Vaginal Fibroblasts Research Articles

3D Printed Mesh Geometry Modulates Immune Response and Interface Biology in Mouse and Sheep Model: Implications for Pelvic Floor Surgery.

Pelvic organ prolapse (POP) is a highly prevalent yet neglected health burden for women. Strengthening thepelvic floor with bioactive tissue-engineered meshes is an emerging concept. The study investigates tissue regenerative design parameters, including degradability, porosity, and angulation, to develop alternative degradable melt electrowritten (MEW) constructs for surgical applications of POP. MEW constructs are fabricated in hierarchical geometries by two-way stacking of the fibers with three different inter layer angles of 90°, 45°, or 22.5°. Implants printed at 22.5° have higher tensile strength under dry conditions and show better vaginal fibroblast (VF) attachment in vitro. In vivo assessment using preclinical mouse and ovine models demonstrates more effective degradation and improved tissue integration in 22.5° angular meshes compared to 90° and 45° meshes, with evidence of neo-collagen deposition within implants at 6 weeks. The pattern and geometry of the layered MEW implants also influence the foreign body response, where in the anti-inflammatory phenotype shows a greater ratio of anti-inflammatory CD206+ M2 macrophages/pro-inflammatory CCR7+ M1 macrophages. This presents an attractive strategy for improving the design and fabrication of next-generation vaginal implants for pelvic reconstructive surgery.

Exosomes from umbilical cord mesenchymal stromal cells promote the collagen production of fibroblasts from pelvic organ prolapse.

Pelvic organ prolapse (POP) involves pelvic organ herniation into the vagina due to pelvic floor tissue laxity, and vaginal structure is an essential factor. In POP, the vaginal walls exhibit abnormal collagen distribution and decreased fibroblast levels and functions. The intricate etiology of POP and the prohibition of transvaginal meshes in pelvic reconstruction surgery present challenges in targeted therapy development. Human umbilical cord mesenchymal stromal cells (hucMSCs) present limitations, but their exosomes (hucMSC-Exo) are promising therapeutic tools for promoting fibroblast proliferation and extracellular matrix remodeling. To investigate the effects of hucMSC-Exo on the functions of primary vaginal fibroblasts and to elucidate the underlying mechanism involved. Human vaginal wall collagen content was assessed by Masson's trichrome and Sirius blue staining. Gene expression differences in fibroblasts from patients with and without POP were assessed via RNA sequencing (RNA-seq). The effects of hucMSC-Exo on fibroblasts were determined via functional experiments in vitro. RNA-seq data from fibroblasts exposed to hucMSC-Exo and microRNA (miRNA) sequencing data from hucMSC-Exo were jointly analyzed to identify effective molecules. In POP, the vaginal wall exhibited abnormal collagen distribution and reduced fibroblast 1 quality and quantity. Treatment with 4 or 6 μg/mL hucMSC-Exo suppressed inflammation in POP group fibroblasts, stimulated primary fibroblast growth, and elevated collagen I (Col1) production in vitro. High-throughput RNA-seq of fibroblasts treated with hucMSC-Exo and miRNA sequencing of hucMSC-Exo revealed that abundant exosomal miRNAs downregulated matrix metalloproteinase 11 (MMP11) expression. HucMSC-Exo normalized the growth and function of primary fibroblasts from patients with POP by promoting cell growth and Col1 expression in vitro. Abundant miRNAs in hucMSC-Exo targeted and downregulated MMP11 expression. HucMSC-Exo-based therapy may be ideal for safely and effectively treating POP.

Exosomes from umbilical cord mesenchymal stromal cells promote the collagen production of fibroblasts from pelvic organ prolapse

BACKGROUND Pelvic organ prolapse (POP) involves pelvic organ herniation into the vagina due to pelvic floor tissue laxity, and vaginal structure is an essential factor. In POP, the vaginal walls exhibit abnormal collagen distribution and decreased fibroblast levels and functions. The intricate etiology of POP and the prohibition of transvaginal meshes in pelvic reconstruction surgery present challenges in targeted therapy development. Human umbilical cord mesenchymal stromal cells (hucMSCs) present limitations, but their exosomes (hucMSC-Exo) are promising therapeutic tools for promoting fibroblast proliferation and extracellular matrix remodeling. AIM To investigate the effects of hucMSC-Exo on the functions of primary vaginal fibroblasts and to elucidate the underlying mechanism involved. METHODS Human vaginal wall collagen content was assessed by Masson’s trichrome and Sirius blue staining. Gene expression differences in fibroblasts from patients with and without POP were assessed via RNA sequencing (RNA-seq). The effects of hucMSC-Exo on fibroblasts were determined via functional experiments in vitro . RNA-seq data from fibroblasts exposed to hucMSC-Exo and microRNA (miRNA) sequencing data from hucMSC-Exo were jointly analyzed to identify effective molecules. RESULTS In POP, the vaginal wall exhibited abnormal collagen distribution and reduced fibroblast 1 quality and quantity. Treatment with 4 or 6 μg/mL hucMSC-Exo suppressed inflammation in POP group fibroblasts, stimulated primary fibroblast growth, and elevated collagen I (Col1) production in vitro . High-throughput RNA-seq of fibroblasts treated with hucMSC-Exo and miRNA sequencing of hucMSC-Exo revealed that abundant exosomal miRNAs downregulated matrix metalloproteinase 11 (MMP11) expression. CONCLUSION HucMSC-Exo normalized the growth and function of primary fibroblasts from patients with POP by promoting cell growth and Col1 expression in vitro . Abundant miRNAs in hucMSC-Exo targeted and downregulated MMP11 expression. HucMSC-Exo-based therapy may be ideal for safely and effectively treating POP.

Silencing of cysteine and serine rich nuclear protein 1 inhibits apoptosis, senescence and collagen degradation in human-derived vaginal fibroblasts in response to oxidative stress or DNA damage

Pelvic organ prolapse (POP) is a group of diseases caused by extracellular matrix (ECM) degradation in pelvic supportive tissues. Cysteine and serine rich nuclear protein 1 (CSRNP1) is involved in cell proliferation and survival regulation, and reportedly facilitates collagen breakdown in human chondrocytes. The present study aimed to probe the effect of CSRNP1 on collagen metabolism in human-derived vaginal fibroblasts. High expression of CSRNP1 was found in POP patient-derived vaginal fibroblasts in comparison to normal-derived vaginal fibroblasts. Following functional experiments revealed that CSRNP1 overexpression led to proliferation inhibition, apoptosis and collagen degradation in normal vaginal fibroblasts. In line with this, silencing of CSRNP1 inhibited hydrogen peroxide (H2O2)-triggered apoptosis, ROS generation and collagen loss in normal vaginal fibroblasts. Silencing of CSRNP1 also reduced the expression of cell senescence markers p21 and γ-H2Ax (the histone H2Ax phosphorylated at Ser139), as well as curbed collagen breakdown in normal vaginal fibroblasts caused by a DNA damage agent etoposide. Transcriptomic analysis of vaginal fibroblasts showed that differentially expressed genes affected by CSRNP1 overexpression were mainly enriched in the Wnt signaling pathway. Treatment with a Wnt pathway inhibitor DKK1 blocked CSRNP1 knockdown-caused collagen deposition. Mechanistically, CSRNP1 was identified to be a target of Snail family transcriptional repressor 2 (SNAI2). Forced expression of CSRNP1 reversed the anti-apoptotic, anti-senescent and anti-collagen loss effects of SNAI2 in normal vaginal fibroblasts exposed to H2O2 or etoposide. Our study indicates that the SNAI2/CSRNP1 axis may be a key driver in POP progression, which provides a potential therapeutic strategy for POP.

SOCS3, Transcriptionally Activated by NR4A1, Induces Apoptosis and Extracellular Matrix Degradation of Vaginal Fibroblasts in Pelvic Organ Prolapse

Pelvic organ prolapse (POP) is a common gynecological chronic disorder. Human vaginal fibroblasts (HVFs) that maintain the integrity of vaginal wall tissues are essential for keeping pelvic organs in place. Apoptosis and the degradation of the extracellular matrix in HVFs contribute to the progression of POP. The cytokine signal transduction inhibitor 3 (SOCS3) exerts significant regulatory effects on cell signal transduction pathways, thereby affecting various pathological processes. To explore the role and mechanism of SOCS3 on HVFs in the context of POP. In vitro cell lines and human-sample study. Anterior vaginal wall tissues were obtained from POP or non-POP patients for the analysis of SOCS3 expression. HVFs were isolated from the vaginal tissues of POP patients, and SOCS3 was either overexpressed or knocked down in HVFs via lentivirus infection. Subsequently, the biological function and mechanism of SOCS3 in HVFs were investigated. SOCS3 was highly expressed in the vaginal tissues of POP patients compared to non-POP patients. Functionally, the overexpression of SOCS3 suppressed cell viability while promoting cell apoptosis in HVFs. The overexpression of SOCS3 also accelerated extracellular matrix degradation (decreasing collagen I, collagen III, and elastin, and increasing MMP2 and MMP9). In terms of mechanism, NR4A1 transcriptionally activated SOCS3 by binding to its promoter. Furthermore, rescue experiments revealed that SOCS3 knockdown hindered NR4A1 overexpression-induced cell apoptosis and extracellular matrix degradation in HVFs. SOCS3 mediated the apoptotic and extracellular matrix degradation effects of NR4A1 on HVFs, underlining that the restraining of the SOCS3 expression may be a promising strategy for POP treatment.

Effect of Functionalization of Texturized Polypropylene Surface by Silanization and HBII-RGD Attachment on Response of Primary Abdominal and Vaginal Fibroblasts.

Soft tissue defects, such as incisional hernia or pelvic organ prolapse, are prevalent pathologies characterized by a tissue microenvironment rich in fragile and dysfunctional fibroblasts. Precision medicine could improve their surgical repair, currently based on polymeric materials. Nonetheless, biomaterial-triggered interventions need first a better understanding of the cell-material interfaces that truly consider the patients' biology. Few tools are available to study the interactions between polymers and dysfunctional soft tissue cells in vitro. Here, we propose polypropylene (PP) as a matrix to create microscale surfaces w/wo functionalization with an HBII-RGD molecule, a fibronectin fragment modified to include an RGD sequence for promoting cell attachment and differentiation. Metal mold surfaces were roughened by shot blasting with aluminum oxide, and polypropylene plates were obtained by injection molding. HBII-RGD was covalently attached by silanization. As a proof of concept, primary abdominal and vaginal wall fasciae fibroblasts from control patients were grown on the new surfaces. Tissue-specific significant differences in cell morphology, early adhesion and cytoskeletal structure were observed. Roughness and biofunctionalization parameters exerted unique and combinatorial effects that need further investigation. We conclude that the proposed model is effective and provides a new framework to inform the design of smart materials for the treatment of clinically compromised tissues.

The Regenerative Capacity of Tissue-Engineered Amniotic Membranes.

Scaffolds can be introduced as a source of tissue in reconstructive surgery and can help to improve wound healing. Amniotic membranes (AMs) as scaffolds for tissue engineering have emerged as promising biomaterials for surgical reconstruction due to their regenerative capacity, biocompatibility, gradual degradability, and availability. They also promote fetal-like scarless healing and provide a bioactive matrix that stimulates cell adhesion, migration, and proliferation. The aim of this study was to create a tissue-engineered AM-based implant for the repair of vesicovaginal fistula (VVF), a defect between the bladder and vagina caused by prolonged obstructed labor. Layers of AMs (with or without cross-linking) and electrospun poly-4-hydroxybutyrate (P4HB) (a synthetic, degradable polymer) scaffold were joined together by fibrin glue to produce a multilayer scaffold. Human vaginal fibroblasts were seeded on the different constructs and cultured for 28 days. Cell proliferation, cell morphology, collagen deposition, and metabolism measured by matrix metalloproteinase (MMP) activity were evaluated. Vaginal fibroblasts proliferated and were metabolically active on the different constructs, producing a distributed layer of collagen and proMMP-2. Cell proliferation and the amount of produced collagen were similar across different groups, indicating that the different AM-based constructs support vaginal fibroblast function. Cell morphology and collagen images showed slightly better alignment and organization on the un-cross-linked constructs compared to the cross-linked constructs. It was concluded that the regenerative capacity of AM does not seem to be affected by mechanical reinforcement with cross-linking or the addition of P4HB and fibrin glue. An AM-based implant for surgical repair of internal organs requiring load-bearing functionality can be directly translated to other types of surgical reconstruction of internal organs.

Promoting cell proliferation and collagen production with ascorbic acid 2-phosphate-releasing poly(l-lactide-co-ε-caprolactone) membranes for treating pelvic organ prolapse.

Pelvic organ prolapse (POP) afflicts millions of women globally. In POP, the weakened support of the pelvic floor results in the descent of pelvic organs into the vagina, causing a feeling of bulging, problems in urination, defaecation and/or sexual function. However, the existing surgical repair methods for relapsed POP remain insufficient, highlighting the urgent need for more effective alternatives. Collagen is an essential component in pelvic floor tissues, providing structural support, and its production is controlled by ascorbic acid. Therefore, we investigated novel ascorbic acid 2-phosphate (A2P)-releasing poly(l-lactide-co-ε-caprolactone) (PLCLA2P) membranes in vitro to promote cell proliferation and extracellular matrix protein production to strengthen the natural support of the pelvic fascia for POP applications. We analysed the mechanical properties and the impact of PLCLA2P on cellular responses through cell culture analysis using human vaginal fibroblasts (hVFs) and human adipose-derived stem/stromal cells (hASCs) compared to PLCL. In addition, the A2P release from PLCLA2P membranes was assessed in vitro. The PLCLA2P demonstrated slightly lower tensile strength (2.2 ± 0.4 MPa) compared to PLCL (3.7 ± 0.6 MPa) for the first 4 weeks in vitro. The A2P was most rapidly released during the first 48 h of in vitro incubation. Our findings demonstrated significantly increased proliferation and collagen production of both hVFs and hASCs on A2P-releasing PLCLA2P compared to PLCL. In addition, extracellular collagen Type I fibres were detected in hVFs, suggesting enhanced collagen maturation on PLCLA2P. Moreover, increased extracellular matrix protein expression was detected on PLCLA2P in both hVFs and hASCs compared to plain PLCL. In conclusion, these findings highlight the potential of PLCLA2P as a promising candidate for promoting tissue regeneration in applications aimed for POP tissue engineering applications.

Injectable polyisocyanide hydrogel as healing supplement for connective tissue regeneration in an abdominal wound model

In pelvic organ prolapse (POP) patients, the uterus, bladder and/or rectum descends into vagina due to weakened support tissues. High recurrence rates after POP surgery suggest an urgent need for improved surgical outcomes. Our aim is to promote connective tissue healing that results in stimulated tissue support functions by surgically applying a hydrogel functionalized with biological cues. We used known vaginal wound healing promoting factors (basic fibroblast growth factor, β-estradiol, adipose-derived stem cells) in the biomimetic and injectable polyisocyanide (PIC) hydrogel, which in itself induces regenerative vaginal fibroblast behavior. The regenerative capacity of injected PIC hydrogel, and the additional pro-regenerative effects of these bioactive factors was evaluated in abdominal wounds in rabbits. Assessment of connective tissue healing (tensile testing, histology, immunohistochemistry) revealed that injection with all PIC formulations resulted in a statistically significant stiffness and collagen increase over time, in contrast to sham. Histological evaluation indicated new tissue growth with moderate to mild immune activity at the hydrogel – tissue interface. The results suggest that PIC injection in an abdominal wound improves healing towards regaining load-bearing capacity, which encourages us to investigate application of the hydrogel in a more translational vaginal model for POP surgery in sheep.

Vaginal Fibroblast Behavior as a Function of Stiffness Changes in a Polyisocyanide Hydrogel for Prolapse Repair.

There is an urgent need for improved outcomes in the treatment of pelvic organ prolapse (POP). Success of primary surgery relies on the load bearing capacity of plicated connective tissue underneath the vaginal wall, which is compromised due to an altered vaginal fibroblast function and collagen composition. There is an important factor in connective tissue repair that relates to changes in stiffness of the vaginal fibroblast microenvironment, which influences cell activity through cellular mechanosensing. The aim of this study is to investigate the effect of stiffness changes on vaginal fibroblast functions that relate to connective tissue healing in prolapse repair. The substrate stiffness was controlled by changing the polymer concentration in the fibrous and strongly biomimetic polyisocyanide (PIC) hydrogel. We analyzed stiffness during cell culture and assessed the consequential fibroblast proliferation, morphology, collagen deposition, and contraction. Our results show that increasing stiffness coincides with vaginal fibroblast alignment, promotes collagen deposition, and inhibits PIC gel contraction. These findings suggest that the matrix stiffness directly influences vaginal fibroblast functionality. Moreover, we observed a buildup in stiffness and collagen, with an enhanced fibroblast and collagen organization on the PIC-substrate, which indicate an enhanced structural integrity of the hydrogel-cell construct. An improved tissue structure during healing is relevant in the functional repair of POP. Therefore, this study encourages future research in the use of PIC gels as a supplement in prolapse surgery, whereby the hydrogel stiffness should be considered.

Molecular Landscape of Pelvic Organ Prolapse Provides Insights into Disease Etiology.

Pelvic organ prolapse (POP) represents a major health care burden in women, but its underlying pathophysiological mechanisms have not been elucidated. We first used a case-control design to perform an exome chip study in 526 women with POP and 960 control women to identify single nucleotide variants (SNVs) associated with the disease. We then integrated the functional interactions between the POP candidate proteins derived from the exome chip study and other POP candidate molecules into a molecular landscape. We found significant associations between POP and SNVs in 54 genes. The proteins encoded by 26 of these genes fit into the molecular landscape, together with 43 other POP candidate molecules. The POP landscape is located in and around epithelial cells and fibroblasts of the urogenital tract and harbors four interacting biological processes-epithelial-mesenchymal transition, immune response, modulation of the extracellular matrix, and fibroblast function-that are regulated by sex hormones and TGFB1. Our findings were corroborated by enrichment analyses of differential gene expression data from an independent POP cohort. Lastly, based on the landscape and using vaginal fibroblasts from women with POP, we predicted and showed that metformin alters gene expression in these fibroblasts in a beneficial direction. In conclusion, our integrated molecular landscape of POP provides insights into the biological processes underlying the disease and clues towards novel treatments.

Absorbable Electrospun Poly-4-hydroxybutyrate Scaffolds as a Potential Solution for Pelvic Organ Prolapse Surgery.

Women with pelvic organ prolapse (POP) have bothersome complaints that significantly affect their quality of life. While native tissue repair is associated with high recurrence rates, polypropylene knitted implants have caused specific implant-related adverse events that have detrimental, often irreversible, effects. We hypothesize that surgical outcome can be improved with a tissue-engineered solution using an absorbable implant that mimics the natural extracellular matrix (ECM) structure, releases estrogen, and activates collagen metabolism by fibroblasts as the main regulators of wound healing. To this aim, we produced electrospun poly-4-hydroxybutyrate (P4HB) scaffolds and biofunctionalized them with estradiol (E2). The cell-implant interactions relevant for POP repair were assessed by seeding primary POP vaginal fibroblasts isolated from patients on electrospun P4HB scaffolds with 1%, 2%, or 5% E2 and without E2. To test our hypothesis on whether ECM mimicking structures should improve regeneration, electrospun P4HB was compared to knitted P4HB implants. We evaluated vaginal fibroblast proliferation, ECM deposition, and metabolism by quantification of collagen, elastin, and matrix metalloproteinases and by gene expression analysis for 28 days. We established effective E2 drug loading with a steady release over time. Significantly higher cell proliferation, collagen-, and elastin deposition were observed on electrospun P4HB scaffolds as compared to knitted P4HB. For this study, physical properties of the scaffolds were more determinant on the cell response than the release of E2. These results indicate that making these electrospun P4HB scaffolds E2-releasing appears to be technically feasible. In addition, electrospun P4HB scaffolds promote the cellular response of vaginal fibroblasts and further studies are merited to assess if their use results in improved surgical outcomes in case of POP repair.

Polyisocyanides as a substrate to trigger vaginal fibroblast functioning in an in vitro model for prolapse repair.

Pelvic organ prolapse (POP) is the descent of the bladder, uterus, and/or rectum into the vagina. POP is associated with altered vaginal fibroblast functionality and connective tissue composition in the vaginal wall. The results of surgical intervention are poor, which may be related to the lack of true restoration of the connective tissue. An innovative treatment addresses tissue repair after surgery by the introduction of a bioactive supplement that enhances the healing process through collagen and elastin deposition. As a novel strategy, we first studied the effects in an in vitro model. Here, we investigate how the presence of cell binding GRGDS (RGD) peptides on the highly biomimetic polyisocyanide (PIC) gel facilitates and promotes the function of primary vaginal fibroblasts isolated from a POP patient. Fibroblast function was analyzed in terms of morphology, proliferation, and extracellular matrix (ECM) deposition and remodeling. RGD modification of the gel facilitated cell spread and proliferation. Quantitative outcomes of the ECM content indicated increased production of collagen and elastin by fibroblasts on gels with the highest RGD density. The in vitro results suggest that PIC-RGD hydrogel application may translate into improved connective tissue healing in the pelvic floor, which is essential for its use as a regeneration promoting additive in surgery.

A physiologically relevant, estradiol‐17β [E2]‐responsive in vitro tissue‐engineered model of the vaginal epithelium for vaginal tissue research

AimsThere are many situations where preclinical models of the human vagina would be valuable for in vitro studies into the pathophysiology of vaginally transmitted diseases, microbicide efficacy, irritability testing, and particularly, for assessing materials to be inserted in the vagina for support of the pelvic floor. The aim of this study is to develop a physiologically relevant, low cost, and ethically suitable model of the vagina using sheep vaginal tissue (SVT) to reduce the need for animal testing in gynecological research.MethodsTissue‐engineered (TE) vaginal models were developed by culturing primary vaginal epithelial cells and vaginal fibroblasts, isolated from the native SVTs on decellularized sheep vaginal matrices at an air–liquid interface. Morphological analyses of the models were conducted by performing hematoxylin and eosin staining and further characterization was done by immunohistofluorescence (IHF) of structural proteins and cytokeratins.ResultsHistological analysis of the models revealed a gradual formation of a stratified epithelium on our decellularized matrices and cell metabolic activity remained high for 21 days as measured by the resazurin assay. Our models showed a dose‐dependent response to estradiol‐17β [E2] with an increase in the vaginal epithelium thickness and cellular proliferation under higher E2 concentrations (100–400 pg/ml). The physiological relevance of these results was confirmed by the IHF analysis of Ki67, and cytokeratins 10 and 19 expression.ConclusionIn this study, we have developed an estradiol‐responsive TE vaginal model that closely mimics the structural and physiological properties of the native SVT.

Induction of Cellular Senescence in Rat Vaginal Fibroblasts and Treatment With Senolytics: An in Vitro Model for the Study of Pelvic Organ Prolapse.

The objective of this study was to develop an in vitro model of cellular senescence using rat vaginal fibroblasts and determine the effects of treatment with senolytics. Rat vaginal tissue biopsies were collected. Primary vaginal fibroblasts were isolated and characterized by immunofluorescence. To induce cellular senescence, fibroblasts were treated with etoposide at 3, 10, and 20 mM for 24 hours, followed by treatment with the senolytics dasatinib (1 mM) and/or quercetin (20 mM). After treatment, RNA was extracted and the expression of selected genes was quantified. Immunostaining of senescence markers was also performed. Fibroblasts were confirmed by positive immunostaining for α-smooth muscle actin and vimentin, and negative immunostaining for pan-cytokeratin. Treatment with etoposide resulted in a dose-dependent increase in expression of the senescence-associated secretory phenotype markers MMP-7, MMP-9, and IL-b1 (P < 0.05) compared with controls. Immunostaining showed increased expression of γ-H2A and p21 after treatment with etoposide. Cells treated with dasatinib and quercetin after etoposide treatment had decreased expression of p21, MMP-7, MMP-9, and IL-1b compared with cells treated only with etoposide (P < 0.05). Upregulation of senescence-associated factors provided evidence that senescence can be induced in vaginal fibroblasts in vitro. Furthermore, treatment with the senolytics dasatinib and quercetin abrogated the senescence phenotype induced by etoposide in rat vaginal fibroblasts. Our findings provide a novel model for the study and development of new therapies targeting the disordered extracellular matrix associated with pelvic organ prolapse.

ATF3 regulates oxidative stress and extracellular matrix degradation via p38/Nrf2 signaling pathway in pelvic organ prolapse

Pelvic organ prolapse (POP) is a common disorder in women, and it is characterized by weakening of pelvic supportive structure with extracellular matrix (ECM) degradation. Activating transcription factor 3 (ATF3) was upregulated in anterior vaginal wall tissues of POP patients. We hypothesized that upregulation of ATF3 might contribute to POP development. This study aims to unveil the role of ATF3 in the pathogenesis of POP using a H2O2-induced in vitro model. Vaginal fibroblasts were isolated from woman with POP-Q stage greater than II and asymptomatic women with normal pelvic floor support. Knockdown of ATF3 enhanced cell viability and decreased cell apoptosis. Flow cytometry and immunnofluorescence showed that ATF3 deficiency inhibited H2O2-induced ROS production and the expression of 8 OHdG and 4-HNE. Western blot and Real-time PCR analysis revealed that ATF3 deficiency attenuated ECM component degradation (increasing collagen I, collagen III and elastin) and MMPs/TIMPs imbalance (decreasing MMP2 and MMP9 and increasing TIMP2). Moreover, knockdown of ATF3 induced the activation of p38/Nrf2/HO-1 signaling pathway. Further treatment with p38 inhibitor SB203580 abolished the protection of ATF3 deficiency against H2O2-induced cell damage, which was reverted by Nrf2 activator TBHQ. Thus, ATF3 likely contributes to POP progression by inducing cell apoptosis, oxidative stress and ECM degradation via regulating p38/Nrf2 pathway, which provides a potential therapeutic target for POP.

Effects of the decrease of β-catenin expression on human vaginal fibroblasts of women with pelvic organ prolapse.

Pelvic organ prolapse (POP) lowers the quality of life in elderly women, and there have been no studies on its role in the pathogenesis of POP. The purpose of this study is to investigate the effect of β-catenin on proliferation and collagen anabolism in human vaginal fibroblasts (HVFs). The adherence and differential adherence methods were used to culture and purify HVFs. RNA interference was applied to knockdown β-catenin and lithium chloride was used to activate Wnt/β-catenin signaling pathway. β-catenin nuclear translocation was tested by immunofluorescence, and HVF proliferation was detected by performing MTT assays. The expression of β-catenin, phosphorylated-β-catenin, phosphorylated-glycogen synthase kinase 3β (p-GSK3β), collagen I, matrix metalloproteinase 2 (MMP2), and tissue-derived inhibitors of metalloproteinases 2 (TIMP2) was assessed by western blot analysis. The expression of β-catenin and collagen I was lower in HVFs of POP group than that of control group. The proliferation rate of HVFs in POP group was lower than that in control group. Knockdown of β-catenin decreased the cell proliferation rate and the expression of collagen I. Lithium chloride can activate the Wnt/β-catenin signaling pathway. β-catenin participates in the proliferation and collagen I synthesis of HVFs. The decrease of β-catenin expression may be closely related to the occurrence, and development of POP. LiCl can activate the Wnt/β-catenin signaling pathway in HVFs and thus increase HVFs proliferation and collagen synthesis.

Small extracellular vesicles secreted by vaginal fibroblasts exert inhibitory effect in female stress urinary incontinence through regulating the function of fibroblasts.

Stress urinary incontinence (SUI) is a common condition in women and associated with extra-cellular matrix (ECM) reconstruction, which is mainly regulated by fibroblasts. However, the underlying mechanism remains obscure. Small extracellular vesicles (sEVs) play fundamental biological roles in various cellular functions. Some studies suggested that the sEVs were involved in the metabolism of ECM and the function of fibroblasts. The purpose of our study was to investigate the effect of sEVs secreted by vaginal fibroblasts on the pathogenesis of SUI. We showed that the fibroblasts of female anterior vaginal wall secreted sEVs. Moreover, fibroblasts of females with SUI had significantly elevated secretion of sEVs. The collagen contents, proliferation and migration capacity of fibroblasts were decreased when fibroblasts were co-cultured with fibroblasts-derived sEVs (fibroblast-sEVs) from SUI patients. Proteomic analysis revealed that fibroblast-sEVs contained various differentially expressed proteins including TIMP2, TGF-β and ABCC4, which were involved in signaling pathways of fibroblasts regulation. Therefore, we suggested that fibroblast-sEVs contributed to the pathogenesis of SUI through various proteins including TIMP2, TGF-β and ABCC4.

Secretomes of human pluripotent stem cell-derived smooth muscle cell progenitors upregulate extracellular matrix metabolism in the lower urinary tract and vagina

BackgroundAdult mesenchymal stem cells (MSCs) have been studied extensively for regenerative medicine; however, they have limited proliferation in vitro, and the long culture time induces cell senescence. MSCs also contribute to tissue repair through their paracrine function. In this study, we sought to examine the paracrine effects of human smooth muscle cell progenitors (pSMC) on the urethra and adjacent vagina of stress urinary incontinence rodents. We use human pluripotent stem cell (PSC) lines to derive pSMCs to overcome the issue of decreased proliferation in tissue culture and to obtain a homogenous cell population.MethodThree human PSC lines were differentiated into pSMCs. The conditioned medium (CM) from pSMC culture, which contain pSMC secretomes, was harvested. To examine the effect of the CM on the extracellular matrix of the lower urinary tract, human bladder smooth muscle cells (bSMCs) and vaginal fibroblasts were treated with pSMC-CM in vitro. Stress urinary incontinence (SUI) was induced in rats by surgical injury of the urethra and adjacent vagina. SUI rats were treated with pSMC-CM and monitored for 5 weeks. Urethral pressure testing was performed prior to euthanasia, and tissues were harvested for PCR, Western blot, and histological staining. Kruskal-Wallis one-way ANOVA test and Student t test were used for statistical comparisons.ResultspSMC-CM upregulated MMP-2, TIMP-2, collagen, and elastin gene expression, and MMP-9 activity in the human bladder and vaginal cells consistent with elastin metabolism modulation. pSMC-CM treatment in the SUI rat improved urethral pressure (increase in leak point pressure compared to intact controls, p < 0.05) and increased collagen and elastin expression in the urethra and the adjacent vagina.ConclusionConditioned media from smooth muscle cell progenitors derived from human pluripotent stem cells improved urethral leak point pressure and collagen and elastin content in the SUI rat. These findings suggest a novel therapeutic potential for PSC-based treatments for SUI and pelvic floor disorders where tissues are affected by collagen, elastin, and smooth muscle loss.

80. Vaginal healing research: from bench to bedside and beyond

<h3>Background</h3> Adolescents undergoing vaginal surgery are often left with scaring and lifelong sequelae that can interfere with their daily lives, including retained menstruation and painful vaginal exams and dyspareunia. While vaginal stents are known to aid in healing, there are no current stents available for young patients and furthermore, we do not fully understand why some girls scar while others do not. It is our goal to understand vaginal healing at the mechanistic level in order to prevent scaring while at the same time provide solutions in the clinic. <h3>Methods</h3> To elucidate fundamental mechanisms of vaginal healing, both in vitro and in vivo methods were developed. Using primary murine fibroblasts, the expression of hyaluronan (HA) related genes and cell migration in response to estrogen and mechanical stretch were evaluated. To model the interplay between vaginal layers, a vagina-on-a-chip is being designed. Murine models, including full thickness wounds and bleomycin instillation plus vaginal trauma, were developed to study regenerative vaginal healing and fibrosis, respectively. In combination with oophorectomy, histologic and gene expression changes were evaluated at various time points after insults. Clinically, vaginal stents were designed and validated in a porcine model and review panels. Virtual reality (VR) technology and 3D imaging is being used for pre-operative training. <h3>Results</h3> We have successfully isolated and rapidly expanded murine primary vaginal fibroblasts, and our ongoing in vitro work suggests that estrogen influences HA via upregulation of HA synthesis genes. Full thickness murine vaginal wounds result in complete epithelial layer closure and resolution of inflammatory changes by 72 hours, which is altered in oopherectomized mice. Bleomycin instillation alone did not lead to fibrosis, but in combination with wire brush trauma led to disrupted collagen organization. The design criteria for a vagina-on-a-chip has been established including sustaining 3D cell culture and matching biomechanical properties. Physical vaginal stent designs to improve retention and patient comfort were validated and two designs were chosen for further benchtop testing in a novel pressurized 3D-printed model and a funded Phase I clinical trial. Resorbable stents were designed with polycaprolactone-based shape memory foams to provide sufficient radial force to maintain vaginal caliber over a target healing time of 4 weeks. Lastly, we are improving clinical training with 3D imaging and VR modeling. <h3>Conclusions</h3> We are advancing vaginal healing research by translating clinical problems into basic science questions and closing the loop ultimately back to bedside improvements.