THE EXPRESSION OF MAJOR ECMPROTEINS-COLLAGEN AND ELASTIN-IS ENHANCED BY LOCAL ESTROGEN THERAPYIN POSTMENOPAUSAL WOMEN WITH SEVERE POP
T. TYAGI1, M. ALARAB 2,S. LYE 3, O. SHYNLOVA 4;
1LunenfeldTanenbaum Res. Inst., Toronto, Canada, 2Obstetrics andGynecology, Division Of Urogynecolo, Mount sinai Hosp., Toronto,Canada, 3Lunenfeld-Tanenbaum Res. Inst., Toronto, Canada,4Mount Sinai Hosp., Toronto, Canada.
Introduction: The severity ofPelvic Organ Prolapse (POP) increases after menopause, mirroringlover level of structural proteins, Collagens and Elastin, in thepelvic tissue of POP patients. It has been hypothesized that thismight be due to the loss of protective effects from ovarian hormonesleading to faster deterioration of pelvic floor extracellular matrix(ECM). Elastic fibres are structural macromolecular components of ECMcomprising of an Elastin core surrounded by a layer of Fibrillin-richmicrofibrils and Fibulin proteins. Local Estrogen Therapy (LET) hasbeen shown to improve vaginal health and the outcomes of urogenitalatrophy. Unlike Systemic Estrogen Therapy, LET imposes lower risk ofendometrial cancer. Previously, we had shown that LET significantlyi) increased the expression of Collagen1,3,5 genes, ii) decreased theprotein level of main tissue-degradation enzymes MMP1,2,3, whileexpression of MMP tissue inhibitors TIMP1,3 were significantlyincreased; iii) enhanced local immune response by inducing cytokineexpression and immune cell infiltration. Here, we hypothesized thatLET will increase the expression of structural proteins, Elastin andCollagens, as well as Elastin-related proteins Firbillin1,2 andFibulin5 in the vaginal tissue of post-menopausal women with severePOP.
Objective: To analyze the effect of LET treatment (inthe form of vaginal conjugated equine estrogen cream or tablets) on:(1) Gene expression of Elastin and Elastin-related proteins involvedin the deposition in mature Elastic fibres; (2) Total Collagen andElastin content; (3) Immunolocalization of Collagen 1, 3, and 5proteins within vaginal biopsy samples of women with POP.
Methods:Postmenopausal women undergoing vaginal hysterectomy or anteriorvaginal wall repair for POP (POPQ=3-4) were recruited. Vaginalbiopsies were collected from patients treated with LET (averageduration 14 months, N=29) and patients not using LET (N=23). Thebiopsies were frozen in liquid nitrogen for biochemical study orfixed in 10% formalin for histological staining andimmunohistochemistry. RNA was isolated, reverse transcribed to cDNAand used for Real Time RT-PCR. To measure protein levels of Collagenand Elastin within vaginal tissue, sections were stained withmodified Masson’s Trichrome histological stain. Visiopharm NewCastSoftware (version 184.108.40.20672) EngineTM and Viewer software module wasused to measure the total Collagen and Elastin content using modified10020-Orcein protocol. Immunolocalization of Collagen 1,3,5 proteinswas examined within vaginal tissues using specific antibodies fromAbcam.
Results: We examined the effect of LET on the mRNAlevels of Elastin (ELN) and proteins involved in its deposition intomature Elastin fibers, Firbillin1 (FBN1), Fibrillin2 (FBN2) andFibulin5 (FBLN5). All genes showed an increase expression levels invaginal biopsy samples from LET-treated patients compared to non-LETpatients with severe POP: FBN1, FBN2, and ELN showed a significantincrease (p<0.05, 1.7, 6.6, and 8.4 fold change correspondingly),while FBLN5 showed a 1.5 fold increase (p=0.24). Immunohistochemicalstaining for Collagen 1, 3, and 5 proteins show that they werelocalized to Lamina Propria layer of vaginal skin biopsy andexpressed a higher staining intensity in LET-treated vs no-LETsamples which directly correlated with their gene expression.Similarly, majority of the Collagen and Elastin histologic stainingwas localized to Lamina Propria. Software analysis detected asignificant increase (1.5-2.3 fold, p<0.05) in the total contentof both major ECM proteins, Collagen and Elastin, in postmenopausalPOP patients treated with LET as compared to non-LET POPpatients.
Conclusions: The increase in Collagen and Elastincontent, along with increased expression of proteins involved inElastin fibers formation, within the vaginal tissue of LET-treatedwomen suggests a beneficial role of LET in remediation of pelvicfloor ECM in postmenopausal patients with severe POP.
References:(1) Obstet. Gynecol 2010 Nov;116(5):1096-1100
Durationof LET (month)
Stageof POP (n)
Gene/Proteinanalysis of ECM, and related proteins (*p<0.05, **p<0.01)
LET(fold change vs control)