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P19 Emerging role for the NLRP3 inflammasome activation in endothelial cells
  1. R Abas,
  2. M Wozniak,
  3. K Herbert
  1. Department of Cardiovascular Science, University of Leicester, Clinical Science Wing, Glenfield Hospital, Groby Road, Leicester, UK

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.

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