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Title: Investigating the effect of plant-derived extracellular vesicles on human placental function
Author: Timms, Katherine
ISNI:       0000 0004 7971 1082
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2018
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A maternal diet high in fruits and vegetables is associated with increased placental size and birthweight and decreased incidence of preeclampsia. This relationship is not explained by maternal socioeconomic status or levels of micro- and macronutrients in the diet, suggesting that an, as yet, unaccounted for nutrient could be contributing to fetal growth. Extracellular vesicles (EVs) are a recently identified component of fruits and vegetables. EVs are nanosized lipid-encased parcels of protein, RNA and other molecules that can alter the function of recipient cells. Fruit and vegetable EVs are internalised into mouse intestinal cells. It was, therefore, hypothesised that EVs from plants consumed as part of the diet would be internalised into human intestinal cells, resulting in the transmission of plant and/or intestinal-derived signals to the placenta, altering placental growth, development or function and thereby linking maternal fruit and vegetable intake to fetal growth. EVs derived from watermelon (WMEVs) were isolated using ultracentrifugation and found to be of a size and morphology consistent with the EV subtype known as exosomes. Mass spectrometry and qPCR arrays were used to identify that WMEVs exhibit protein, lipid and miRNA profiles that differ from watermelon cells, suggesting that their contents are likely actively loaded. Further analysis of the WMEV proteome predicted a possible dual origin from the endosome and plastid for these WMEVs and suggested they may have a role in watermelon fruit ripening. WMEVs were labelled with a lipid (PKH26) or RNA (SYTO RNAselect) dye and incubated apically with Caco-2 cells differentiated on transwell membranes into small intestinelike epithelial cells. Flow cytometry determined that PKH26-labelled WMEVs were internalised into Caco-2 cells in a time (49.45% of cells at 180 minutes; p < 0.0001) and dose-dependent (R2 = 0.99; p < 0.0001) manner, resulting in increased Caco-2 proliferation (7.12-fold increase; p < 0.05). WMEV RNAs, including microRNAs (analysed by qPCR array), were transferred by WMEVs into Caco-2 cells (96.16% of cells at 240 minutes; p < 0.0001) and through the Caco-2 tight barrier to the basal media compartment (16.36% apical-basal crossover; p < 0.05). Proteomic analysis of this conditioned basal media (WMEV-CBM) revealed alterations to the Caco-2 secretome (5 proteins > 1.5-fold upregulated; 13 proteins > 1.5-fold downregulated) in response to WMEVs. Caco-2 secretion of fetuin-A, an inhibitor of extravillous trophoblast (EVT) migration and trophoblast viability, was decreased (4.3-fold lower; p < 0.01) by exposure to WMEVs. Proteomic analysis of placental trophoblast BeWo cells exposed to WMEV-CBM predicted beneficial alterations to several trophoblast parameters, which were investigated experimentally. WMEV-CBM induced expression of 6 syncytialisation genes and increased cell-cell fusion in BeWo cells (1.44-fold increase; p < 0.01) and primary cytotrophoblasts (1.75-fold increase; p < 0.001). WMEV-CBM was also found to decrease apoptosis (1.98-fold; p < 0.01 in BeWo cells) and increase EVT migration (2.17- fold; p < 0.001) from first trimester placental explants. These data suggest that plant EVs, such as WMEVs have the potential to impact upon placental development, growth and function, potentially linking maternal fruit and vegetable intake to improved fetal growth. With further research, this could inform dietary advice in pregnancy and the development of plant EV-based therapies for pregnancies at risk of fetal growth restriction or preeclampsia.
Supervisor: Day, Anil ; Mclaughlin, John ; Westwood, Melissa ; Forbes, Karen Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: diet ; nutrition ; extracellular vesicle ; trophoblast ; pregnancy ; placenta ; watermelon