Abstract 15426: Inflammasome-induced Signalling Mechanisms via Microparticles

Abstract

Introduction: The inflammasome, a multi-protein signaling platform, has been shown to be an important mediator in the development of atherogenesis. Microparticles (MPs) are small membrane vesicles, which are released from apoptotic cells. Whether inflammasome activation leads to the release of MPs by vascular cells is unknown. Hypothesis: Inflammasome-activation in vascular cells leads to microparticle release. These microparticles contain components of the inflammasome and exert functional effects on other vascular cells. Methods: EMP (endothelial microparticles) and MMP (monocytic microparticles) were quantified by flow cytometry using TruCount tubes. Inflammasome-activation was investigated by IL-1β ELISA and immunoblotting. The latter was also used to determine EMP content. Viability Assay was performed to test cell-viability and Scratch assay to examine effects of EMP on migration and proliferation of vascular cells. Results: Human coronary artery endothelial cells (HCAEC) and monocytes were primed with LPS and subsequently stimulated with either ATP or Nigericin, two established inflammasome activators. MP release was quantified by flow cytometry using TruCount tubes. Stimulation of HCAEC and monocytes with both Nigericin and ATP resulted in a time-dependent release of MPs. Inflammasome-activation in HCAEC was confirmed by the release of IL-1β into the cell supernatant and immunoblotting against Caspase-1. Immunoblotting also showed that inflammasom-activated HCAEC and the associated EMP contain pro-Caspase-1, active Caspase-1 and NALP-3. Inoculation of HCAEC with EMP, derived from HCAEC stimulated with Nigericin, lead to a rapid and significant cell death and reduced cell migration/ proliferation shown by Viability Assay and Scratch Assay, respectively. Conclusions: We show for the first time that Nigericin and ATP, two established inflammasome activators, lead to inflammasome activation and release of microparticles by vascular cells. Furthermore, we could demonstrate that these microparticles contain pro-Caspase-1, mature-Caspase-1 and NALP-3 and that they cause cell death accompanied with reduced cell migration and proliferation.