Abstract

The NLRP3 inflammasome may be involved in atherosclerosis by activation of inflammatory processes in response to danger-associated molecular patterns and pattern-associated molecular patterns. NLRP3 inflammasome requires two steps for activation: signal 1 or priming and signal 2 for activation. This process results in active caspase-1 which in turn cleaves the pro-forms of IL-1β and IL-18 to their active pro-inflammatory forms and also mediates pyroptosis. The aim of this study is to investigate NLRP3 inflammasome activation in human endothelial cells. Differentiated THP-1 cells (positive control), EA.hy926 and human umbilical vein endothelial (HUVEC) were ‘primed’ with lipopolysaccharide (LPS) for 24 hours and then activated by exposure to ATP (300 µM) for 1 hour. Phorbol 12-myristate 13-acetate (PMA) (100 nM) which was used to differentiate THP-1 cells was also investigated as an EA.hy926 cell priming agent. Using Western blotting, NLRP3, pro-IL-1β and processed IL-1β were observed in THP-1 cells following LPS and PMA demonstrating priming and activation of the inflammasome as expected. NLRP3 protein was also upregulated in human endothelial cells following LPS priming but pro-IL-1β expression was not readily observed. However, following PMA treatment of EA.hy926 cells, a substantial synthesis of pro-IL-1β was detected suggesting effective inflammasome priming. Interestingly, pro-IL-1β expression was reduced by high levels of LPS (5 µg/ml) Further work is aimed at defining whether active IL-1β is produced in endothelial cells and defining the pyroptotic response in these cells.