TGF-beta 1 is a potential regulator of vaginal tropoelastin production.

INTRODUCTION AND HYPOTHESIS: Our aims were to correlate transforming growth factor (TGF)-ß1 and elastin mRNA expression in the vagina of women and to measure the effects of TGF-ß1 on vaginal smooth muscle cell (SMC) proliferation and tropoelastin production. METHODS: Vaginal walls were sampled in women (n = 20). TGF-ß1 and elastin mRNA expression was assessed by RT-PCR. SMC cultures were performed from vaginal wall biopsies. SMC were incubated with TGF-ß1, and cell proliferation was assessed by MTT-assay. Tropoelastin production was measured by the Fastin Elastin Assay. RESULTS: There was a significant positive correlation between TGF-ß1 and elastin mRNA (r = 0.784, P < 0.01). SMC proliferation was significantly increased by 10 ng/mL TGF-ß1 [relative cell number, mean ± SD, 198% ± 32% of control (P = 0.01)]. Tropoelastin production was significantly increased by TGF-ß1 [mean ± SD, 645% ± 180% of control (P = 0.01)]. CONCLUSIONS: There is a positive correlation between TGF-ß1 and elastin mRNA expression in the vaginal wall. In vitro, TGF-ß1 increases vaginal tropoelastin production in vaginal SMC.

The effect of biological and synthetic meshes on vaginal smooth muscle cell proliferation.

AIMS: To compare the effects of polypropylene mesh or porcine dermal acellular collagen matrix mesh with and without estradiol supplementation on vaginal smooth muscle cells (VaSMC) proliferation. RESULTS: Under in vitro culture conditions, VaSMC proliferation was significantly higher in the porcine dermal acellular collagen matrix mesh exposed cells compared to the type I polypropylene mesh exposed cells (relative cell number, mean ± SD, 0.27 ± 0.03 vs. 0.21 ± 0.01, P = 0.03). Under estradiol supplementation cell proliferation in the porcine mesh exposed cells remained significantly higher compared to the polypropylene mesh exposed cells (relative cell number, mean ± SD, 0.27 ± 0.04 vs. 0.15 ± 0.03, P < 0.01). CONCLUSION: The decreased rate of erosion found with the utilization of biological absorbable mesh in vaginal reconstructive surgery may partially be explained by the significantly increased VaSMC proliferation with porcine dermal acellular collagen mesh compared to polypropylene mesh.

Cellular proliferation in female pelvic organ prolapse: a pilot study.

PURPOSE: To assess cell proliferation in pelvic organ prolapse (POP). METHODS: Tissue samples of the anterior vaginal wall and uterosacral ligaments (USLs) were obtained from eight women with combined anterior vaginal wall and uterine prolapse and from eight women without POP in a standardized fashion. Immunohistochemistry against Ki-67 was used to assess cell proliferation in vaginal and USL biopsies. RESULTS: There were no significant differences in age, parity, menopausal status or hormone replacement therapy between the two groups. The POP-Q stage of uterine and anterior vaginal wall prolapse was significantly higher in the group of women with prolapse compared to the group without prolapse [median (range) 3 (3-4) vs. 0 (0), <0.01]. There was no significant difference between Ki-67 expressions in women with or without prolapse. CONCLUSION: There were no significant differences in cell proliferation between samples from women with or without POP.

Differential expression of fibulins in the uterosacral ligaments of women with uterine prolapse.

PURPOSE: To compare fibulin-3 (FIB-3) and fibulin-5 (FIB-5) expressions in uterosacral ligaments (USL) of women with and without uterine prolapse. STUDY DESIGN: USL were sampled in a standardized fashion from women with (n = 8) or without (n = 8) uterine prolapse. Quantitative Real-Time PCR was performed to measure mRNA and immunohistochemistry to assess protein expression. RESULTS: FIB-3 mRNA expression and FIB-3 staining intensity was similar in the USL of women with and without uterine prolapse {[(FIB-3 mean +/- SD mRNA relative units) 0.45 +/- 0.41 vs. 0.46 +/- 0.82, NS] and [Intensity score, median (range), 1(0-1) vs. 1(0-1), NS]}. Both FIB-5 mRNA expression and FIB-5 staining intensity was significantly decreased in USL from women with uterine prolapse compared to women without prolapse {[(FIB-5 mean +/- SD mRNA relative units) 0.07 +/- 0.02 vs. 0.26 +/- 0.20, P = 0.02] and [Intensity score, median (range), 0(0-2) vs. 3(2-3), P = 0.002]}. CONCLUSION: FIB-5 expression is decreased in USL of women with uterine prolapse.

Levormeloxifene inhibits vaginal tropoelastin and transforming growth factor beta 1 production.

PURPOSE: To measure the effects of levormeloxifene on vaginal smooth muscle cell (SMC) proliferation, tropoelastin and transforming growth factor (TGF)-ß1 production. METHODS: Primary SMC cultures were performed from vaginal wall biopsies. SMC were incubated with levormeloxifene (0.1 µM, 1 µM), in 96-well plates and cell proliferation was assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide (MTT) assay at 24 hours. Tropoelastin production was measured by the Fastin Assay kit and TGF-ß1 levels were assessed by ELISA. RESULTS: SMC proliferation was significantly increased by levormeloxifene [relative cell number, mean ± SE, levormeloxifene 0.1 µM 130 ± 13% of control (P=NS), 1 µM 151 ± 19% of control (P<0.05)]. Tropoelastin production was significantly decreased by levormeloxifene [mean ± SE, levormeloxifene 0.1 µM 75 ± 4% of control (P=NS), 1 µM 64 ± 2% of control (P<0.05)]. In addition, TGF-ß1 production was significantly decreased [mean ± SE, levormeloxifene 0.1 µM 79 ± 11% of control (P=NS), 1 µM 72 ± 14% of control (P<0.05)]. CONCLUSIONS: Levormeloxifene increases vaginal SMC proliferation, inhibits tropoelastin and TGF- ß1 production.

Normal vulvovaginal, perineal, and pelvic anatomy with reconstructive considerations.

A thorough insight into the female genital anatomy is crucial for understanding and performing pelvic reconstructive procedures. The intimate relationship between the genitalia and the muscles, ligaments, and fascia that provide support is complex, but critical to restore during surgery for correction of prolapse or aesthetic reasons. The external female genitalia include the mons pubis, labia majora and minora, clitoris, vestibule with glands, perineal body, and the muscles and fascia surrounding these structures. Through the perineal membrane and the perineal body, these superficial vulvar structures are structurally related to the deep pelvic muscle levator ani with its fascia. The levator ani forms the pelvic floor with the coccygeus muscle and provides vital support to all the pelvic organs and stability to the perineum. The internal female genital organs include the vagina, cervix, uterus, tubes, and ovaries with their visceral fascia. The visceral fascia also called the endopelvic fascia, surrounds the pelvic organs and connects them to the pelvic walls. It is continuous with the paraurethral and paravaginal fascia, which is attached to the perineal membrane. Thus, the internal and external genitalia are closely related to the muscles and fascia, and work as one functioning unit.

CD1d degradation in Chlamydia trachomatis-infected epithelial cells is the result of both cellular and chlamydial proteasomal activity.

Chlamydia trachomatis is an obligate intracellular pathogen that can persist in the urogenital tract. Mechanisms by which C. trachomatis evades clearance by host innate immune responses are poorly described. CD1d is MHC-like, is expressed by epithelial cells, and can signal innate immune responses by NK and NKT cells. Here we demonstrate that C. trachomatis infection down-regulates surface-expressed CD1d in human penile urethral epithelial cells through proteasomal degradation. A chlamydial proteasome-like activity factor (CPAF) interacts with the CD1d heavy chain, and CPAF-associated CD1d heavy chain is then ubiquitinated and directed along two distinct proteolytic pathways. The degradation of immature glycosylated CD1d was blocked by the proteasome inhibitor lactacystin but not by MG132, indicating that degradation was not via the conventional proteasome. In contrast, the degradation of non-glycosylated CD1d was blocked by lactacystin and MG132, consistent with conventional cellular cytosolic degradation of N-linked glycoproteins. Immunofluorescent microscopy confirmed the interruption of CD1d trafficking to the cell surface, and the dislocation of CD1d heavy chains into both the cellular cytosol and the chlamydial inclusion along with cytosolic CPAF. C. trachomatis targeted CD1d toward two distinct proteolytic pathways. Decreased CD1d surface expression may help C. trachomatis evade detection by innate immune cells and may promote C. trachomatis persistence.

Distinct defensin profiles in Neisseria gonorrhoeae and Chlamydia trachomatis urethritis reveal novel epithelial cell-neutrophil interactions.

Defensins are key participants in mucosal innate defense. The varied antimicrobial activity and differential distribution of defensins at mucosal sites indicate that peptide repertoires are tailored to site-specific innate defense requirements. Nonetheless, few studies have investigated changes in peptide profiles and function after in vivo pathogen challenge. Here, we determined defensin profiles in urethral secretions of healthy men and men with Chlamydia trachomatis- and Neisseria gonorrhoeae-mediated urethritis by immunoblotting for the epithelial defensins HBD1, HBD2, and HD5 and the neutrophil defensins HNP1 to -3 (HNP1-3). HBD1 was not detectable in secretions, and HBD2 was only induced in a small proportion of the urethritis patients; however, HD5 and HNP1-3 were increased in C. trachomatis infection and significantly elevated in N. gonorrhoeae infection. When HNP1-3 levels were low, HD5 appeared mostly as the propeptide; however, when HNP1-3 levels were >10 microg/ml, HD5 was proteolytically processed, suggesting neutrophil proteases might contribute to HD5 processing. HD5 and HNP1-3 were bactericidal against C. trachomatis and N. gonorrhoeae, but HD5 activity was dependent upon N-terminal processing of the peptide. In vitro proteolysis of proHD5 by neutrophil proteases and analysis of urethral secretions by surface-enhanced laser desorption ionization substantiated that neutrophils contribute the key convertases for proHD5 in the urethra during these infections. This contrasts with the small intestine, where Paneth cells secrete both proHD5 and its processing enzyme, trypsin. In conclusion, we describe a unique defensin expression repertoire in response to inflammatory sexually transmitted infections and a novel host defense mechanism wherein epithelial cells collaborate with neutrophils to establish an antimicrobial barrier during infection.