Trauma induces overexpression of Cx43 in human fetal membrane defects.

David W Barrett,Christopher Thrasivoulou,Alvaro Mata,Aumie Kethees,Anna L David,Neil J Sebire,David L Becker,Tina T Chowdhury,Alex Virasami,Alex C Engels,Jan A Deprest

doi:10.1002/pd.5104

Abstract

ObjectiveWe developed an in vitro model to examine whether trauma induces connexin 43 (Cx43) expression and collagen organisation in the amniotic membrane (AM) of fetal membrane (FM) defects.MethodTerm human FM was traumatised in vitro. Cell morphology and Cx43 were examined in the wound edge AM by immunofluorescence (IMF) confocal microscopy and compared to control AM. Collagen microstructure was examined by second harmonic generation (SHG) imaging. Cell viability was assessed with calcein and ethidium staining.ResultsAfter trauma, the AM showed a dense region of cells, which had migrated towards the wound edge. In wound edge AM, Cx43 puncta was preferentially distributed in mesenchymal cells compared to epithelial cells with significant expression in the fibroblast layer than epithelial layer (p < 0.001). In the fibroblast layer, the collagen fibres were highly polarised and aligned in parallel to the axis of the wound edge AM. There was an absence of cell migration across the defect with no healing after 168 h. Cell viability of the FM after trauma was maintained during culture.ConclusionCx43 overexpression in wounded AM drives structural changes in collagen that slows down efficacy of cell migration across the FM defect. © 2017 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.

Highlights

Iatrogenic preterm premature rupture of the fetal membranes is a major complication after invasive fetal diagnostic or therapeutic fetal interventions and is a significant threat to fetal survival and well-being if the membranes rupture preterm, before 37 weeks of gestation. iPPROM occurs in 6 to 45% of cases after fetoscopic procedures with complications related to the number of fetoscopic ports required, defect size, amniotic fluid (AF) leakage and membrane separation.[1,2,3,4] The high prevalence of iPPROM after fetal interventions reduces the effectiveness of interventions to treat fetal abnormalities.Several research groups have explored potential therapies to prevent iPPROM or to repair the fetal membrane (FM) defect after established rupture
After trauma, the amniotic membrane (AM) showed a dense region of cells, which had migrated towards the wound edge
Representative images of connexin 43 (Cx43) in the wound edge AM show that the protein was distributed in a scattered and punctate fashion, with greater localisation in cells in the fibroblast layer compared to the epithelial layer (Figure 1C, E)

Summary

Introduction

Several research groups have explored potential therapies to prevent iPPROM or to repair the fetal membrane (FM) defect after established rupture. In pre-clinical models, development of methods to seal the FM defect using collagen plugs or maternal platelets mixed with fibrinogen to promote adhesiveness of the amniopatch therapy was reported, but neither approach prolonged pregnancy with favourable perinatal outcome.[5,6,7,8] Other authors used a variety of tissue sealants to plug defects within explant or in vivo models albeit with varying success.[9,10,11,12,13,14,15,16] In contrast, studies of FM healing in animal models revealed limited repair of tissues in large mammals such as rhesus monkey or sheep.[17,18] This is in contrast to rats, where adhesion of the AM and chorionic membrane (CM) was observed 5 days after FM wounding, with fibroblast proliferation and clot formation, and in rabbits, restoration of tissue integrity was induced by matrixmetalloproteinase (MMP) -2 and -9 activity.[19,20] Whilst the in vitro models have adopted a variety of tissue engineering approaches to induce healing after trauma and assess treatment efficacy using monolayer, explant or decellularised FM models, the evidence of healing the FM defect is variable with conflicting data on repair.[21,22]

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Results

Conclusion