Prolyl-isomerase-1 (pin1) mediates hyperglycemia-induced oxidative stress, endothelial function and vascular inflammation: insights in patients with type 2 diabetes

Purpose: Prolyl-isomerase-1 (Pin1) regulates function of protein substrates through isomerization of peptide bonds that link phosphoserine or phosphothreonine to proline. Pin1 triggers reactive oxygen species (ROS) production and inflammation in human cancer. Whether Pin1 is involved in cardiovascular disease remains largely unknown. This study investigates the role of Pin1 in diabetes-related vascular dysfunction. Methods: Human aortic endothelial cells (HAECs) were exposed to normal (NG, 5mmol/L) or high glucose concentrations (HG, 25 mmol/L) in the presence or in the absence of Pin1 pharmacological inhibitor Juglone or siRNA-mediated knockdown. Diabetes was induced in C57/B6 male mice (aged 4-6 months) by streptozocin and animals were treated i.v. with Pin1 siRNA or Juglone i.p for 30 days. Endothelial function was assessed by dose-response curve with acetylcoline. Protein expression was assessed by immunoblotting. Mitochondrial ROS were measured by ESR spectroscopy. Immunoprecipitation was performed to show the interaction of Pin1 with phosphorylated p66Shc and NFkB subunit p65. In parallel, Pin1 gene expression was assessed in peripheral blood monocytes (PBM) of 30 patients with type 2 diabetes (T2DM) and 18 healthy age-matched controls and correlated with flow-mediated vasodilation (FMD) of the brachial artery, urinary 8-isoprostaglandinF2α (8-isoPGF2α) as a marker of oxidative stress, and plasma adhesion molecules VCAM-1, ICAM-1 and MCP-1. Results: Pin1 expression markedly increased in HAECs exposed to HG (289±22% vs NG, p<0.01) and aortas of diabetic mice (216±32% vs controls, p<0.05). Immunoprecipitation showed that Pin1 recognizes phosphoserine motifs of the pro-oxidant mitochondrial adaptor p66Shc as well as NFkB subunit p65. Interestingly, Juglone or Pin1siRNA prevented p66Shc-induced ROS production and suppressed upregulation of adhesion molecules VCAM-1, ICAM-1 and MCP-1 via inhibition of p65 nuclear translocation. In vivo knockdown of Pin1 or Juglone in diabetic mice protected against hyperglycemia-induced endothelial dysfunction, ROS production and vascular inflammation. Of note, Pin1 mRNA was significantly upregulated in PBM of T2DM patients as compared with healthy controls (370±97 vs. 25±28, p<0.01) and correlated with glycated haemoglobin (r=0.44, p<0.05), FMD (r=-0.36,p<0.01), urinary 8-isoPGF2α (r=0.39, p<0.05), as well as VCAM-1 (r=0.56, p<0.05) and ICAM-1 (r=0.53, p<0.05). Conclusions: This study shows for the first time that Pin1 may critically contribute to oxidative stress, endothelial dysfunction and vascular inflammation in patients with type 2 diabetes.