Thiol reducing agents modulate induced apoptosis in porcine endothelial cells.

When cultured porcine endothelial cells are exposed first to endotoxin (lipopolysaccharide (LPS)) followed by standard inducers of the heat shock response in vitro (heat or sodium arsenite), these cells aberrantly execute programmed cell death. This cell death is dependent upon two distinct events: the LPS-priming step and the heat shock-induced activation step. Prior work demonstrated that the LPS-priming step could be blocked by the prior application of cell-permeable hydroxyl radical scavengers, suggesting a role for this reactive oxygen species as an important intracellular signal mediating the first step. In these present experiments, we evaluated the potential role of reduction-oxidation mechanisms in the heat shock activation step. The thiol reducing agents reduced glutathione (GSH), n-acetylcysteine (NAC), and dithiothreitol (DTT) were evaluated for their ability to block programmed cell death in LPS-primed porcine aortic endothelial cells. Both DTT and NAC, agents that augment intracellular reduced glutathione levels, were protective against cell death when applied prior to heat shock induction with sodium arsenite (As) in endothelial cells treated previously with LPS. The less cell permeable agent GSH was not protective. Delayed application of DTT or NAC could block progression to cell death for up to 1.5 h after initiation of the heat shock response with As. These data show that heat shock-induced programmed cell death in LPS-primed endothelial cells can be arrested, at least in its early stages, by agents that augment or stabilize the reducing potential of the cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Antioxidants modulate induction of programmed endothelial cell death (apoptosis) by endotoxin.

OBJECTIVE: To evaluate the potential role of reactive oxygen metabolites as signals for endothelial cell apoptosis. DESIGN: A series of antioxidants were evaluated for their ability to block apoptosis in cultured porcine aortic endothelial cells in vitro. RESULTS: Scavenging of the hydroxyl radical with the membrane-permeable scavenger dimethyl sulfoxide or blocking its generation via the Fenton reaction by the chelation of iron with o-phenanthroline blocked apoptosis, whereas the cell membrane-impermeable scavengers superoxide dismutase and catalase did not block apoptosis. Inhibition of xanthine oxidase with enzyme-inhibitory levels of allopurinol also failed to block apoptosis, whereas high levels of allopurinol, which also scavenge the hydroxyl radical in vitro, conferred protection. In each case (dimethyl sulfoxide, o-phenanthroline, and high-dose allopurinol), hydroxyl radical ablation was only effective when administered before the priming step (lipopolysaccharide) and was ineffective when administered later, prior to the activation step (heat shock). CONCLUSIONS: These findings suggest a novel role for the hydroxyl radical as a nonlethal intracellular signal in endothelial cell apoptosis. Moreover, the results support a role for programmed cell death in the pathogenesis of multiple organ dysfunction syndrome and suggest novel strategies for prophylaxis and therapy of the most common cause of death in surgical intensive care units.