Targeting Prolyl-isomerase-1 protects against hyperglycemia-induced endothelial dysfunction and vascular inflammation: alterations in patients with type 2 diabetes

Introduction: Prolyl-isomerase-1 (Pin1) regulates function of protein substrates through isomerization of peptide bonds that link phosphoserine or phosphothreonine to proline. Recent studies showed that Pin1 favours cancerogenesis through reactive oxygen species (ROS) production and inflammation. Whether Pin1 partecipates to vascular disease is unknown. Hypothesis: 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 by streptozotocin in C57/B6 mice and animals treated with Pin1 siRNA i.v or Juglone i.p for 30 days. Endothelial function was assessed by dose-response curve with acetylcoline (10-9-10-6 mol/L). Mitochondrial ROS were measured by ESR spectroscopy. Pin1 gene expression was assessed in peripheral blood monocytes (PBM) of 37 patients with type 2 diabetes (T2DM) and 20 age-matched controls and correlated with flow-mediated vasodilation (FMD) of the brachial artery, urinary oxidative marker 8-isoprostaglandinF2α (8-isoPGF2α) and plasma adhesion molecules VCAM-1, ICAM-1 and MCP-1. Results: Pin1 expression 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 and NF-kB p65. Interestingly, Juglone or Pin1siRNA prevented p66Shc-induced ROS production and suppressed NF-kB-dependent upregulation of adhesion molecules. In diabetic mice, silencing of Pin1 or Juglone prevented endothelial dysfunction, ROS production and vascular inflammation. Of note, Pin1 mRNA was significantly upregulated in PBM of T2DM patients as compared with controls (370±97 vs. 25±28,p<0.01) and correlated with FMD (r=-0.33, p<0.01), urinary 8-isoPGF2α (r=0.46, p<0.05), VCAM-1 (r=0.58, p<0.05) and ICAM-1 (r=0.55, p<0.05). Conclusions: Pin1 may critically participate to vascular disease in T2DM patients.