Effects of Klotho protein on proliferation, migration, adhesion and vascular endothelial growth factor expression of human umbilical vein cells

Abstract

To explore the effects of anti-aging Klotho protein in the proliferation, migration and adhesiveness of human umbilical vein endothelial cell (HUVEC) and its influence of vascular endothelial growth factor (VEGF) expression in HUVEC. HUVEC were treated for 24 h with various concentrations of Klotho protein (final concentrations of 0.1, 1, 10 nmol/L respectively). At the same time, HUVEC treated with PBS, 10 nmol/L VEGF and 10 nmol/L Klotho protein plus AKT inhibitors served as blank control, positive control and AKT signaling pathway inhibition group respectively. The effects of Klotho proteins on proliferation, migration and adhesiveness of HUVEC were determined by thiazolyl blue tetrazolium bromide (MTT), scratches, Transwell and cell adhesion assays respectively. After HUVEC exposed to Klotho protein, the induced function of VEGF expression was measured by Western blot analysis. The MTT results showed that the A values of 0.1, 1, 10 nmol/L Klotho protein group increased respectively to 0.63 ± 0.03, 0.71 ± 0.04, 0.80 ± 0.04, control to the blank group (0.59 ± 0.03, F = 9.32, all P < 0.05). The scratches tests showed that compared with the blank control (9.40 ± 2.07)%, the cell migration rates of 0.1, 1, 10 nmol/L Klotho protein group increased significantly to (12.28 ± 0.62)%, (31.66 ± 1.50)%,(36.69 ± 0.79)%, (F = 9.50, all P < 0.05). Transwell assay found that cell migration count of 0.1, 1, 10 nmol/L Klotho protein group increased to 95.88 ± 9.54, 143.13 ± 7.83, 178 ± 12.77, higher than the blank control group (80.13 ± 12.19), the difference was statistically significant (F = 11.51, all P < 0.05). In cell adhesion assays, the number of cell adhesion increased to 59.60 ± 5.13, 78.40 ± 7.16, 114.60 ± 5.55 at 0.1, 1, 10 nmol/L Klotho protein group versus the blank control (49.40 ± 6.23) and the difference was also statistically significant (F = 9.75, all P < 0.05). Using Western blot, the expressions of VEGF were significantly induced to 0.46 ± 0.02,0.71 ± 0.12,0.81 ± 0.16 at 0.1, 1, 10 nmol/L Klotho protein group versus blank control group (0.35 ± 0.08, F = 8.95, all P < 0.05). Klotho protein can promote the proliferation, migration and adhesiveness of HUVEC and induce a significant expression of VEGF, and its functions are related with the AKT signaling pathway.