Critical Regulation of Angiogenesis by Nrf2 Signaling Is Absent in Diabetic Wound Healing

Abstract

Diabetes-associated ulcers account for 11% of non-traumatic limb and extremity amputations. Tailoring therapies for chronic diabetic wounds is challenging, however, as specific molecular/cellular events responsible for the extreme delays or lack of wound healing mostly remain at large. We previously identified dysfunction of the cytoprotective Nrf2/Keap1 signaling pathway induced by chronic hyperglycemia and diabetes. In the diabetic wound bed, we found detrimental impact on angiogenesis which is a requisite for tissue repair. Exogenous activation of Nrf2 in the whole diabetic wound bed accelerated healing and illustrated the promise of targeting Nrf2 activity for reversing diabetes-associated impairments. Here, we explored the specific role of Nrf2 in endothelial cells of the wound tissue repair niche, and corresponding deficiencies in Nrf2 activity during diabetic wound healing. We generated Cadherin5-CreER;Nrf2fl/fl mice to deplete Nrf2 specifically in endothelial cells, upon tamoxifen administration (Nrf2KO mice). Stented excisional wounds (10mm diameter) on Nrf2KO mice, induced prior to wounding, required 33±1.23 days for closure. This delayed repair phenotype was significantly higher than that of wild type wounds requiring 14.5±0.29 days (p<0.05), but very similar to the 31.5±0.65 days required by wounds on Leprdb/db diabetic mice. Successful inhibition of Nrf2 in microvasculature-derived endothelial cells in vitro demonstrated compromised proliferation (7.12±1.19 units), compared to cells transfected with non-sense siRNA (11.37±0.96 units, p<0.05). Endothelial cells with silenced Nrf2 also demonstrated less angiogenic capacity in vitro, unable to branch or form nodes. Our data prove that Nrf2 activity regulates endothelial cell proliferation and angiogenesis among features that are vital during wound repair. Our results reinforce the necessity of Nrf2 activation in skin for wound repair and underscore the impact of its deficiency in diabetic skin. Disclosure P.S. Rabbani: None. J.A. David: None. D.L. Sultan: None. A.P. Villarreal-Ponce: None. J. Kwong: None. J. Lee: None. C. Shen: None. S.A. Abdou: None. D. Ceradini: None.