{"paper_id":"327191d8-d1eb-4659-947d-f0da7b9a1d5b","body_text":"Mechanisms responsible for ‘scarless’ tissue repair in the endometrium\nItem Status\nEmbargo End Date\nDate\nAuthors\nKirkwood, Phoebe Maud\nAbstract\nThe endometrium is the inner lining of the uterine cavity, composed of distinct\nepithelial and stromal cell compartments with the latter containing fibroblasts, a\nvascular compartment and fluctuating populations of immune cells. The endometrium\nis a highly dynamic tissue that undergoes cycles of proliferation and stromal cell\ndifferentiation (decidualisation) followed by tissue shedding (menstruation) and rapid\nrepair/remodelling, all under control of fluctuating concentrations of steroid hormones\nsecreted from the ovaries. This is known as the menstrual cycle.\nIn response to the ‘injury’ inflicted as a consequence of decidual breakdown and\nshedding, the endometrium exhibits a unique capacity to restore tissue architecture by\nrapid tissue repair. This repair process is tightly regulated to ensure that the\nendometrium heals consistently every month throughout a woman’s reproductive\nlifespan, without the accumulation of fibrotic scar tissue which could have a negative\nimpact on fertility.\nThe initiation of menstruation is triggered by the withdrawal of progesterone as a\nconsequence of the demise of the corpus luteum within the ovaries which precipitates\nan increase in production of inflammatory mediators, focal hypoxia and activation of\nmatrix metalloproteinase enzymes culminating in endometrial shedding. In contrast\nthe cellular and molecular mechanisms responsible for the rapid and scar-free tissue\nrepair of endometrium remain poorly understood. Parallels can be drawn between the\nrepair process of the endometrium and that of the foetal skin and oral mucosa which\nalso exhibit ‘scarless’ healing, including rapid reepithelialisation, widespread cellular\nproliferation and migration and a short time to wound closure. However endometrial\nrepair also shares key features of the wound healing experienced by adult tissues that\nexhibit scarring including extensive angiogenesis and a substantial inflammatory\nresponse. The endometrium appears to be unique, fitting in a gap between tissues that\ntypically undergo ‘scarless’ or ‘scarring’ tissue repair.\nIn women, endometrial shedding is considered an inflammatory event and the\nculmination of a cascade of inflammatory signals result in the accumulation of a\ndiverse population of immune cells within the tissue. Whilst we believe immune cells\nplay a key role in regulating spatial and temporal tissue breakdown and shedding their\nrole in repair and restoration of tissue homeostasis remains poorly understood. One\nprocess essential for endometrial repair is restoration of the luminal epithelial cell layer\n(re-epithelialisation) and imaging studies have demonstrated that this appears not only\nto be rapid but also to occur synchronously with tissue degeneration and shedding.\nRe-epithelialisation was previously thought to be governed by proliferation and\nmigration of glandular epithelial cells in the basal (unshed) tissue compartment,\nhowever new data suggest a role for trans-differentiation of stromal cells into epithelial\ncells which merits further investigation. In addition to role(s) for immune and stromal\ncells in regulation of endometrial tissue function, a role for somatic stem/progenitor\ncells capable of differentiating into mature endometrial cells to regenerate the tissue\nhas also been claimed.\nIn summary, whilst progress has been made in understanding the processes governing\nendometrial decidualisation, breakdown and shedding the regulation and roles of the\ndifferent cell types that participate in scar-free repair of the tissue remain poorly\ndefined. The studies in this thesis set out to address this gap by addressing three key\naims:\nAim 1. To investigate the phenotype and location of immune cell populations during\nscarless tissue repair.\nAim 2. To identify and characterise a putative population of mesenchymal\nstem/progenitor cells in endometrium.\nAim 3. To investigate the contribution of putative mesenchymal stem/progenitor cells\nto endometrial tissue repair.\nThe aims were addressed using a recently refined and extensively characterised mouse\nmodel in which endometrial shedding (‘menstruation’) and repair occurs over a 48\nhour period following removal of a progesterone stimulus. Importantly the Saunders’\ngroup have already demonstrated that this model recapitulates the key features of\nhuman menses including overt vaginal bleeding, immune cell influx, tissue necrosis,\ntransient hypoxia, re-epithelialisation and most importantly simultaneous breakdown\nand repair. Uterine tissues recovered 12, 24, and 48hrs after removal of progesterone\nand were investigated using immunohistochemistry (spatial organisation), flow\ncytometry (quantitation of cell subpopulations), FACS sorting (isolation of\nsubpopulations) and molecular profiling (qPCR, RNAseq and single cell sequencing)\nwith additional insights from bioinformatic analysis.\nTo address Aim 1 endometrial shedding and repair was studied in Macgreen® mice:\nin this transgenic line all the cells of the mononuclear phagocyte lineage (monocytes,\nmonocyte-derived macrophages) express green fluorescent protein.\nImmunohistochemistry revealed striking spatio-temporal changes in both numbers and\nlocation of GFP+ cells during endometrial breakdown and repair, the most prominent\nchanges occurring 24hrs after removal of progesterone. Flow Cytometry quantified\nseveral immune cell populations with a significant increase in GFP+ cells during\nrepair, the majority of which were GR1+F4/80- (inflammatory monocytes). These\nnovel data provided compelling evidence to support a role for inflammatory\nmonocytes in endometrial repair and provide the platform for future studies on the role\nof these cells in scarless healing.\nTo address Aims 2 and 3 Pdgfrβ-BAC-eGFP® transgenic mice in which GFP was\nexpressed under control of promoter elements of the Pdgfrβ gene was used to identify\nputative mesenchymal progenitor cells and investigate their role in endometrial repair.\nGFP+ cells were located exclusively within the endometrial stromal compartment and\nexamination of tissue sections revealed that two subpopulations could be distinguished\nbased on the both the intensity of GFP and expression of CD146 (Mcam).\nCharacterisation by immunohistochemistry, flow cytometry and qPCR identified a\nGFPbright subpopulation located adjacent to CD31+ endothelial cells that were\nclassified as pericytes based on location and phenotype (NG2+, CD146+, CD31-).\nWhen menstruation was stimulated in Pdgfrβ-BAC-eGFP® mice detailed analysis\nusing flow cytometry revealed an increase in the perivascular pericyte subpopulation\nduring active healing (24hrs) and also identified a new previously unidentified subpopulation\nof GFP+ cells which had a unique phenotype during repair. Evidence that\nGFP+ cells contribute to restoration of epithelial repair was obtained with an increase\nin expression of the epithelial cell marker EpCAM and GFP+ cells in the renewed\nepithelial cell layer. RNAseq and single cell sequencing combined with bioinformatics\ncomplemented these findings by identifying novel changes in gene expression in both\nendometrial fibroblasts and pericyte populations consistent with induction of novel\npathways and trans-differentiation of stromal cells by a mesenchymal-to-epithelial\ntransition (MET).\nIn conclusion, using a mouse model of endometrial breakdown and repair a\nheterogeneous population of myeloid cells and a putative population of endometrial\nprogenitors (pericytes) have been characterised, quantified and novel changes in gene\nexpression identified. Adaptation of these cell types to the insult of endometrial\nshedding appears to play a key role in temporal and spatial regulation of rapid, scar-free\nendometrial tissue repair. These novel findings may inform the development of\nnew approaches to treating gynaecological disorders associated with aberrant\nendometrial repair such as heavy menstrual bleeding, Asherman’s syndrome and\nendometriosis as well as other disorders associated with excessive fibrosis and scar\nformation.\nThis item appears in the following Collection(s)","source_license":"CC0","license_restricted":false}