Endothelial cell ER stress and Ca^<2+> signaling pathway

内皮细胞（ECs）的凋亡被认为是动脉粥样硬化的第一步。最近的研究报道了内质网Ca^<2+>储存的耗竭在细胞凋亡中起重要作用。Caspase-12是内质网应激诱导细胞凋亡的关键信号。然而，目前尚不清楚ER Ca^<2+>的耗尽是否与ECs中的caspase-12信号传导有关。在此，我们研究了Ca^<2+>信号和caspase-12切割在猪主动脉内皮细胞凋亡中的相互作用。用fura-2/AM测定胞质Ca^<2+>浓度（[Ca^<2+>]i）。DNA阶梯形成法检测细胞凋亡，Western blotting检测caspase-12的裂解。Thapsigargin （6μM）是ER相关Ca^<2+>- atp酶的抑制剂，可使[Ca^<2+>]i升高（F340/F380比值为0.717±0.137至6.133±0.710,p<0.001），诱导ER钙持续缺失、caspase-12的切割和细胞凋亡。缓激肽（10 nM）增加了[Ca^<2+>]i (F340/F380比值为0.717±0.053 ~ 6.133±0.528:p<0.001)，但未诱导caspase-12的裂解和细胞凋亡。然而，当BAPTA/AM （100μM）处理ECs时，BK导致ER的Ca^<2+>耗竭和凋亡，而caspase-12没有被切割。此外，非选择性caspase抑制剂zVAD-fmk （100μM）抑制tg刺激的ECs的凋亡和caspase-12的切割，calpain抑制剂MDL 28170 （120μM）抑制caspase-12的切割，但不抑制细胞凋亡。这些结果表明，[Ca^<2+>]i的增加在ECs诱导凋亡中没有重要作用，ER Ca^<2+>的缺失诱导凋亡，不依赖于caspase-12相关的信号通路。

Apoptosis of endothelial cells (ECs) is now regarded to be an initial step inducing atherosclerosis. Recent studies have reported that the depletion of endoplasmic reticulum (ER) Ca^<2+> stores plays an important role in apoptosis. Caspase-12 is a key signal to lead ER stress-induced apoptosis. However, it is not known whether the depletion of ER Ca^<2+> is linked to caspase-12 signaling in ECs. Here we have investigated the interaction of Ca^<2+> signaling and caspase-12 cleavage in apoptosis of cultured porcine aortic ECs. Cytosolic Ca^<2+> concentration ([Ca^<2+>]i) was measured using fura-2/AM. Apoptosis was assessed by DNA ladder formation, and cleavage of caspase-12 by Western blotting. Thapsigargin (6μM), an inhibitor of the ER-associated Ca^<2+>-ATPase, increased [Ca^<2+>]i (F340/F380 ratio 0.717±0.137 to 6.133±0.710 : p<0.001) and induced persistent ER calcium depletion, cleavage of caspase-12 and apoptosis. Bradykinin (10 nM) increased [Ca^<2+>]i (F340/F380 ratio 0.717±0.053 to 6.133±0.528 : p<0.001) but induced neither cleavage of caspase-12 nor apoptosis. However, when ECs were treated with BAPTA/AM (100μM), BK caused the Ca^<2+> depletion of ER and apoptosis without the cleavage of caspase-12. Moreover non-selective caspase inhibitor, zVAD-fmk (100μM), inhibited apoptosis and cleavage of caspase-12 in TG-stimulated ECs, and calpain inhibitor, MDL 28170 (120μM), inhibited cleavage of caspase-12 but not inhibited apoptosis. These results suggested that the increase of [Ca^<2+>]i did not play an important role in inducing apoptosis in ECs, and ER Ca^<2+> depletion induced apoptosis, which is independent from caspase-12 linked signaling pathway.