Inhibition of Fas-Fas Ligand Interaction Attenuates Microvascular Hyperpermeability Following Hemorrhagic Shock

Abstract

Hemorrhagic shock (HS) induced microvascular hyperpermeability poses a serious challenge in the management of trauma patients. Microvascular hyperpermeability occurs mainly due to the disruption of endothelial cell adherens junctions, where the 'intrinsic' apoptotic signaling plays a regulatory role. The purpose of this study was to understand the role of the 'extrinsic' apoptotic signaling molecules, particularly Fas-Fas ligand interaction in microvascular endothelial barrier integrity. Rat lung microvascular endothelial cells (RLMECs) were exposed to HS serum in the presence or absence of the Fas ligand inhibitor, FasFc. The effect of HS serum on Fas receptor and Fas ligand expression on RLMECs was determined by flow cytometry. Endothelial cell permeability was determined by monolayer permeability assay and the barrier integrity by β-catenin immunofluorescence. Mitochondrial ROS formation was determined using dihydrorhodamine 123 probe by fluorescent microscopy. Mitochondrial transmembrane potential (MTP) was studied by fluorescent microscopy as well as flow cytometry. Caspase-3 enzyme activity was assayed fluorometrically. RLMECs exposed to HS serum showed increase in Fas receptor and Fas ligand expression levels. FasFc treatment showed protection against HS serum induced disruption of the adherens junctions, and monolayer hyperpermeability (p < 0.05), in the endothelial cells. Pretreatment with FasFc also decreased HS serum induced increase in mitochondrial ROS formation, restored HS serum induced drop in MTP, and reduced HS serum induced caspase-3 activity in RLMECs. These findings open new avenues for drug development to manage HS induced microvascular hyperpermeability by targeting the Fas-Fas ligand mediated pathway.

In vitro endothelial hyperpermeability occurs early following traumatic hemorrhagic shock

“…Animal studies showed an early increase in endothelial permeability following hemorrhagic shock [14,15], which even further increased following fluid resuscitation [16][17][18]. This was confirmed by a study showing that rodent plasma, collected after fluid resuscitation, induced in vitro rodent endothelial hyperpermeability [19]. However, evidence regarding the effect of human plasma from patients following traumatic hemorrhagic shock on endothelial barrier is limited.…”

“…Current knowledge regarding hemorrhagic shock-induced hyperpermeability is restricted to animal studies [14][15][16][17][18] or in vitro studies investigating the effect of rodent plasma on endothelial barrier function [19]. Animal studies showed an early increase in endothelial permeability following hemorrhagic shock [14,15], which even further increased following fluid resuscitation [16][17][18].…”