|dc.contributor.author||Kirkwood, Phoebe Maud||en
|dc.description.abstract||The endometrium is the inner lining of the uterine cavity, composed of distinct
epithelial and stromal cell compartments with the latter containing fibroblasts, a
vascular compartment and fluctuating populations of immune cells. The endometrium
is a highly dynamic tissue that undergoes cycles of proliferation and stromal cell
differentiation (decidualisation) followed by tissue shedding (menstruation) and rapid
repair/remodelling, all under control of fluctuating concentrations of steroid hormones
secreted from the ovaries. This is known as the menstrual cycle.
In response to the ‘injury’ inflicted as a consequence of decidual breakdown and
shedding, the endometrium exhibits a unique capacity to restore tissue architecture by
rapid tissue repair. This repair process is tightly regulated to ensure that the
endometrium heals consistently every month throughout a woman’s reproductive
lifespan, without the accumulation of fibrotic scar tissue which could have a negative
impact on fertility.
The initiation of menstruation is triggered by the withdrawal of progesterone as a
consequence of the demise of the corpus luteum within the ovaries which precipitates
an increase in production of inflammatory mediators, focal hypoxia and activation of
matrix metalloproteinase enzymes culminating in endometrial shedding. In contrast
the cellular and molecular mechanisms responsible for the rapid and scar-free tissue
repair of endometrium remain poorly understood. Parallels can be drawn between the
repair process of the endometrium and that of the foetal skin and oral mucosa which
also exhibit ‘scarless’ healing, including rapid reepithelialisation, widespread cellular
proliferation and migration and a short time to wound closure. However endometrial
repair also shares key features of the wound healing experienced by adult tissues that
exhibit scarring including extensive angiogenesis and a substantial inflammatory
response. The endometrium appears to be unique, fitting in a gap between tissues that
typically undergo ‘scarless’ or ‘scarring’ tissue repair.
In women, endometrial shedding is considered an inflammatory event and the
culmination of a cascade of inflammatory signals result in the accumulation of a
diverse population of immune cells within the tissue. Whilst we believe immune cells
play a key role in regulating spatial and temporal tissue breakdown and shedding their
role in repair and restoration of tissue homeostasis remains poorly understood. One
process essential for endometrial repair is restoration of the luminal epithelial cell layer
(re-epithelialisation) and imaging studies have demonstrated that this appears not only
to be rapid but also to occur synchronously with tissue degeneration and shedding.
Re-epithelialisation was previously thought to be governed by proliferation and
migration of glandular epithelial cells in the basal (unshed) tissue compartment,
however new data suggest a role for trans-differentiation of stromal cells into epithelial
cells which merits further investigation. In addition to role(s) for immune and stromal
cells in regulation of endometrial tissue function, a role for somatic stem/progenitor
cells capable of differentiating into mature endometrial cells to regenerate the tissue
has also been claimed.
In summary, whilst progress has been made in understanding the processes governing
endometrial decidualisation, breakdown and shedding the regulation and roles of the
different cell types that participate in scar-free repair of the tissue remain poorly
defined. The studies in this thesis set out to address this gap by addressing three key
Aim 1. To investigate the phenotype and location of immune cell populations during
scarless tissue repair.
Aim 2. To identify and characterise a putative population of mesenchymal
stem/progenitor cells in endometrium.
Aim 3. To investigate the contribution of putative mesenchymal stem/progenitor cells
to endometrial tissue repair.
The aims were addressed using a recently refined and extensively characterised mouse
model in which endometrial shedding (‘menstruation’) and repair occurs over a 48
hour period following removal of a progesterone stimulus. Importantly the Saunders’
group have already demonstrated that this model recapitulates the key features of
human menses including overt vaginal bleeding, immune cell influx, tissue necrosis,
transient hypoxia, re-epithelialisation and most importantly simultaneous breakdown
and repair. Uterine tissues recovered 12, 24, and 48hrs after removal of progesterone
and were investigated using immunohistochemistry (spatial organisation), flow
cytometry (quantitation of cell subpopulations), FACS sorting (isolation of
subpopulations) and molecular profiling (qPCR, RNAseq and single cell sequencing)
with additional insights from bioinformatic analysis.
To address Aim 1 endometrial shedding and repair was studied in Macgreen® mice:
in this transgenic line all the cells of the mononuclear phagocyte lineage (monocytes,
monocyte-derived macrophages) express green fluorescent protein.
Immunohistochemistry revealed striking spatio-temporal changes in both numbers and
location of GFP+ cells during endometrial breakdown and repair, the most prominent
changes occurring 24hrs after removal of progesterone. Flow Cytometry quantified
several immune cell populations with a significant increase in GFP+ cells during
repair, the majority of which were GR1+F4/80- (inflammatory monocytes). These
novel data provided compelling evidence to support a role for inflammatory
monocytes in endometrial repair and provide the platform for future studies on the role
of these cells in scarless healing.
To address Aims 2 and 3 Pdgfrβ-BAC-eGFP® transgenic mice in which GFP was
expressed under control of promoter elements of the Pdgfrβ gene was used to identify
putative mesenchymal progenitor cells and investigate their role in endometrial repair.
GFP+ cells were located exclusively within the endometrial stromal compartment and
examination of tissue sections revealed that two subpopulations could be distinguished
based on the both the intensity of GFP and expression of CD146 (Mcam).
Characterisation by immunohistochemistry, flow cytometry and qPCR identified a
GFPbright subpopulation located adjacent to CD31+ endothelial cells that were
classified as pericytes based on location and phenotype (NG2+, CD146+, CD31-).
When menstruation was stimulated in Pdgfrβ-BAC-eGFP® mice detailed analysis
using flow cytometry revealed an increase in the perivascular pericyte subpopulation
during active healing (24hrs) and also identified a new previously unidentified subpopulation
of GFP+ cells which had a unique phenotype during repair. Evidence that
GFP+ cells contribute to restoration of epithelial repair was obtained with an increase
in expression of the epithelial cell marker EpCAM and GFP+ cells in the renewed
epithelial cell layer. RNAseq and single cell sequencing combined with bioinformatics
complemented these findings by identifying novel changes in gene expression in both
endometrial fibroblasts and pericyte populations consistent with induction of novel
pathways and trans-differentiation of stromal cells by a mesenchymal-to-epithelial
In conclusion, using a mouse model of endometrial breakdown and repair a
heterogeneous population of myeloid cells and a putative population of endometrial
progenitors (pericytes) have been characterised, quantified and novel changes in gene
expression identified. Adaptation of these cell types to the insult of endometrial
shedding appears to play a key role in temporal and spatial regulation of rapid, scar-free
endometrial tissue repair. These novel findings may inform the development of
new approaches to treating gynaecological disorders associated with aberrant
endometrial repair such as heavy menstrual bleeding, Asherman’s syndrome and
endometriosis as well as other disorders associated with excessive fibrosis and scar
|dc.contributor.sponsor||Medical Research Council (MRC)||en
|dc.publisher||The University of Edinburgh||en
|dc.relation.hasversion||Cousins, F. L. & Kirkwood, P. M., Saunders, P. T. & Gibson, D. A. (2016) Evidence for a dynamic role for mononuclear phagocytes during endometrial repair and remodelling. Sci Rep, 6, 36748||en
|dc.relation.hasversion||Cousins, F.L., Kirkwood, P. M., Murray, A. A., Collins, F. Gibson, D.A. & Saunders, P.T (2016) Androgens regulate scarless repair of the endometrial “wound” in a mouse model of menstruation. FASEB J, 30, 2802-11.||en
|dc.relation.hasversion||Haideri. S.S., McKinnon, A.C., Taylor, A.H., Kirkwood, P.M., Starkey-Lewis, P.J., O’Duibhir, E., Vernay, B., Forbes, S. & Forrester, L.M. (2017) Injection of embryonic stem cell derived macrophages ameliorates fibrosis in a murine model of liver injury. Npj Regenerative Medicine, 2, 14||en
|dc.subject||scarless endometrial healing||en
|dc.title||Mechanisms responsible for ‘scarless’ tissue repair in the endometrium||en
|dc.type||Thesis or Dissertation||en
|dc.type.qualificationname||PhD Doctor of Philosophy||en