Abstract
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 ([Ca2+]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 μg/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 [Ca2+]i in HUVECs in a time-dependent manner, which was associated with increased activity of the Ca2+-dependent protease calpain. Amiloride (1−10 μmol/L) significantly suppressed LPS-induced increases in NHE1 activity, [Ca2+]i. and calpain activity. In the presence of the Ca2+ 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 μmol/L) or the calpain inhibitor ZLLal (50 μmol/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 [Ca2+]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.
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Acknowledgements
We thank Dr Humphrey (Department of Medicine, University of Oklahoma Health Sciences Center) for critically reading this manuscript. This work was funded by Jilin University grant (450060445662, 430504001043, and 430505010272 to Qi-sheng PENG; 4305050102Q1 to Wan-chun SUN).
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Cui, Gm., Zhao, Yx., Zhang, Nn. et al. Amiloride attenuates lipopolysaccharide-accelerated atherosclerosis via inhibition of NHE1-dependent endothelial cell apoptosis. Acta Pharmacol Sin 34, 231–238 (2013). https://doi.org/10.1038/aps.2012.155
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DOI: https://doi.org/10.1038/aps.2012.155
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