SUMMARY:Mouse lung endothelial cells (MLEC) and HUVEC were used under serum withdrawal (SW) conditions as a model of endothelial cell (EC) apoptosis. Apoptosis was quantified by time-lapse video microscopy. Mouse lung ECs from caspase-1 Ϫ/Ϫ mice had significantly reduced rates of SW-induced apoptosis compared with wild-type mice, specifically implicating caspase-1 in proapoptotic signaling in ECs. SW conditions induced HUVEC apoptosis with concomitant activation of caspase-1. Further studies demonstrated that the caspase-1 inhibitors z-VAD and z-YVAD significantly reduced the rate of SW-induced HUVEC apoptosis. HUVEC, when transfected with caspase-1, showed a highly significant increase in apoptosis. SW was associated with increases in reactive oxygen species production that were significantly inhibited by the antioxidant N-acetyl-L-cysteine, although rates of apoptosis and caspase-1 activation were unaffected. These results demonstrate the involvement of caspase-1 in SW-induced EC apoptosis, independently of reactive oxygen species production. (Lab Invest 2003, 83:1497-1508.A poptosis, the process of innate cellular death, is an accepted part of cell biology. The cellular mechanisms of apoptosis are complex and diverse, with considerable cell-type specificity.The activation of intracellular cysteine proteases (caspases) that have a unique cleavage site involving an aspartate residue is crucial to the process of apoptosis (Whyte, 1996;Wolf and Green, 1999). Once activated, caspases cleave cellular substrates, leading to characteristic morphologic hallmarks of apoptosis (Nicholson and Thornberry, 1997). There is a "hierarchy" of caspases in the control of apoptosis, with regulatory caspases (eg, caspase-2, -8, -9, and -10), which have long prodomains, initiating cleavage of downstream executioner caspases (eg, caspase-3, -6, and -7). The prototypic caspase, caspase-1, however, was originally identified as the enzyme responsible for processing IL-1␤ to its 17-kDa mature form (Cerretti et al, 1992). Overexpression of caspase-1, as with most other caspases, produces apoptosis (Miura et al, 1993). However, caspase-1 is not generally regarded as a component of the cell death machinery; together with caspases-4 and -5, it is regarded as an "inflammatory caspase" that functions only in cytokine processing (Martinon et al, 2002;Wolf and Green, 1999).The vascular endothelium has a central role in inflammation, the response to infection, and the pathogenesis of vascular disease. Endothelial cells (EC) can be induced to undergo apoptosis by a number of treatments, including TNF-␣ in the presence of protein synthesis inhibitors (Polunovsky et al, 1994), radiation (Haimovitz-Freidman et al, 1994), certain snake venoms (Araki et al, 1993), lipopolysaccharide (LPS) (Choi et al, 1998), and oxidized low-density lipoprotein (Harada-Shiba et al, 1998;Sata and Walsh, 1998). EC apoptosis occurs as a widespread event in thrombotic thrombocytopenic purpura (Laurence et al, 1996) and may occur in systemic sclerosis induced by anti-EC a...

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