Method: Floxed FOXO1 was deleted in keratinocytes in vivo by keratin-14 driven Cre recombinase and by RNAi in vitro. Type 1 diabetes was induced by multiple low-dose streptozotocin. Healing in FOXO1 deleted experimental mice (K14.Cre+/FOXO1L/L) was compared to matched control (K14.Cre-/FOXO1L/L) diabetic mice. Small 2mm excisional wounds were created in the scalp and histomorphometric analysis was performed on histologic sections as well as the TUNEL assay, expression of PCNA and detection of 8-OHdG, a marker of oxidative DNA damage. Closure of an in vitro “scratch wound” examined the effect of FOXO1 siRNA compared to scrambled siRNA on keratinocyte migration, apoptosis (TUNEL assay) and expression of selected genes by real-time PCR in the presence of high glucose. Significance was determined by one-way ANOVA.
Result: Wound closure was 55% faster in FOXO1-deficient diabetic mice compared with control diabetic mice. FOXO1 deletion reduced keratinocyte apoptosis by 77% and increased proliferation 66% in healing diabetic wounds in vivo (P<0.05). FOXO1 deletion reduced oxidative damage by 60% comparing with control diabetic mice (P<0.05). In vitro, high glucose increased FOXO1 activation by 40% and decreased keratinocyte migration by 40% (p<0.05). Deletion of FOXO1 by siRNA rescued the negative impact of high glucose on keratinocyte migration. High glucose also stimulated keratinocyte apoptosis in vitro, which was reversed by FOXO1 siRNA (P<0.05). Potential FOXO1 target genes were significantly downregulated by FOXO1 siRNA by 40-89% compared to scrambled siRNA and included SPERINB2, CCL20, MMP9 and IL-23A (p<0.05).
Conclusion: FOXO1 mediates some of the detrimental effects of diabetes on wound healing. FOXO1 dysregulation in diabetic wounds impairs closure by increasing keratinocyte apoptosis and oxidative damage and decreasing keratinocyte migration and proliferation.