Estrogen alters remodeling of the vaginal wall after surgical injury in guinea pigs

Abstract

Loss of pelvic organ support (i.e., pelvic organ prolapse) is common in menopausal women. Surgical reconstruction of pelvic organ prolapse is plagued with high failure rates. The objective of this study was to determine the effects of estrogen on biomechanical properties, lysyl oxidase (LOX), collagen content, and histomorphology of the vagina with or without surgical injury. Nulliparous ovariectomized guinea pigs were treated systemically with either 50 μg/kg/day estradiol (E2,) or vehicle. After 2 wk, vaginal surgery was performed, and animals were treated with either beta-aminopropionitrile (BAPN, an irreversible LOX inhibitor), or vehicle to determine the role of LOX in recovery of the vaginal wall from injury with or without E2. Estradiol resulted in (i) significant growth, increased smooth muscle, and increased thickness of the vagina, (ii) increased distensibility without compromise of maximal force at failure, and (iii) increased total and cross-linked collagen. In the absence of E2, BAPN resulted in decreased collagen and vaginal wall strength in the area of the injury. In contrast, in E2-treated animals, increased distensibility, maximal forces, and total collagen were maintained despite BAPN. Interestingly, LOX mRNA was induced dramatically (9.5-fold) in the injured vagina with or without E2 at 4 days. By 21 days, however, LOX levels declined to near baseline in E2-deprived animals. LOX mRNA levels remained strikingly elevated (12-fold) at 21 days in the estrogenized vagina. The results suggest that prolonged E2 induced increases in LOX, and collagen cross-links may act to sustain a matrix environment that optimizes long-term surgical wound healing in the vagina.

Keywords: collagen; estradiol/estradiol receptor; lysyl oxidase; matrix remodeling; menopause; pelvic organ prolapse; vagina.

FIG. 1

Vaginal surgical injury (posterior colpoperineorrhaphy) in the guinea pig model. A) Stay sutures placed in posterior vaginal wall at perineum and midvagina. B) Perineum incised and vaginal wall undermined. C) Full-thickness strip of posterior vaginal wall excised (arrow). D) Primary reapproximation of wound.

FIG. 2

Effect of estrogen on growth of the female reproductive tract in ovariectomized guinea pigs. A) Vulva of ovariectomized guinea pigs treated with vehicle was characterized by a membrane covering the vaginal introitus (arrow). The membrane was dissolved in estrogen-treated animals. B) Vaginal (vag, light gray bar), uterine (ut, dark gray bar), and cervical (cvx, black bar) weight were increased significantly in estrogen-treated animals compared with vehicle-treated controls. Bladder (Bl, white bar) weights were similar between the two groups. Each bar represents mean ± SEM from six animals in each group. *P < 0.01 compared with corresponding organ from controls. C) Gross appearance of female reproductive tract in vehicle and estrogen-treated animals.

FIG. 3

Effect of E2 on histology of the vaginal wall. A) Representative photographs of Masson (a, b) and Hart (c, d) -stained vaginal rings from ovariectomized animals treated with vehicle (a, c) or E2 (b, d) for 21 days. Note increased thickness of epithelium (epi) and muscularis (mus) in E2-treated animals. Sections were taken at midvagina. Dotted line delineates epithelium (epi) from underlying lamina propria (l.p.) and muscularis (mus). Arrows indicate elastic fibers. Bar = 200 μm for a, b; 50 μm for c, d. B) Thickness of epi, l.p., mus, and total wall (Total) in control (n = 6, open bar) and E2-treated (n = 6, closed bar) animals. *P < 0.05 C. Elastin density in subepithelium (within 500 μm of basal lamina) and muscularis of control (open bar) and E2-treated (closed bar) animals. Data represent mean ± SEM of noninjured animals with similar results 21 days after the injury. *P < 0.05 compared with control.

FIG. 4

Effect of injury and E2 on collagen and tropoelastin mRNA in the vaginal wall. Col1A2 (A), Col3A1 (B), and tropoelastin (C) mRNA levels were quantified in vaginal tissues from noninjured (NI) or injured (Inj) ovariectomized vehicle- (Veh) or E2-treated animals at 4 and 21 days after injury. Data were normalized to B2M and represent the mean ± SEM of six animals in each group. *P < 0.05 compared with NI. **P < 0.05 compared with controls, two-way ANOVA.

FIG. 5

Effect of injury and E2 on TGFβ1 and LOX gene expression in the vaginal wall. TGFβ1 (A) and LOX (B) mRNA levels were quantified in vaginal tissues from noninjured (NI) or injured (Inj) ovariectomized vehicle- (Veh) or E2-treated animals at 4 and 21 days after injury. Data were normalized to B2M and represent mean ± SEM of six animals in each group. *P < 0.05 compared with NI. **P < 0.05 compared with controls, two-way ANOVA.

FIG. 6

Effect of E2 and injury on collagen content of the vaginal wall. A) Effect of E2 on total collagen content in the vagina from ovariectomized vehicle- (Veh) or E2-treated noninjured (NI) or injured (Inj) animals after 21 days. *P < 0.05 compared with NI. **P < 0.05 compared with NI Veh. B) Effect of E2 on immature (open bar), denatured (grey bar), or mature (solid bar) collagen content in the vagina from ovariectomized Veh- or E2-treated NI or Inj animals. *P < 0.05 compared with NI, paired analysis; **P < 0.05 compared with controls. Data represent mean ± SEM of six animals in each group.

FIG. 7

Effect of E2 and BAPN on biomechanical properties of the vaginal wall in noninjured and injured animals 21 days after the surgery. Stiffness and maximal force were determined in vaginal rings from noninjured (NI) or injured (Inj) ovariectomized vehicle- or E2-treated animals treated with control (CTL) or BAPN as described in Materials and Methods. Data represent mean ± SEM of six animals in each group. *P < 0.05 compared with corresponding control t-test.