Keap1/Nrf2 Dysfunction Impairs Wound Healing in Diabetes
Joshua A. David, BS, William J. Rifkin, BA, Zachary Borab, BA, Sophia Hameedi, BA, Piul S. Rabbani, PhD, Daniel J. Ceradini, M.D..
New York University Langone Medical Center, New York, NY, USA.
PURPOSE:Diabetes mellitus is the leading cause of non-healing wounds and lower extremity amputation in the US. We have previously implicated dysfunction of the Nrf2/Keap1 anti-oxidant pathway in this pathology, but the relative contribution of Nrf2 to physiologic and diabetic wound healing remains unknown.
METHODS:Excisional wounds were created in the dorsum of wild-type, diabetic, Nrf2-/-, and Nrf2+/- mice, and stented to prevent secondary contraction. Wound tissue was analyzed for oxidative damage (8-OhdG), gene expression, and re-epithelialization (epithelial gap).
RESULTS:Oxidative damage was greater in both diabetic (2.8 ng/mL) and Nrf2-/- (2.5 ng/mL) skin post-wounding as compared to wild-type (1.1 ng/mL) or Nrf2+/- (1.8 ng/mL, p<.05). Intact skin of diabetic mice had 20% lower levels of Nrf2 gene expression relative to wild-type, and the differences persisted post-wounding (day 5: 27% day 7: 58%). Nrf2+/- mice had intermediate expression levels (day 5: 60%, p<.02, day 7: 76%). Keap1, the physiologic repressor of Nrf2, was overexpressed in diabetic mice as compared to wild-type (intact: 171%, day 5: 253%, day 7: 118%) while it remained constant or decreased in Nrf2-/- mice (intact: 103%, day 5: 61%, day 7: 107%). Gene expression of NAD(P)H quinone dehydrogenase (NQO-1), an antioxidant enzyme activated by Nrf2, peaked later in diabetic (day 10) and Nrf2-/- (day 7) mice compared to wild-type and Nrf2+/- mice (day 5). NQO-1 was also decreased in both diabetic and Nrf2 -/- intact (2% and 30%, respectively p<.05) and wounded tissue (11% and 8%, respectively p<.05) as compared to wild-type, while Nrf2+/- displayed an intermediate phenotype (intact: 37%). At day 7 post-wounding, diabetic and Nrf2+/-wounds displayed the largest epithelial gap (9.7mm and 7.6mm, respectively). Wild-type mice had the smallest epithelial gap (4.1mm), while Nrf2-/- mice displayed an intermediate phenotype (7.0mm).
CONCLUSION:Here, we demonstrate the role of Keap1-mediated Nrf2 loss in impaired diabetic redox handling and wound-healing in an animal model. Our findings further validate this pathway as a promising target for therapies aimed at improving both diabetic and physiologic wound healing.
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