Role of ice-like proteases in endothelial cell hypoxic and reperfusion injury

Because of its location between blood and tissue, the endothelium is particularly vulnerable to hypoxic/reperfusion injury, but the mechanisms responsible for this injury are not known. A number of recent findings suggest that hypoxia and reperfusion injures neuronal cells via apoptosis. Apoptosis has recently been shown to depend on the activation of a class of proteases with homology to Interleukin-1β converting enzyme (ICE) protease. Therefore, we examined the effect of specific inhibitors of ICE-like proteases on hypoxic and reperfusion injury in cultured EAhy926 endothelial cells. Pretreatment of cells with ICE inhibitor II (Ac-YVAD-CMK), ICE inhibitor III (Z-Asp-2,6-dichlorobenzoyloxymethylketone-Z-Asp-CH₂-DCB), or ICE inhibitor IV (Ac-YVKD-CHO) (all at 10-100 μM) did not protect cells from hypoxic injury. However, pretreatment of cells with ICE inhibitor III and to a lesser extent with ICE inhibitor II, but not with ICE inhibitor IV, protected cells from reperfusion injury. The protective effect of ICE inhibitor III was not dependent upon pH, but was associated with decreased release of arachidonic acid from cells. These findings suggest that reperfusion injury to EAhy926 endothelial cells involves ICE-like proteases. The identity of the protease(s) is not known but it does not appear to be a YAMA-type protease based upon ICE inhibitor specificity. Our data also indicate that a potential target of this protease is phospholipase A₂ (PLA₂).