Amiloride attenuates lipopolysaccharide-accelerated atherosclerosis via inhibition of NHEl-dependent endothelial cell apoptosis

• Published in: 中国药理学报：英文版 2013 (2), p.231-238
• Main Author: Gui-mei CUI Yu-xi ZHAO Na-na ZHANG Zeng-shan LIU Wan-chun SUN Qi-sheng PENG
• Format: Article
• Language: English
• Subjects: Bcl-2蛋白; 人脐静脉内皮细胞; 动脉粥样硬化; 细胞凋亡; 脂多糖; 蛋白酶活性; 血管内皮; 钙蛋白酶
• ISSN: 1671-4083; 1745-7254

Aim： To investigate the effects of the potassium-sparing diuretic amiloride on endothelial cell apoptosis during lipopolysaccharide （LPS）-accelerated atherosclerosis. Methods： Human umbilical vein endothelial cells （HUVECs） were exposed to LPS （100 ng/mL） in the presence of drugs tested. The activity of Na^＋/H^＋ exchanger 1 （NHE1） and calpain, intracellular free Ca2＋ level （[Ca^2＋]i）, as well as the expression of apoptosis-related proteins in the cells were measured. For in vivo study, ApoE-deficient （ApoE^-/-） mice were fed high-fat diets with 0.5% （w/w） amiloride for 4 weeks and LPS （10 pg/mouse） infusion into caudal veins. Afterwards, atherosclerotic lesions, NHE1 activity and Bcl-2 expression in the aortic tissues were evaluated. Results： LPS treatment increased NHE1 activity and [Ca^2＋]i in HUVECs in a time-dependent manner, which was associated with increased activity of the Ca^2＋ -dependent protease calpain. Amiloride （1-10 pmol/L） significantly suppressed LPS-induced increases in NHE1 activity, [Ca^2＋]i. and calpain activity. In the presence of the Ca^2＋ chelator BAPTA （0.5 mmol/L）, LPS-induced increase of calpain activity was also abolished. In LPS-treated HUVECs, the expression of Bcl-2 protein was significantly decreased without altering its mRNA level. In the presence of amiloride （10 pmol/L） or the calpain inhibitor ZLLal （50 pmol/L）, the down-regulation of Bcl-2 protein by LPS was blocked. LPS treatment did not alter the expression of Bax and Bak proteins in HUVECs. In the presence of amiloride, BAPTA or ZLLal, LPS-induced HUVEC apoptosis was significantly attenuated. In ApoE-/- mice, administration of amiloride significantly suppressed LPS-accelerated atherosclerosis and LPS-induced increase of NHE1 activity, and reversed LPS-induced down-regulation of Bcl-2 expression. Conclusion： LPS stimulates NHE1 activity, increases [Ca^2＋]i, and activates calpain, which leads to endothelial cell apoptosis related to decreased Bcl-2 expression. Amiloride inhibits NHE1 activity, thus attenuates LPS-accelerated atherosclerosis in mice.