ROLE OF ICE-LIKE PROTEASES IN ENDOTHELIAL-CELL HYPOXIC AND REPERFUSION INJURY

Because of its location between blood and tissue, the endothelium is p articularly vulnerable to hypoxic/reperfusion injury, but the mechanis ms responsible for this injury are not known. A number of recent Bindi ngs suggest that hypoxia and reperfusion injures neuronal cells via ap optosis. Apoptosis has recently been shown to depend on the activation of a class of proteases with homology to Interleukin-1 beta convertin g 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 in hibitor II (Ac-YVAD-CMK), ICE inhibitor III (Z-asp-2,6-dichlorobenzoyl oxy-methylketone- Z-Asp-CH2-DCB), or ICE inhibitor IV (Ac- YVKD-CHO) ( all at 10-100 mu M) 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 slot with ICE inhibitor PV, protected cells from reperfusion injury. The protective effect of ICE inhibitor III w as 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 i dentity 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 als o indicate that a potential target of this protease is phospholipase A (2)(PLA(2)). (C) 1997 Academic Press.