Newman-Keuls test) vs. other treatment groups.

Figure 5

Ki67 and von Willebrand factor (vWF) immunocytochemical labeling of endometrial microvessel endothelial cells in baboons at 0 h (A and B) and 6 h after the administration of estradiol (E₂) (C and D) or estradiol plus VEGF Trap (E and F). G and H, Isotype-matched secondary Ig IgM controls. Magnification, ×400.

Figure 6

Proliferation of endometrial microvessel endothelial cells, expressed as percentage of Ki67⁺/Ki67 ⁻ cells, in baboons 0, 2, and 6 h after administration of estradiol (E₂) (n = 5), estradiol plus VEGF Trap (n = 3), or saline/hFc vehicle (n = 5). *, Different (P < 0.05, ANOVA and Newman-Keuls test) from time 0 h.