Abstract 430: Impairment of Instant Membrane Resealing by Lipid Raft Clustering in Endothelial Cells: Role in NLRP3 Inflammmasome Activation

Abstract

Instant membrane resealing importantly contributes to the functional and structural integrity of the endothelial cells (ECs) that are exposed to various physical and chemical stimuli in blood stream. The present study was designed to explore the molecular mechanisms mediating this endothelial membrane resealing with a focus on the role of lipid rafts (LR) clustering. Using high energy Laser gun, a tiny hole was made in cultured EC bathed with FM1-43, and the rapid entry of this FM1-43 to produce fluorescence was used to measure membrane resealing. We demonstrated that ECs exhibited a Ca2+-dependent instant membrane resealing, as shown by a significant reduction of fluorescence appearance within these cells compared to ECs bathed with Ca2+ free solution. This Ca2+-dependent instant membrane resealing was also observed in ECs up stimulation of Lactobacillus casei cell wall fragments (LCWE), which was commonly used to produce arteritis. When ECs were pretreated with LRs clustering stimulators such as C-16 ceramide, ASMase or FasL, EC membrane resealing was substantially abolished and membrane wounding areas are enriched with LR clusters. By dual wavelength high speed wavelength switching microscopy, we found that a small Ca2+ influx (fura-2 fluorescence) was recorded at beginning of LCWE treatment of ECs (about 10 min), but membrane was immediately resealed (no PI fluorescence). However, if the LR stimulator FasL was used to pretreat these ECs, a large flushing of both PI and Ca2+ into ECs was monitored. By calculation of df/dt and time of PI fluorescence reaching the maximum, it was shown that FasL largely increased LCWE-induced PI fluorescence to a level equal to ECs with Ca2+-free solution over a 20-min period (maximum df/dt=1.17 fold/sec±0.22 at 14 min with FasL+Ca2+, 0.87 fold/sec±0.21 at 19 min with Ca2+ free, and 0.03 fold/sec±0.01 at 20 min with Ca2+ in bath). In addition, it was shown that impaired EC membrane resealing significantly enhanced the production of active caspase-1, a marker of NLRP3 inflammasome activation (a ~3-fold increase). These results strongly suggest that ceramide-associated LR clustering impairs the instant membrane resealing in ECs, which may activate EC inflammasomes leading to vascular inflammation and injury.