Vitamin D Prevents High Glucose-Induced Endothelial Apoptosis through Inhibition of Prolyl Isomerase-1 Mediated Mitochondrial Oxidative Stress

Aim: To observe the effects of vitamin D on endothelial apoptosis, Pin1 protein expression and activity, mitochondrial translocation of p66Shc, and reactive oxygen species (ROS) levels in high glucose-cultured human umbilical vein endothelial cells (HUVECs) , and explore the role of VDR in these processes.
Methods: HUVECs were treated with high glucose (33 mmol/L) in the presence or absence of vitamin D or Pin1 inhibitor Juglone; Cell apoptosis were measured by flow cytometry and TUNEL staining method; Intracellular ROS levels were examined by flow cytometry and fluorescence microscopy; Protein levels of Pin1, p66Shc, p-p66Shc, mitochondria to cytoplasm ratio of p66Shc, and caspase3 in HUVECs were measured by Western blotting; Pin1 activity in HUVECs lysate was assessed by using a commercially available kit; Knockdown of VDR by siRNA was used to evaluated the role of VDR in the regulatory effects of vitamin D on Pin1 protein expression and activity in HUVECs under hyperglycemia conditions.
Results: 1)Vitamin D suppressed HUVECs apoptosis and intracellular ROS generation induced by high glucose in a dose-dependent (10^-8～10^-6 M) manner (P < 0.01); 2) Vitamin D inhibited high glucose-induced upregulation of Pin1 protein expression and activity in a dose-dependent (10^-8～10^-6 mmol/L) manner (P<0.01); 3) Vitamin D inhibited phosphorylation and mitochondrial translocation of p66Shc and caspase3 protein expression induced by high glucose (P< 0.01); 4) Knockdown of VDR by siRNA abolished the inhibitory effects of vitamin D on high glucose-induced upregulation of Pin1 protein expression and activity.
Conclusions: Vitamin D alleviated high glucose-induced endothelial apoptosis through inhibition of Pin1 protein expression and activity, and consequently p66Shc-mediated mitochondrial oxidative stress, which were dependent on VDR activation.