A constitutive cytoprotective pathway protects endothelial cells from lipopolysaccharide-induced apoptosis

Lipopolysaccharide (LPS) has been implicated as the bacterial component res ponsible for much of the endothelial cell injury/dysfunction associated wit h Gramnegative bacterial infections. Protein synthesis inhibition is requir ed to sensitize the endothelium to lipopolysaccharide-induced apoptosis, su ggesting that a constitutive or inducible cytoprotective protein(s) is requ ired for endothelial survival. We have identified two known endothelial ant i-apoptotic proteins, c-FLIP and Mcl-1, the expression of which is decrease d markedly in the presence of cycloheximide. Decreased expression of both p roteins preceded apoptosis evoked by lipopolysaccharide + cycloheximide, Ca spase inhibition protected against apoptosis, but not the decreased express ion of c-FLIP and Mcl-1, suggesting that they exert protection upstream of caspase activation. Inhibition of the degradation of these two proteins wit h the proteasome inhibitor, lactacystin, prevented lipopolysaccharide + cyc loheximide-induced apoptosis, Similarly, lactacystin protected against endo thelial apoptosis induced by either tumor necrosis factor-alpha or interleu kin-1 beta in the presence of cycloheximide. That apoptosis could be blocke d in the absence of new protein synthesis by inhibition of the proteasome d egradative pathway implicates the requisite involvement of a constitutively expressed protein(s) in the endothelial cytoprotective pathway. Finally, r eduction of FLIP expression with antisense oligonucleotides sensitized endo thelial cells to LPS killing, demonstrating a definitive role for FLIP in t he protection of endothelial cells from LPS-induced apoptosis.