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Nrf2 Mediates Angiogenic Function Of Endothelial Cells Through Regulation Of Cxcr4/sdf-1 Axis
Jasmine Lee, BA1, Alvaro P. Villareal-Ponce, PhD1, Joseph F. Kuhn, MD1, Joshua A. David, MD2, Piul S. Rabbani, PhD1, Daniel J. Ceradini, MD1.
1NYU Langone Medical Center, New York, NY, USA, 2University of Pittsburgh Medical Center, Pittsburgh, PA, USA.

Purpose: Diabetes mellitus is the leading cause of non-healing wounds, with chronic wounds costing the US healthcare system up to 31.7 billion dollars annually. Diabetes negatively affects the soft tissue healing via dysregulations of redox homeostasis and angiogenesis. We have previously shown that inadequate signaling of Nrf2, a master transcriptional regulator of antioxidant response signaling, contributes to the oxidative stress underlying pathologic diabetic wounds. Delayed wound closure may be a result of neo-vascularization deficits from dysfunctional endothelial cells. Restoration of Nrf2 signaling in diabetic wounds decreases wound closure time and increases neovascularization of the wound bed. The membrane protein CXCR4 and its ligand, SDF-1, have been implicated in modulating the neo-angiogenesis required to revascularize wounded tissue. Here, we identify a possible role of Nrf2 dysregulation in endothelial cells impairing angiogenesis in wound neovascularization.
Methods: To study the functional role of endothelial Nrf2, we crossed Cadherin5 (Cdh5 )CreER and Nrf2flox/flox mice to generate Cdh5-CreER;Nrf2flox/flox double transgenic mice and administered tamoxifen 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), heterozygous, and KO mice. 10 days post wounding, wound endothelial cells were isolated using flow cytometry and angiogenesis assay was performed.In order to elucidate a relationship between Nrf2 and the CXCR4/SDF-1 axis, C166 mouse endothelial cell line was transfected with Nrf2 siRNA to create Nrf2 knockdown (KD) cells. After transfection, cells were then placed in a 5% oxygen hypoxic incubator for 48 hours to model a hypoxic wound environment. QT-PCR for Nrf2, CXCR4, SDF-1, and NQO1 was performed.
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 was similar to 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. 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). We found that selective Nrf2 KO in endothelial cells reduced their capacity to form tubular networks, indicating impaired angiogenic activity. QT-PCR of the C166 endothelial cells after transfection and incubation in a hypoxic environment demonstrated reduced transcription of Nrf2 compared to control of 76.5% (p=0.001) and 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 in a hypoxic environment. These findings suggest that the Nrf2 dysregulation associated with diabetes may contribute to the impaired angiogenesis and decreased revascularization present in chronic diabetic wounds.


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