Nrf2 Dysfunction is the Basal Epidermis Coincides with Delayed Healing in Type II Diabetes
Joseph Kuhn, M.D., Joshua A. David, B.S., Alvaro Villarreal-Ponce, PhD, Darren Sultan, B.S., Salma A. Abdou, B.A., Jennifer Kwong, B.A., Piul S. Rabbani, PhD, Daniel Ceradini, M.D..
NYU Langone, New York, NY, USA.
Purpose: Despite optimal medical management of type II diabetes, unmitigated oxidative stress is an enduring force underlying delayed wound healing, a complication that leads to amputation, causes disability, and robs patients of their quality of life. Nrf2, the master transcriptional regulator of redox homeostasis, holds the potential to be a powerful diagnostic tool and therapeutic target against overwhelming reactive oxygen species in diabetic wounds. However, the spatial dimensions of this derangement are not well characterized. Here, we localize lost Nrf2 function to discrete locations in human wounds in tandem with dysregulated redox homeostasis.
Methods: Primary human and diabetic epidermal keratinocytes (NHEK and DHEKs, respectively) underwent real time QT-PCR, protein quantification, and immunocytochemistry, for Nrf2 and its downstream effectors. Human surgical specimens from wounded and unwounded tissues in diabetic and nondiabetic patients underwent parallel biomolecular analysis. Keap1, a repressor of Nrf2, was silenced (siKeap1) in DHEKs, with siNonsense (siNS) as a control. siKeap1-transfected DHEKs underwent functional assays.
Results: QT-PCR of DHEKs demonstrates reduced transcription of Nrf2 downstream antioxidant genes like GSR, GPX3, GSTP1, and MnSOD, and an unfavorable cytokine profile compared to NHEKs (increased TNFa and decreased TGFb). siKeap1 in DHEKs rescues the expression of GSR, GPX3, and HO-1 by 25%, 383% and 237% respectively, and restores a cytokine profile favorable to healing (5-fold increase in TGF-b, p=0.0251, and 2-fold reduction in TNF-a, p=0.019), vs that in DHEK-siNS. Compared to siNS, siKeap1 DHEKs also demonstrate decreased oxidative DNA damage as measured by 8-hydroxydeoxyguanosine (8-OHdG) assay and enhanced proliferation by 25% (54.6 vs. 32.6%, p=0.0034) in a BrdU incorporation assay. Keap1-silenced-DHEKs exhibit significant improved migration as early as 2 hours post-injury and at all following time points versus siNS. Intact diabetic skin demonstrates a 2-fold increase in 8-OHdG levels (p=0.0390), with corresponding increases in protein levels of Nrf2-P, the translocated intranuclear form of Nrf2, and downstream effector MnSOD. Compared to that of nondiabetic wounds, Keap1 expression persists at pre-wounded levels in diabetic wounds, which correlates with low Nrf2-P. Downstream expression of antioxidant genes in diabetic wounds remains suppressed compared to normal wounded skin leading to increased 8-OHdG levels. Nrf2-P is found throughout the epidermis in unwounded tissue but becomes localized to the basal epithelium in the wounded state. However, in diabetic wounds, there is a 77% (p=<0.0001) overall decrease in Nrf2-P expression with near complete absence of Nrf2-P in all but the most basal layer of the epidermis.
Conclusion: Dysregulation of Nrf2 leads to profound disruption of redox homeostasis in Type II diabetes. Nrf2 dysfunction in diabetic epidermal cells in vitro parallels that of the epidermal compartment in vivo. This dysfunction manifests in a loss of antioxidant gene expression, the accumulation of oxidative damage in DNA, and increased pathological wound burden among diabetic patients. Loss of active Nrf2 from the basal epithelium of the epidermis may be a sentinel event in the pathogenesis of delayed wound healing. Manipulating the Nrf2 pathway holds great promise as a novel therapeutic strategy to restore cellular redox homeostasis and the healing potential of diabetic skin.
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