Microvascular network remodeling in dura mater of ovariectomized pigs: role for angiopoietin-1 in estrogen-dependent control of vascular stability

Abstract

Estrogen is a key regulator of vascular responses and angioadaptation in multiple organs and tissues, including brain. However, the consequences of a loss of ovarian steroid hormone secretion on the status of microvascular networks in brain and meninges are largely unknown. Here, using the perfused dura mater model coupled with high-resolution digital epifluorescence and laser scanning confocal microscopy and computer-assisted morphometric analysis, we demonstrate that cessation of ovarian hormone production causes dramatic vascular remodeling in meningeal microvascular networks characterized by a threefold decrease in microvessel density and capillary rarefaction and an almost fourfold increase in vascular permeability. These changes were accompanied by a significant decrease in angiopoietin-1 (Ang-1) expression and Ang-1/Tie-2 ratio (1.4-fold, P < 0.01, and 1.5-fold, P < 0.05, respectively) in ovariectomized animals compared with intact females, but no changes were detected in the expression of estrogen receptors (ER)-alpha and -beta. We conclude that estrogen-dependent control of Ang-1 expression plays an important role in stabilizing meningeal microvessel and maintaining healthy microvascular networks.

Fig. 1

Gradual remodeling of the terminal dura mater microvascular networks in pigs following ovariectomy (OVX). A–D: microvascular networks of intact female (IF; A) and OVX animals 1 mo (B), 1.5 mo (C), and 2 mo (D) post-OVX. Note the apparent loss of capillaries, increase in average microvessel size, and enhanced dye accumulation in perivascular space with time following OVX. Scale bar shown in D, 100 μm. E–G: response to OVX on a tissue volume occupied by terminal microvascular networks (E), blood vessel surface-to-volume ratio (F), and microvessel permeability (G). Bar graphs (E–G, top) show means ± SE, and box diagrams (E–G, bottom) show statistical distribution of data as lowest value, 25th percentile, median, 75th percentile, and highest value. H: immunohistochemical analysis of capillary density using antibody directed against endothelium-specific von Willebrand factor. Scale bar, 50 μm. Bar graph, means ± SE.

Fig. 2

Changes in estradiol (E₂) plasma levels and estrogen receptor (ER) expression following OVX. A: plasma levels of E₂ in IF (n = 9) and OVX (n = 5) pigs. In IF animals, in addition to the overall E₂ level (hatched bar), estrous (E; n = 3) and diestrous (DE; n = 6) E₂ levels are shown (open bars). The gray area indicates the magnitude of physiological fluctuations of plasma E₂ levels as per the literature. B: Western blot analysis of ER-α and ER-β expression in dura mater of IF and OVX Yucatan miniature pigs 2 mo post-OVX. In porcine ovary extract (OV), anti-ER-α antibody recognizes a full-length (∼66 kDa) receptor (red arrow) as well as higher (∼80 kDa) molecular weight (blue arrow) and truncated (∼50–52 kDa; black arrow) species. In dura mater samples (IF and OVX), however, truncated (∼50–52 kDa) ER-α-immunoreactive bands are clearly present, whereas ∼66 and ∼80 kDa are barely detectable. In contrast, the anti-ER-β antibody recognizes a single ∼59-kDa immu-noreactive band corresponding to a full-length receptor. C–E: immunohistochemical analysis of ER-α (C and E) and ER-β (D) expression in dura mater of IF and OVX Yucatan miniature pigs 2 mo post-OVX. The brown color represents ER-α (C and E) and ER-β (D) immunoreactivity. In E, note cytoplasmic (arrows) and nuclear (arrowheads) expression of ER-α in both vascular smooth muscle (black arrows and black arrowheads) and endothelial (white arrows and white arrowheads) cells in arteries (A) and veins (V) of various diameters. Scale bars, 50 μm.

Fig. 3

Western blot and immunohistochemical analysis of angiopoietin-1 (Ang-1; A, C, and F) and Tie-2 (B, D, and G) expression in dura mater of IF and OVX Yucatan miniature pigs 2 mo post-OVX. Note the 1.4-fold decrease in Ang-1-normalized relative expression (C) and 1.5-fold decrease in Ang-1-to-Tie-2 ratio (E) in OVX animals compared with IF, whereas Tie-2 expression (D) remains unchanged. C–E: bar graphs show means ± SE. F and G: note vascular expression of Ang-1 (F) and Tie-2 (G). The Ang-1 immunoreactivity (F) is confined to the vascular smooth muscle cells (black arrows) in both IF (left) and OVX (right) animals and is markedly reduced in OVX pigs compared with IF. In contrast, there is no difference in the immunoreactivity of the Ang-1 receptor Tie-2 between the groups (G), and Tie-2 expression is evident in both smooth muscle cells (black arrows) and endothelial cells (white arrows). Scale bars, 50 μm.

Fig. 4

Schematic representation of a proposed mechanism of the ovarian-dependent control of vascular stability. Loss of ovarian E₂ production results in a reduced ER-α-dependent Ang-1 synthesis, leading to a decrease in vessel-stabilizing Tie-2 signaling, microvessel destabilization, and post-OVX vascular remodeling.