Phenotype and role of the endometrial macrophage in regulating angiogenesis
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Introduction: The uterine endometrium is a dynamic tissue that undergoes cycles of proliferation, differentiation breakdown and repair in response to fluctuations in the ovarian-derived sex steroids oestrogen (E) and progesterone (P4). During the P4- dominated secretory phase there is an influx of leukocytes which further increases during the menstrual phase. The second most populous leukocyte within the endometrium is the macrophage. Previous studies have postulated a role for the macrophage in breakdown and repair occurring during the menstrual phase associated with production of MMPs and both pro- and anti-inflammatory cytokines. However the mechanisms responsible for the impact of the endometrial subpopulation on endometrial function remain poorly understood. The specific aims of the studies described in this thesis were to examine: 1) the phenotype of the endometrial macrophage during the different phases of the menstrual cycle. 2) the relationship, if any, between the macrophage and endometrial vasculature and 3) whether platelet factor 4 may be one of the factors involved in the interplay between macrophages and other endometrial cells (endothelium, stroma). Methods/Results: Transmission electron microscopy (TEM) identified macrophages carrying out phagocytosis at all stages of the menstrual cycle. Single and double immunocytochemistry revealed that macrophages were proliferating (Ki67+, PH3+) during the late secretory and menstrual phases; TEM and immunohistochemistry identified macrophages in close proximity to the endometrial vasculature throughout the menstrual cycle. Macrophages were immunopositive for glucocorticoid receptor (GR). They expressed anti-angiogenic factors (ANG-2, THBS-1, TWEAK) during the second half of the menstrual cycle preceding endometrial breakdown and pro-angiogenic factors (ANG-1, CTGF, IL-8) during phases characterised by repair processes. When endometrial endothelial capillary networks were incubated in vitro with peripheral monocyte derived macrophages (PMDM) that were incubated in media promoting differentiation into proinflammatory or anti-inflammatory macrophages ‘breakdown’ of capillaries was observed. Further studies revealed media from cortisol(F)-exposed PMDM significantly upregulated expression of pro-angiogenic factors (CTGF, IL-8 and VEGFC) by human endometrial endothelial cells (HEEC) When human endometrial stromal cells (hESC) were incubated with media conditioned by P4-exposed PMDM they increased expression of the anti-angiogenic factor IL-12a and ANGPTL4, a pro-angiogenic factor reported to be upregulated by hypoxia. Notably expression of platelet factor 4 (PF4) by hESC was significantly upregulated by incubation with media from GM-CSF and IFNγ-treated, E-treated or P4-treated PMDM. Further research into the role of PF4 within the human endometrium found the protein increased as the cycle progressed from the proliferative to the menstrual phase, when levels were maximal. Expression was co-localised with CD68, the macrophage marker, throughout the menstrual cycle, as well as in other endometrial cell types. PF4 was significantly increased within HEECs after treatment with F and hESCs after experiencing “P4 withdrawal” (treatment of P4 and cAMP for 6 days then washed and incubated with treatment-free media for 48 hours). Both conditions occur locally within the endometrium during menstruation. Interestingly, PF4 was found to be chemotactic to macrophages that had been exposed to F. Addition of PF4 to HEEC capillary networks resulted in a significant breakdown of the network; PF4 was found to downregulate bFGF RNA expression within HEECs and hESCS. MMP-1 RNA expression was also downregulated by PF4 after P4 withdrawal within HEECs and hESCs. MMP-3 was downregulated by PF4 within F-primed hESCs. This regulation of bFGF indicates a role in anti-angiogenesis; inhibition of MMP-1 and MMP-3 in these culture conditions proposes a role for PF4 in the downregulation of MMPs at menstruation in order enable regrowth of the endometrium and the start of the next menstrual cycle. Conclusions: These data shed new insights into the importance of macrophages in regulating key events in endometrial tissue function during the normal cycle with strong evidence they play a key role in regulating the vasculature during the breakdown and repair of endometrium at menstruation. Notably, new evidence suggesting PF4 may control expression of genes encoding MMP-1 and MMP-3 during menstruation. Further study on the crosstalk between tissue resident populations of macrophages found within the endometrium and other endometrial cell types may provide novel targets for therapies for reproductive disorders associated with inflammation and aberrant angiogenesis including heavy menstrual bleeding and endometriosis.