839.9 - Enhanced Vascular Endothelial NLRP3 Inflammasome Activation by Plasma Exosomes from Liver-specific Acid Ceramidase Gene Knockout Mice on the High Fat Diet

Xinxu Yuan (Virginia Commonwealth University), Xiaoyuan Wu (Virginia Commonwealth University), Pin-Lan Li (Virginia Commonwealth University)

Circulatory exosomes have been reported to induce vascular endothelial injury and neointima proliferation in mice with liver-specific deletion of acid ceramidase gene (Asah1fl/fl/Albcre, Asah1 is mouse code of this gene). However, the precise mechanism mediating this exosome-induced endothelial injury remains unknown. The present study tested a hypothesis that plasma exosomes from Asah1fl/fl/Albcre mice on the high fat diet (HFD) activate endothelial NLRP3 inflammasome activation leading to endothelial dysfunction. To test this hypothesis, wild type (WT/WT) mice and Asah1fl/fl/Albcre mice were fed with normal diet (ND) or HFD for 2 months. Using immunofluorescence confocal microscopy, we confirmed that ceramide levels significantly increased in the liver of Asah1fl/fl/Albcre mice compared to its WT/WT littermates and that HFD further enhanced lysosome ceramide levels in the liver as shown by the colocalization of ceramide with lysosome marker, lamp-1. Then, isolated exosomes from the plasma of these mice were used to treat the primary cultured WT/WT endothelial cells from the carotid artery. It was found that exosomes isolated from HFD-treated mice (HFD-Exo) even from WT/WT mice significantly stimulated NLRP3 inflammasome formation and activation as shown by the significant increases in the colocalization of NLRP3 with Caspase-1 or ASC, caspase-1 activity and interleukin 1 beta (IL-1β) level in the endothelial cells compared to exosomes from ND treated mice (ND-Exo). This inflammasome formation and activation were remarkably enhanced if endothelial cells were treated by HFD-Exo isolated from Asah1fl/fl/Albcre mice. To confirm whether lysosomal ceramide contributes to HFD-Exo-induced endothelial NLRP3 inflammasome activation, we further treated endothelial cells with C(2)-ceramide, which was shown similar effects comparable to that induced by HFD-Exo. Using dibutyryl-cAMP, a blocker of ceramide-induced action significantly attenuated C(2)-ceramide- or HDF-Exo-induced enhancement of  endothelial NLRP3 inflammmasome activation. Together, these results suggest that liver-specific deletion of lysosomal Asah1 gene promotes release of HFD-Exos that may contain high ceramide, activating endothelial NLRP3 inflammasome and thereby resulting in endothelial dysfunction during HFD-induced liver steatosis.

Supported by R01 HL075316 and HL075316