Nrf2 Dysfunction Impairs Wound Healing in Diabetes Via Impairments in Re-Epithelialization and Angiogenesis
Joshua A. David, BS, Salma A. Abdou, BA, Darren L. Sultan, BA, William J. Rifkin, BA, Jennifer Kwong, BA, Piul S. Rabbani, PhD, Daniel J. Ceradini, MD.
New York University Langone Medical Center, New York, NY, USA.
PURPOSE: Chronic diabetic wounds are the leading cause of lower extremity amputations in the United States. Despite advances in detection, glucose control, and wound therapy, this remains a challenging clinical dilemma. We have previously implicated dysfunction of the Nrf2 anti-oxidant pathway in diabetic wound healing in mice, but the spatial features of Nrf2 dysregulation, and whether this relationship extends to diabetic wound healing in humans, have yet to be elucidated.
METHODS: 10 mm excisional dorsal wounds were created in wild-type (WT), diabetic (db/db), conventional nrf2 knockout (Nrf2 -/-), K14-CreER-flox (epithelial specific, inducible), and Cdh5-CreER-flox (endothelial specific, inducible) mice. Wounds were analyzed grossly and histologically for time to closure (days until healing), wound burden (excisional wound area), epithelial gap (marker of re-epithelialization), and granulation tissue formation. Additionally, wound tissue was obtained from non-diabetic and diabetic patients and analyzed for histology, immunoreactivity of fluorescent stains (Nrf2, K14, CD31), oxidative stress (8-OhdG, a marker of reactive oxygen species (ROS) end-products), and expression of Nrf2-downstream anti-oxidant enzymes (western blot).
RESULTS: In mice, time to wound closure was delayed in Nrf2-/- beyond WT (19d vs. 17d), but not as long as K14-CreER-flox or Cdh5-CreER-flox mice (30d), whose phenotype more closely resembled that of db/db (32d). Pathological wound burden was significantly increased in db/db, K14-CreER-flox, and Cdh5-CreER-flox mice as compared to Nrf2-/- and db/db, p<0.0001. Histologic wound analysis revealed that epithelial gap in both K14-CreER-flox (day 7: 8.7mm, day 10: 6.03mm) and Cdh5-CreER-flox (8.55mm, 6.889) mice more closely resembled db/db (10.07mm, 7.55mm), than WT (6.088mm, 4.346) or Nrf2-/- (5.89mm, 5.558mm), p<0.0003. Granulation tissue was with significantly decreased formation in db/db (.4561mm2), K14CreER-flox (1.083mm2), and Cdh5-CreER-flox (1.873mm2) as compared to WT (4.827mm2) and Nrf2 -/- (2.98mm2) mice, p<0.02. In CD31+ cells within human diabetic wounds, Nrf2 expression was decreased 55% compared to non-diabetic wounds (1.6x10-6 vs. 1.9x10-6 corrected total cell fluorescence (CTCF)). Nrf2 expression was slightly decreased in wound-edge keratin-14+ keratinocytes in human diabetic wounds as compared to non-diabetic wounds (7.6x10-5 vs. 6.9x10-5 CTCF). Further histologic analysis revealed 52.4% decreased blood vessel diameter (152.2 vs. 319.3um), 30% decreased epidermal thickening (283.8um vs 402um), and 28.1% decreased granulation tissue (481014 mm2 vs. 668788mm2) in human diabetic wounds as compared to non-diabetic wounds. Despite more oxidative stress within diabetic wounds as compared to non-diabetic wounds (4.941 ng/mL vs 1.572 ng/mL, p=0.008), expression of Nrf2-downstream anti-oxidant enzyme MnSOD increased 10-fold in non-diabetic wounds, while only a 3-fold increase was seen in diabetic wounds (p<0.001).
CONCLUSION: Here, we demonstrate that tissue-specific loss of Nrf2 in epithelial and endothelial cells recapitulates the diabetic wound-healing phenotype in mice via impairments in epithelial and endothelial cell function. Furthermore, we demonstrate attenuation of Nrf2 expression and function in endothelial and epithelial cells of human diabetic wounds, resulting in impaired regenerative capacity within wounds. Together, these results implicate Nrf2-associated dysfunction of re-epithelialization and angiogenesis - critical functions of wound healing - in the impaired diabetic phenotype. Our findings further validate the Nrf2-antioxidant pathway as a potential therapeutic target or biomarker for improving pathologic diabetic wound healing.
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