Instant membrane resealing in nlrp3 inflammmasome activation of endothelial cells.

The present study explored the molecular mechanisms by which instant cell membrane resealing (CMR) controls the activation of NOD-like receptor pyrin domain containing 3 (Nlrp3) inflammasomes. Using wavelength-switching fluorescent microscopy with PI and fura-2 as indicators, we monitored instant CMR simultaneously with (Ca(2+))i in mouse microvascular endothelial cell (MVECs). LCWE or saponin wad found to produce membrane injury, which was resealed in a Ca(2+)-dependent manner, but abolished by FasL, a membrane raft (MR) clustering stimulator. Even in the presence of Ca(2+), FasL prolonged the CMR time as shown by an earlier onset of PI influx (48±12 sec vs. 17±3 min. of control). These effects of FasL were substantially blocked by an MR disruptor, methyl-beta-cyclodextrin (MCD). The failure of CMR upon FasL activated Nlrp3 inflammasomes, which was blocked by MCD, a membrane resealing compound, VA64 or siRNA of an MR-facilitating enzyme, acid sphingomyelinase. This inflammasome activation was due to increased lysosomal permeability and cathepsin B release. It is concluded that an MR-associated CMR protects ECs from Nlrp3 inflammasome activation induced by membrane injury.