An investigation into the mechanisms of the endothelial cell inflammatory responses to trophoblast debris

Abstract

Background: Preeclampsia is a maternal disease characterised by hypertension and proteinuria which could progress to seizures and death if not treated by the delivery of the placenta. Increased quantities of necrotic trophoblast debris (NTD) shed from the placenta could accumulate in the small blood vessels of the maternal lungs during preeclampsia. Here they may be cleared by endothelial cells (ECs) which phagocytose them, leading to EC activation and the production of pro-inflammatory cytokines. However, the mechanism by which ECs detect and respond to NTD is unknown. In macrophages the NALP3 inflammasome is responsible for the detection and pro-inflammatory response to necrotic cells. The primary aim of this thesis was to investigate whether the NALP3 inflammasome is activated in ECs during the response to NTD. In addition, other mechanisms which may be mediating the EC response to NTD were investigated. Methods: Human microvascular endothelial cells-1 (HMEC-1s) were treated in vitro with necrotic JAR cells or NTD from first trimester placentae and expression of the components of the NALP3 inflammasome were investigated by RT-PCR, qRT-PCR and western blotting. Cytokine production, secretion or release from HMEC-1s and NTD was analysed by RT-PCR, qRT-PCR and ELISA. A preliminary quantitative proteomic analysis of proteins which may be involved in the endothelial cell response to NTD was performed by iTRAQ mass spectrometry. Results: Endothelial cells non-inducibly express the components of the NALP3 inflammasome at the mRNA level but expression was not induced by NTD. However data on the expression at the protein level was inconclusive. ECs do not secrete IL-1β, one of the canonical markers of NALP3 inflammasome activation in response to NTD while NTD released IL-1β. ECs did produce and secrete significantly more IL-6 and IL-8, this may be due to NTD derived IL-1β. Seven proteins involved in cell death and immune responses were inducible in endothelial cells after treatment with NTD.