Obstetrics and Gynaecology - Theses
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ItemBiological Pathways Used by Decidual Mesenchymal Stem Cell-Derived Extracellular Vesicles in Repairing Dysfunctional Endothelium( 2022)Preeclampsia (PE), a common pregnancy-related disorder, is characterised by endothelial cell dysfunction and oxidative stress. Oxidative stress is a consequence of reduced blood flow to the placenta and results in the secretion of anti-angiogenic factors that cause systemic damage to maternal vascular endothelial cells. Endothelial cell dysfunction culminates in the clinical symptoms of PE, including elevated blood pressure, proteinuria, and HELLP syndrome. Human cell culture models variously use damaging agents such as lipopolysaccharide (LPS), hydrogen peroxide (H2O2), and serum derived from preeclamptic women (PE serum) to model the damage to endothelial cells in PE. However, the three methods have not been compared. Several studies reported that decidual mesenchymal stem cells (DMSCs) and the extracellular vesicles derived from them (DMSC_EVs) have beneficial effects on the cell growth profile of endothelial cells. However, the biological pathways by which DMSC_EVs exert their beneficial effects are poorly understood. The damaging agents (LPS, H2O2, and PE serum) were used to treat human umbilical vein endothelial cells (HUVEC), which are a common model for human endothelial cells. Damaged HUVEC were treated with or without DMSC_EVs. HUVEC growth profiles were measured by xCELLigence real-time functional assays. HUVECs were collected by centrifugation (i.e. cell pellets) as was as the conditioned medium (CM) containing secreted proteins. The general hypothesis for the study was that DMSC_EVs repair endothelial cell damage in human cell culture models of PE, and that the biological pathways involved in repair can be identified. The first aim determined the optimal concentration of each damaging agent and xCELLigence assays showed that all damaging agents had a detrimental effect on HUVEC growth phases of attachment and proliferation. Following exposure of HUVEC to the damaging agent and treatment with various concentrations of DMSC_EVs (0, 50, 100, 150 microgram/ml), the growth phases of HUVEC were assessed by xCELLigence real-time analysis. In each of the three models of endothelial cell damage, adding 100 microgram/ml DMSC_EVs to damaged HUVECs had a consistent stimulatory effect compared to untreated cells with respect to both cell attachment (LPS P<0.01, H2O2 P<0.001, PE serum P<0.01) and proliferation (LPS P<0.001, H2O2 P<0.001, PE serum P<0.001). PE serum was chosen for subsequent analyses as this damaging agent most reflects the in vivo situation in PE. Following xCELLigence analysis, cell pellets and conditioned media were collected from PE serum-damaged HUVEC treated with, or without, 100 microgram/ml DMSC_EVs. These were assessed by mass spectrometry to determine which biomolecules were involved in the effect of DMSC_EVs on PE-serum damaged HUVEC attachment and proliferation. Following DMSC_EV treatment of PE serum-damaged HUVEC, the most abundant and differentially expressed HUVEC proteins during the attachment phase were NADH-ubiquinone oxidoreductase chain 4 (1.91 fold) and Interferon-induced transmembrane protein 3 (-2.14 fold), while in the proliferation phase, cellular proteins YIF1B (3.58 fold) and Brain acid soluble protein 1 (-2.15 fold) were the most abundant and differentially expressed. The most abundant and differentially expressed secreted proteins after DMSC_EV treatment of PE serum-damaged HUVEC treatment in the attachment phase were Cytochrome c1 (6.54 fold) and Midasin (-4.21 fold), while in the proliferation phase, Collagen alpha-1(IV) chain (6.6 fold) and cAMP-dependent protein kinase catalytic subunit alpha (-3.76 fold) were the most abundant and differentially expressed. Analysis of relevant biological processes revealed the most affected processes were cell organization and biogenesis. Kegg pathway analysis revealed that DMSC_EV treatment altered pathways relevant to cell attachment included focal adhesion, crosslinking of collagen fibrils and extracellular matrix organization. Kegg pathway analysis showed cell proliferation pathways of extracellular matrix organization and degradation were altered by DMSC_EV treatment. Finally, literature searches showed many of the proteins that displayed the highest abundance and differential expression levels following DMSC_EV treatment were implicated directly or indirectly either in the pathogenesis of the PE placenta or showed altered levels in PE patient blood. This study shows that in a cell culture model of PE, treatment with DMSC_EVs had beneficial effects on important cell growth phases of damaged endothelial cells (i.e. attachment and proliferation), that relevant biological processes and biological pathways were altered and that many of the proteins involved were also altered in human PE. This work lays the groundwork for future PE animal model studies to determine whether DMSC_EVs improve endothelial cell function and whether the biological processes and pathways of endothelial repair identified in the human cell culture models of PE in this study are relevant in vivo.