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Nrf2 Mediates Angiogenic Function Of Endothelial Cells Through Regulation Of CXCR4/SDF-1 Axis
Jasmine Lee1, Alvaro Villareal Ponce1, Joshua A. David2, Joseph F. Kuhn1, Daniel J. Ceradini1.
1NYU Langone Medical Center, New York, NY, USA, 2University of Pittsburgh, Pittsburgh, PA, USA.

BACKGROUND: The purpose of this study is to propose a mechanism by which Nrf2 directly regulates endothelial cell angiogenic potential in tissue regeneration via CXCR4/SDF-1 axis.
METHODS: Cadherin5 (Cdh5)-CreER mice were crossed with Nrf2flox/flox mice to generate Cdh5-CreER;Nrf2flox/flox double transgenic mice. Tamoxifen was administered to induce conditional deletion of Nrf2 in cadherin 5 expressing endothelial cells (KO). Ten mm diameter full-thickness stented excisional wounds were created on the dorsum of 6-8-week-old wildtype (WT), diabetic, and KO mice. After 10 days, wound endothelial cells were isolated and ex vivo angiogenesis assays performed. Additionally, C166 mouse endothelial cell line was transfected with Nrf2 siRNA to create Nrf2 knockdown (KD) cells, which were placed in a 5% oxygen hypoxia to model a hypoxic wound environment. The treated endothelial cells underwent real time QT-PCR for Nrf2, CXCR4, SDF-1, and NQO1.
RESULTS: In the Nrf2 KO mice, time to wound closure was delayed compared to wildtype mice (KO 33.01.73 days vs. WT 14.00 days) and more closely approximated time to closure in diabetic mice (KO 33.01.73 days vs. Diabetic 31.01.41 days). Primary endothelial cells from the wounds of KO mice demonstrated significantly decreased angiogenic functionality when compared to heterozygous controls. Nrf2 KO endothelial cells formed significantly fewer branches (KO 64.7514.81 branches/cm2 vs. control 282.846.7 branches/cm2, p=0.014) and fewer branching points (KO 39.05.18 branching points/cm2 vs. Control 150.815.34 branching points/cm2, p=0.0031). QT-PCR of C166 endothelial cells after Nrf2 KD revealed reduced transcription of Nrf2 compared to control of 76.5% (p=0.001). NQO1, a downstream target of Nrf2, decreased 66.4% (p=0.02). Expression of CXCR4 and SDF-1 decreased 61.7% (p=0.001) and 47.6% (p=0.02), respectively.
CONCLUSIONS: Nrf2 expression in endothelial cells is necessary for physiologic wound healing. Nrf2 knockdown results in increased time to wound closure and decreased angiogenic function. Decreased Nrf2 expression also results in decreased transcription of the neo-vascularization related proteins CXCR4 and SDF-1. These findings suggest that the Nrf2 dysregulation associated with diabetes may contribute to the impaired angiogenesis present in chronic diabetic wounds.


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