APE1/Ref-1 redox function prevents VSMC phenotypic switching through KLF4 suppression

APE1/Ref-1, a multifunctional protein with redox activity, plays a pivotal role in maintaining cellular redox homeostasis and regulating gene expression. This study explored the role of APE1/Ref-1 deficiency on vascular smooth muscle cell (VSMC) phenotypic switching and its underlying mechanisms. Aorta with heterozygous APE1/Ref-1 deficiency (APE1/Ref-1^+/-) exhibited increased oxidative stress and impaired antioxidant defenses, as indicated by elevated reactive oxygen species (ROS) levels, significant 8-hydroxy-2'-deoxyguanosine (8-OHdG) accumulation and reduced expression of antioxidant enzymes. APE1/Ref-1^+/- VSMCs showed a significant reduction in contractile markers (SM22α and α-SMA), alongside an increase in Kruppel-like factor 4 (KLF4) and the proliferative marker PCNA. Treatment with hydrogen peroxide (H₂O₂) increased KLF4 expression and decreased contractile marker expression in VSMC. Furthermore, KLF4 knockdown restored the expression of contractile markers in VSMC. Chemical inhibition of KLF4 using kenpaullone also reversed the downregulation of contractile markers in APE1/Ref-1^+/- VSMCs. Overexpression of wild-type APE1/Ref-1, but not a redox-deficient mutant, rescued contractile marker expression and reduced KLF4 levels. In vivo, APE1/Ref-1^+/- mice displayed enhanced neointimal formation after carotid artery ligation. These findings suggest that APE1/Ref-1 deficiency promotes VSMC phenotypic switching and pathological vascular remodeling via oxidative stress-mediated KLF4 upregulation, and suggesting the critical role of APE1/Ref-1's redox function in vascular homeostasis.