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 ([Ca²⁺]_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 μM), an inhibitor of the ER Ca²⁺-ATPase, depleted ER Ca ²⁺ 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 μM), bradykinin caused ER Ca²⁺ depletion and apoptosis without accompanying caspase-12 cleavage. A non-selective caspase inhibitor, z-VAD.fmk (100 μM), inhibited apoptosis and cleavage of caspase-12 stimulated by thapsigargin, while a calpain inhibitor, MDL 28170 (120 μM), 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.