Static Mechanical Loading Influences the Expression of Extracellular Matrix and Cell Adhesion Proteins in Vaginal Cells Derived From Premenopausal Women With Severe Pelvic Organ Prolapse.

INTRODUCTION: Primary human vaginal cells derived from women with severe pelvic organ prolapse (POP-HVCs) demonstrate altered cellular characteristics as compared to cells derived from asymptomatic women (control-HVCs). Using computer-controllable Flexcell stretch unit, we examined whether POP-HVCs react differently to mechanical loading as compared to control-HVCs by the expression of extracellular matrix (ECM) components, cell-ECM adhesion proteins, and ECM degrading and maturating enzymes. METHODS: Vaginal tissue biopsies from premenopausal patients with Pelvic Organ Prolapse Quantification System stage ≥3 (n = 8) and asymptomatic controls (n = 7) were collected during vaginal hysterectomy or repair. Human vaginal cells were isolated by enzymatic digestion, seeded on collagen (COLI)-coated plates, and stretched (24 hours, 25% elongation). Total RNA was extracted, and 84 genes were screened using Human ECM and Adhesion Molecules polymerase chain reaction array; selected genes were verified by quantitative reverse transcription-polymerase chain reaction. Stretch-conditioned media (SCM) were collected and analyzed by protein array, immunoblotting, and zymography. RESULTS: In mechanically stretched control-HVCs, transcript levels of integrins (ITGA1, ITGA4, ITGAV, and ITGB1) and matrix metalloproteinases (MMPs) 2, 8, and 13 were downregulated (P < .05); in POP-HVCs, MMP1, MMP3, and MMP10, ADAMTS8 and 13, tissue inhibitor of metalloproteinases (TIMPs) 1 to 3, ITGA2, ITGA4, ITGA6, ITGB1, contactin (CNTN1), catenins (A1 and B1), and laminins (A3 and C1) were significantly upregulated, whereas COLs (1, 4, 5, 6, 11, and 12) and LOXL1 were downregulated. Human vaginal cells massively secrete MMPs and TIMPs proteins; MMP1, MMP8, MMP9 protein expression and MMP2 gelatinase activity were increased, whereas TIMP2 decreased in SCM from POP-HVCs compared to control-HVCs. CONCLUSIONS: Primary human vaginal cells derived from women with severe pelvic organ prolapse and control-HVCs react differentially to in vitro mechanical stretch. Risk factors that induce stretch may alter ECM composition and cell-ECM interaction in pelvic floor tissue leading to the abatement of pelvic organ support and subsequent POP development.

Comparative Characterization of Vaginal Cells Derived From Premenopausal Women With and Without Severe Pelvic Organ Prolapse.

BACKGROUND: This study tested a hypothesis that primary human vaginal cells derived from tissue of premenopausal women with severe pelvic organ prolapse (POP-HVCs) would display differential functional characteristics as compared to vaginal cells derived from asymptomatic women with normal pelvic floor support (control-HVCs). METHODS: Vaginal tissue biopsies were collected from premenopausal patients with POP (n = 8) and asymptomatic controls (n = 7) during vaginal hysterectomy or repair. Primary vaginal cells were isolated by enzymatic digestion and characterized by immunocytochemistry. Cell attachment and proliferation on different matrices (collagen I, collagen II, collagen IV, fibronectin, laminin, tenascin, and vitronectin) were compared between POP-HVCs and control-HVCs. RNA was extracted, and the expression of 84 genes was screened using Human Extracellular Matrix and Adhesion Molecules RT(2) Profiler PCR array. The expression of selected genes was verified by quantitative reverse transcription-polymerase chain reaction. RESULTS: (1) Control-HVCs attached to collagen IV more efficiently than POP-HVCs; (2) control-HVCs and POP-HVCs show a similar proliferation rate when plated on proNectin and collagen I; (3) when seeded on collagen I, resting POP-HVCs expressed significantly (P < .05) increased transcript levels of collagen VII, multiple matrix metalloproteinases (MMP3, MMP7, MMP10, MMP12, MMP13, and MMP14), integrins (ITGA1, ITGA4, ITGA6, ITGA8, ITGB1, ITGB2, and ITGB3), and cell adhesion molecules as compared to control-HVCs. Collagen XV and tissue inhibitors of MMPs (TIMP1 and TIMP2) as well as genes involved in the biogenesis and maturation of collagen and elastin fibers (LOX, LOXL1-LOXL3, BMP1, and ADAMTS2) were significantly downregulated in POP-HVCs versus control-HVCs (P < .05). CONCLUSIONS: Resting primary POP-HVCs in vitro show altered cellular characteristics as compared to control-HVCs, which may influence their dynamic responses to external mechanical or hormonal stimuli.

Attachment of Primary Vaginal Fibroblasts to Absorbable and Nonabsorbable Implant Materials Coated With Platelet-Rich Plasma: Potential Application in Pelvic Organ Prolapse Surgery.

OBJECTIVES: Pelvic organ prolapse (POP) is a common condition in women. The lifetime risk of undergoing surgery to correct POP is 11%, with 30% recurrence rate. Various types of vaginal implants, absorbable and nonabsorbable, that have been introduced in pelvic floor reconstructive surgeries have numerous serious adverse effects. Platelet-rich plasma (PRP) is an autologous product that accelerates tissue healing and regeneration. We hypothesized that autologous PRP will promote human vaginal fibroblast (HVF) attachment to vaginal implants and increase their healing potential. METHODS: Vaginal tissue biopsies were collected from postmenopausal patients with POP (n = 10) and asymptomatic control subjects (n = 4) during vaginal hysterectomy or repair. Primary cells were isolated and characterized by immunocytochemistry. Cell attachment and proliferation were compared between POP HVFs and control HVFs (n = 4/group). Twelve weeks after the surgery, blood samples were collected from 6 POP patients to obtain autologous PRP. Two meshes, absorbable (Vicryl) and nonabsorbable (Restorelle), were coated in PRP or control media; autologous POP HVFs (n = 6) were seeded on meshes for 2 hours. Cells attached to the meshes were fixed, stained with DAPI (4,6-diamidino-2-phenylindole dihydrochloride), and counted. RESULTS: Pelvic organ prolapse HVFs were similar to control HVFs in attachment to different matrix substrates and in proliferation rate. Attachment of POP HVFs to both meshes was significantly increased after coating with PRP versus Dulbecco modified Eagle medium (Vicryl: 9875 vs. 1006 cells/cm, Restorelle: 3724 vs. 649 cells/cm; P < 0.001 for both). CONCLUSIONS: In vitro, primary POP HVFs show better attachment to implant materials when treated with PRP, which may lead to reduced mesh-related complications in vivo, indicating its great potential for urogynecologic surgeries.

Expression of extracellular matrix-remodeling proteins is altered in vaginal tissue of premenopausal women with severe pelvic organ prolapse.

AIM: The molecular etiology of pelvic organ prolapse (POP) is complex and not well understood. We compared the expression/activity of extracellular matrix (ECM)-processing (procollagen I N-proteinase/ a disintegrin and metalloproteinase with thrombospondin motifs [ADAMTS]-2,-3,-14) and ECM-degrading (matrix metalloproteinase [MMP]-1, -2, -7, -8, -9, -12) enzymes and their natural tissue inhibitors (tissue inhibitors of metalloproteinase [TIMP]-1,-2,-3,-4) in vaginal tissues from premenopausal women with advanced POP (POP-Q stage ≥ 3) and asymptomatic controls (POP-Q = 0). STUDY DESIGN: We sampled the anterior vaginal wall of 36 premenopausal women (17 patients with POP and 19 controls) undergoing total hysterectomy. Exclusion criteria include steroid therapy, malignancy, previous pelvic surgery, and connective tissue diseases. Total RNAs and proteins were quantified by real-time polymerase chain reaction, immunoblotting, and Luminex assay; MMPs activity was analyzed by zymography and tissue localization by immunohistochemistry. RESULTS: The MMP-2 gelatinase activity as well as expression of 58-kDa isoform of ADAMTS-2 was upregulated in patients with POP, irrespective of menstrual phase status, secretory or proliferative, when compared to controls (P < .05). The TIMP-1-4 gene and TIMP-1 protein expression were significantly (P < .05) reduced, whereas protein expression of MMP-12 (pro and active forms) was significantly increased in vaginal biopsies of patients with POP in the proliferative phase of the menstrual cycle compared to corresponding controls. Analyses of MMP-12, TIMP-1, and ADAMTS-2 tissue immunostaining indicate similar localization in the vaginal specimens from control and patients with POP. CONCLUSION: Expression of ECM-remodeling proteins is altered in the vagina of premenopausal patients with severe POP. We speculate that dysregulation of MMP/TIMP complexes and ADAMTS-2 proteins may cause connective tissue defects, which result in weakened vaginal wall support and POP development.