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

BACKGROUND: 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 (H(2)O(2))-induced apoptosis and endoplasmic reticulum oxidative stress in human umbilical vein endothelial cells (HUVECs). MATERIAL/METHODS: HUVECs were cultured in vitro and treated with H(2)O(2). The MTT assay evaluated cell viability of H(2)O(2)-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 H(2)O(2)-treated HUVECs and reduced the expression of NO, TNF-α, and IL-6. Klotho protein reduced the rate of apoptosis of H(2)O(2)-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.