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

内皮细胞凋亡被认为是动脉粥样硬化形成的第一步。最近的研究表明，内质网（ER）钙库的耗竭在细胞凋亡中起重要作用。Caspase-12是导致内质网应激诱导凋亡的关键信号。然而，目前还不清楚ER Ca^<2+>的耗竭是否与EC中的caspase-12信号转导有关。在此，我们研究了Ca^2+信号转导和caspase-12切割在培养的猪主动脉内皮细胞凋亡中的相互作用。用Fura-2/AM测定胞浆Ca^2+浓度（[Ca^2+]i）。DNA梯状条带形成法检测细胞凋亡，Western印迹法检测caspase-12的裂解。Thapsigargin（6μM）是一种ER相关Ca^<2+>-ATP酶的抑制剂，可增加[Ca^<2+>]i（F340/F380比值为0.717±0.137至6.133±0.710：p<0.001），并诱导持续的ER钙耗竭、caspase-12裂解和细胞凋亡。缓激肽（10 nM）增加[Ca^<2+>]i（F340/F380比值为0.717±0.053至6.133±0.528：p<0.001），但既不诱导caspase-12裂解，也不诱导凋亡。然而，当用BAPTA/AM（100μM）处理EC时，BK引起ER的Ca^<2+>耗竭和凋亡，而不引起caspase-12的裂解。此外，非选择性caspase抑制剂zVAD-favor（100μM）可抑制TG刺激的EC凋亡和caspase-12的裂解，而钙蛋白酶抑制剂MDL 28170（120μM）可抑制caspase-12的裂解，但不抑制凋亡。上述结果提示，[Ca^<2+>]i升高在诱导内皮细胞凋亡中不起重要作用，ER Ca^<2+>耗竭诱导内皮细胞凋亡不依赖于caspase-12相关信号通路。

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.