Endoplasmic reticulum Ca2+ depletion induces endothelial cell apoptosis independently of caspase-12

Objective: Apoptosis of endothelial cells is considered an initial step in the development of atherosclerosis. Recent studies have indicated that depletion of the endoplasmic reticulum (ER) Ca2+ content plays an important role in apoptosis. Caspase-12 is a key signal in ER stress-induced apoptosis. However, it is not known whether the depletion of ER Ca2+ is linked to caspase-12 signalling in endothelial cells. Here we have investigated the interaction of Ca2+ signalling and caspase-12 cleavage in apoptosis of endothelial cells. Methods: Cytosolic Ca2+ concentration ([Ca 2+](i)) of primary porcine aortic endothelial cells was measured using fura-2/AM. Apoptosis was assessed by DNA fragmentation, and cleavage of caspase-12 using Western blotting techniques. Results: Thapsigargin (5 mu M), an inhibitor of the ER Ca2+-ATPase, depleted ER Ca2+ content, increased [Ca2+](i) cleaved caspase-12, and induced apoptosis. Bradykinin (10 nM) also increased [Ca2+](i) but did not cleave caspase-12 or induce apoptosis. However, when intracellular Ca2+ was chelated with BAPTA/AM (100 mu M), bradykinin caused ER Ca2+ depletion and apoptosis without accompanying caspase-12 cleavage. A non-selective caspase inhibitor, z-VAD.fmk (100 mu M), inhibited apoptosis and cleavage of caspase-12 stimulated by thapsigargin, while a calpain inhibitor, MDL 28170 (120 mu M), inhibited caspase-12 cleavage but not apoptosis. Conclusions: Thus, increases in intracellular Ca2+ concentration are not sufficient for the induction of apoptosis in endothelial cells, and ER Ca2+ depletion appears to induce apoptosis independently of caspase-12. (c) 2005 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.