Background:

Atherosclerosis as a chronic inflammatory disease is at least partly based on inflammasome activation and circulating microparticles. Activation of the inflammasome occurs as a response to danger signals and stimulates inflammatory processes that cause the activated cells to undergo pyroptosis. Microparticles are extracellular vesicles that are released by activated or apoptotic cells and serve as a mechanism of intercellular communication. 

Methods and results:

With this study, we sought to elucidate the role of endothelial inflammasome activation on microparticle release and the subsequent functional effects. We found that stimulation of HCAEC (human coronary artery endothelial cells) with the established inflammasome activators LPS (Lipopolysaccharide) and nigericin led to activation of the NLRP3 (NACHT, LRR and PYD domains-containing protein 3) inflammasome. Inflammasome-activation was detected by increased expression of NLAP3, IL-1b and Caspase-1 in RT-PCR and western blot experiments, cleavage of Gasdermin-D in western blot experiment, pyroptosis shown by light microscopic imaging  and release of LDH in LDH assay. Beyond this, examination of cellular supernatants by flow cytometry, electron microscopic imaging, nano tracking analysis and western blot experiments revealed a time-dependent release of endothelial microparticles containing cleaved Caspase-1. Stimulation of HCAEC with inflammasome-induced endothelial microparticles was followed by microparticle incorporation proven by fluorescent microscopic imaging. Microparticle uptake led to NLRP3-inflammasome activation, inflammatory response, pyroptosis and reduced endothelial migration and proliferation shown by viability assay, cytotoxity assay, scratch assay , RT-PCR experiments, western blots. Observed effects could be attenuated by treatment of the inflammasome-induced microparticles with the inflammasome-inhibitor ILG, heat-inactivation and freezing. Similar to endothelial cells microparticle uptake, inflammasome-activation after incorporation with inflammasome-induced microparticles and attenuation of inflammasome activation by inflammasome inhibitors could be observed for vascular smooth muscle cells.

Discussion:

These data show that the endothelial NLRP3-inflammasome and associated microparticles are involved in the innate immune response of the vascular wall, thereby contribute to the development of vascular disease and could be a therapeutic target for the treatment of atherosclerosis.