Targeting mechanotransduction mechanisms and tissue weakening signals in the human amniotic membrane

David W Barrett,Rebecca K John,David L Becker,Alvaro Mata,Tina T Chowdhury,Anna L David,Christopher Thrasivoulou,Jan A Deprest

doi:10.1038/s41598-019-42379-4

Abstract

Mechanical and inflammatory signals in the fetal membrane play an important role in extracellular matrix (ECM) remodelling in order to dictate the timing of birth. We developed a mechanical model that mimics repetitive stretching of the amniotic membrane (AM) isolated from regions over the placenta (PAM) or cervix (CAM) and examined the effect of cyclic tensile strain (CTS) on mediators involved in mechanotransduction (Cx43, AKT), tissue remodelling (GAGs, elastin, collagen) and inflammation (PGE2, MMPs). In CAM and PAM specimens, the application of CTS increased GAG synthesis, PGE2 release and MMP activity, with concomitant reduction in collagen and elastin content. Co-stimulation with CTS and pharmacological agents that inhibit either Cx43 or AKT, differentially influenced collagen, GAG and elastin in a tissue-dependent manner. SHG confocal imaging of collagen fibres revealed a reduction in SHG intensity after CTS, with regions of disorganisation dependent on tissue location. CTS increased Cx43 and AKT protein and gene expression and the response could be reversed with either CTS, the Cx43 antisense or AKT inhibitor. We demonstrate that targeting Cx43 and AKT prevents strain-induced ECM damage and promotes tissue remodelling mechanisms in the AM. We speculate that a combination of inflammatory and mechanical factors could perturb typical mechanotransduction processes mediated by Cx43 signalling. Cx43 could therefore be a potential therapeutic target to prevent inflammation and preterm premature rupture of the fetal membranes.

Highlights

To characterise the direction of collagen alignment, an orientation distribution analysis using the Directionality ImageJ plug-in (v2) was performed
The placenta was separated from the uterus by gentle cord traction and rinsed with Earle’s Balanced Salt Solution (EBSS) for 3 min to remove excess maternal blood (Sigma-Aldrich, Fancy Road, Poole, UK)
The AM was separated from the chorionic membrane (CM) and placenta tissue using gentle traction

Summary

Methods

Term human placentas were collected after women gave informed consent from women undergoing elective caesarean section (n = 28 separate donors, 37 to 42 weeks of gestation) at University College London Hospital. At Caesarean section after delivery of the baby but before delivery of the placenta, a sterile Babcock tissue clip was placed on the lower edge of the AM within the uterine incision to provide a landmark. The placenta was separated from the uterus by gentle cord traction and rinsed with Earle’s Balanced Salt Solution (EBSS) for 3 min to remove excess maternal blood (Sigma-Aldrich, Fancy Road, Poole, UK). The AM was separated from the chorionic membrane (CM) and placenta tissue using gentle traction. The orientation of the membrane to the placenta, incision line and cervix was noted throughout the procedure and the AM nearest the cervix was identified using a clip. AM specimens (30 × 30 mm) from the cervix (CAM) and placenta (PAM) regions were dissected from the tissue as described previously (Chowdhury B, 2014) and cultured with 1 ml Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5 μg/ml penicillin, 5 μg/ml streptomycin, 15 μg/ml ascorbate and 20% Fetal Calf Serum (FCS) prior to mechanical loading experiments

Results

Discussion

Conclusion