Mechanical stretching promotes the differentiation of BMSC into pelvic floor ligament fibroblasts

Bing Zhao,Mengcai Hu,Chenchen Ren,Lulu Chen,Luwen Wang,Junmin Wang,Xiaojun Liu,Juan Chen,Feng Xu,Xueqin Zheng,Shihong Cui,Huiyan Wu

doi:10.18632/oncotarget.24439

Abstract

// Bing Zhao 1, * , Junmin Wang 2, 1, * , Chenchen Ren 1 , Mengcai Hu 1 , Huiyan Wu 1 , Lulu Chen 1 , Xiaojun Liu 3 , Luwen Wang 1 , Feng Xu 1 , Xueqin Zheng 1 , Juan Chen 1 and Shihong Cui 1 1 Third Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province 450052, China 2 Department of Human Anatomy, Basic Medical College of Zhengzhou University, Zhengzhou, Henan Province 450052, China 3 Henan Medical Equipment Inspection Institute, Zhengzhou, Henan Province 450052, China * These authors have contributed equally to this work Correspondence to: Shihong Cui, email: shihongcui@126.com Keywords: mechanical stretch; BMSC; KDM4B; LOX; elastin Received: December 28, 2016 Accepted: July 24, 2017 Published: February 07, 2018 ABSTRACT Pelvic floor dysfunction (PFD) is a prevalent and debilitating condition in aging women that is associated with weakened pelvic connective tissue. Fibroblasts are the main component of pelvic connective tissue and play a vital role in the maintenance of the pelvic organ. Mechanical stretching is a known modulator leading to the proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs). In this paper, we measured the lysyl oxidase (LOX), elastin and KDM4B expression in cultured BMSCs under different cyclic mechanical stretch (CMS) conditions via Western blot assays. Our findings indicated that the expression of these proteins was significantly higher in the CMS group than in the normal control group, and 10% CMS at a 0.5 Hz frequency had the best stimulatory effect. We further evaluated the influence of KDM4B on LOX and elastin expression via knockdown and overexpression of KDM4B in BMSCs. Chromatin immunoprecipitation (ChIP)-PCR analysis was performed to determine how H3K9me3 and KDM4B affect the regulatory regions of LOX and elastin in BMSCs subjected to 10% CMS combined with KDM4B knockdown. Finally, we verified the expression of KDM4B under CMS in vivo by subcutaneously transplanting KDM4B- overexpressing BMSCs into mice via immunofluorescence assays. The results showed that activating KDM4B by mechanical stretching increased the protein and gene expression levels of LOX and elastin and induced more LOX-positive cells in the BMSCs. These data suggest that CMS contributes to the differentiation of mesenchymal stem cells (MSCs) into fibroblasts, which indicates its potential use as a cell-based therapy for PFD.

Highlights

Pelvic floor dysfunction (PFD), mainly including stress urinary incontinence (SUI), pelvic organ prolapse (POP), and fecal incontinence (FI), is common in women and more prevalent in the aging population [1,2,3]
Western blot assays were used to evaluate the effects of 1%, 2%, 4% and 10% cyclic mechanical stretch (CMS) with 0.5 Hz mechanical stretching on the elastin, Lox and KDM4B protein levels in the bone marrow mesenchymal stem cells (BMSCs) (Figure 1)
The results showed that the elastin and lysyl oxidase (LOX) protein levels were decreased after KDM4B shRNA transfection, suggesting that silencing KDM4B expression led to a loss of elastin and LOX in BMSCs, possibly through weakening the bonding at their promotor sites

Summary

Introduction

Pelvic floor dysfunction (PFD), mainly including stress urinary incontinence (SUI), pelvic organ prolapse (POP), and fecal incontinence (FI), is common in women and more prevalent in the aging population [1,2,3]. PFD is a complex disease that involves many factors, and the majority of research suggests that the total collagen content is decreased in pelvic connective tissue and that the pelvic support tissues (the ligament, muscle and fascia) are relaxed because of reduced collagen fibers, which eventually leads to PFD [4, 5]. The occurrence and development of PFD is closely related to the structural function of the uterosacralcardinal ligament, which provides the main support for the pelvic floor.

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