Abstract 564: Inflammasome-induced Endothelial Microparticles Exert Detrimental Effects on Recipient Vascular Cells

Abstract

Background: The inflammasome, a multi-protein signaling platform, is an important mediator of vascular inflammation resulting in atherosclerosis. Microparticles (MPs) are small membrane vesicles, specifically packaged and released from apoptotic cells for intercellular communication. Endothelial cell derived MPs have recently been linked to atherogenesis. Whether inflammasome activation in endothelial cells mediates MP release and thereby exert biological effects on recipient vascular cells is unknown. Methods and results: Human coronary artery endothelial cells (HCAEC) were primed with 1μg/ml LPS and subsequently stimulated with 20μM Nigericin leading to a specific inflammasome activation as detected by significant upregulation of Caspase-1 mRNA in RT-PCR and protein in Westernblots (Figure1). Inflammasome activation in vascular cells lead to formation of endothelial microparticles (EMP) in a time- and dosis-dependend manner (Figure 2). Comparison of EMP with beads of known size using flow cytometric and electron microscopic imaging could point out an EMP-size between 0,1 – 1 μm (Figure 3). EMP-uptake by recipient vascular cells could be illustrated by fluorescence-microscopic imaging of PKH26 labeled EMP (Figure 4). Viability assay and scratch assay showed detrimental effects of EMPs from inflammasome activated cells on recipient vascular cells. Viability, proliferation and migration were reduced significantly 4h after treatment (Figure 5). Conclusions: We show for the first time that Nigericin, an established inflammasome activator, leads to inflammasome activation and release of microparticles by endothelial cells. Furthermore, that these microparticles are taken up by recipient vascular cells and thereby cause cell death accompanied with reduced cell migration and proliferation.