The global cost of diabetes in the U.S. is 1.3 trillion dollars, with chronic diabetic foot ulcers accounting for up to 13 billion dollars annually. An underlying factor that contributes to the chronicity of these wounds is poor vascular flow, which impedes physiologic wound healing. Here, using an in vivo murine model of wound healing, we demonstrate that Nrf2 expression in endothelial cells is necessary for physiologic neovascularization. Full-thickness 10 mm stented excisional wounds were created on the dorsa of double transgenic mouse with inducible conditional deletion of Nrf2 in cadherin 5 expressing endothelial cells (KO), wildtype (WT), and heterozygous mice. Time to wound closure was delayed in the KO compared to WT mice (KO 33.0±1.73 days vs. WT 14.0±0 days) and Nrf2 KO healed similarly to diabetic mice (KO 33.0±1.73 days vs. Diabetic 31.0±1.41 days). Endothelial cells isolated from the wounds of KO mice via flow cytometry demonstrated significantly decreased angiogenic functionality when compared to heterozygous controls. KO endothelial cells formed significantly fewer branches (KO 64.75±14.81 branches/cm2 vs. control 282.8±46.7 branches/cm2, p=0.014) and fewer branching points (KO 39.0±5.18 branching points/cm2 vs. Control 150.8±15.34 branching points/cm2, p=0.0031), indicating impaired angiogenic activity. Immunofluorescent staining of endothelial cells within the wound beds of KO also demonstrate decreased vascularity compared to WT controls. We looked at regulation of the CXCR4/SDF-1 axis, key to angiogenesis, using C166 mouse endothelial cells. Nrf2 knockdown cells in hypoxia, results in reduced Nrf2 levels by 76.5% (p=0.001) and that of downstream marker NQO1 by 66.4% (p=0.02). Expression of CXCR4 and SDF-1 decreased 61.7% (p=0.001) and 47.6% (p=0.02) respectively. These findings suggest that the Nrf2 dysregulation associated with diabetes may contribute to the impaired angiogenesis and decreased revascularization present in chronic diabetic wounds.
J. Lee: None. A.P. Villarreal Ponce: None. J.A. David: None. J. Kuhn: None. J. Kwong: None. D. Ceradini: None.
American Diabetes Association/Pathway to Stop Diabetes (1-16-ACE-08 to D.C.)