Suppression of Apoptosis in Human Umbilical Vein Endothelial Cells (HUVECs) by Klotho Protein is Associated with Reduced Endoplasmic Reticulum Oxidative Stress and Activation of the PI3K/AKT Pathway
Wei Cui, Bin Leng, Wei Liu, GaoPin Wang
Department of Key Laboratory of Cardiovascular and Cerebrovascular Drug Research of Liaoning Province, Jinzhou Medical University, Jinzhou, Liaoning, China (mainland)
Med Sci Monit 2018; 24:8489-8499
Klotho protein has been shown to act as a hormone on the cardiovascular system, and to have specific protective effects on vascular endothelial cells. The aim of this study was to investigate the mechanisms of the anti-oxidative and anti-apoptotic effects of klotho protein on hydrogen peroxide (H₂O₂)-induced apoptosis and endoplasmic reticulum oxidative stress in human umbilical vein endothelial cells (HUVECs).
MATERIAL AND METHODS: HUVECs were cultured in vitro and treated with H₂O₂. The MTT assay evaluated cell viability of H₂O₂-treated HUVECs, and flow cytometry measured cell apoptosis. An enzyme-linked immunosorbent assay (ELISA) measured the levels of nitric oxide (NO), tumor necrosis factor-alpha (TNF-α), and interleukin (IL)-6. Western blot was used to detect the expression of the proteins, 78 kD glucose-regulated protein (GRP78), CCAAT-enhancer-binding protein homologous protein (CHOP), caspase-3, caspase-9, caspase-12, and AKT. The effects of LY294002, a pharmacological inhibitor of PI3K, were evaluated.
RESULTS: Klotho protein increased the viability of H2O2-treated HUVECs and reduced the expression of NO, TNF-α, and IL-6. Klotho protein reduced the rate of apoptosis of H₂O₂-treated HUVECs and downregulated the expression of proteins associated with endoplasmic reticulum oxidative stress, GRP78 and CHOP, and the expression of the apoptotic proteins, caspase-3, caspase-9, and caspase-12, and activated the phosphorylation of AKT. The addition of LY294002 inhibited klotho protein downregulation of GRP78, CHOP, caspase-3, caspase-9, and caspase-12 expression.
CONCLUSIONS: In HUVECs, klotho protein suppressed apoptosis mediated by endoplasmic reticulum oxidative stress by activation of the PI3K/AKT pathway.
Keywords: Apoptosis, Endoplasmic reticulum stress, Human Umbilical Vein Endothelial Cells