Nrf2 Deficiency is Associated with Endothelial Dysfunction in Pathologic Wound Environments
Darren Sultan, BA, Joshua A. David, BS, Salma Abdou, BA, Jennifer Kwong, BA, Jasmine Lee, BA, Chen Shen, BA, Piul S. Rabbani, PhD, Daniel J. Ceradini, MD.
NYU School of Medicine, New York, NY, USA.
Chronic wounds are among the most common complications of diabetes mellitus with 5-10% of diabetics suffering from foot ulcers. Physiologic wound healing involves the complex interplay of multiple cellular components, including endothelial cells as a major angiogenic workhorse. We have previously shown that the absence of effective Nrf2 signaling contributes to pathologic diabetic wounds. The purpose of this study is to analyze the detrimental effects to endothelial cell function that result from a relative loss of Nrf2 within in vitro and in vivo models.
Mouse endothelial C166 cells were transfected with siRNA specific to Nrf2 or a nonsense control. RNA was isolated from cell lysates, and knockdown was confirmed with quantitative RT-PCR. Cell growth was determined by MTS assay comparing transfected populations to appropriate controls over 3 days. In vitro angiogenesis measures were carried out by plating cells on Matrigel to induce tube formation under stress conditions. In vivo studies were performed by histologic analysis of tissue sections taken from humanized murine wounds.
To recapitulate an environment of Nrf2 dysfunction akin to that seen in chronic diabetic wounds, assays were designed to mimic pathophysiologic defects. Quantitative RT-PCR demonstrated between 70-77% knockdown of Nrf2 expression compared to non-sense control cohorts (p<0.01). Similarly transfected populations were employed for further in vitro study. Nrf2 knockdown was associated with a 25% reduction in relative cell growth as compared to non-sense controls (p<0.05). Untreated cohorts showed a 60% increase in growth, and cells treated with 0.5% triton-x showed a 95% decrease (p<0.01). Plating endothelial cells on Matrigel was shown to induce tube formation akin to primitive capillary sprouting. In hyperglycemic conditions, transfected cohorts displayed poor migration and fewer ring structures per high-powered-field as compared to silencer controls (average 4.7 versus 13/HPF 10x, p<0.01). Tissue sections were taken from 10-day-old wounds in wild-type and Nrf2 knock-out mice and were stained for CD31 to identify microvessels. Nrf2 KO tissue sections showed fewer than half the number of vessels as compared to the wild-type sections (average 16 versus 34/HPF, p<0.01).
Employing a number of experimental methodologies, we have highlighted a significant role of Nrf2 as it relates to endothelial cell function and angiogenic potential in wound environments. Our evidence demonstrates that a lack of Nrf2 is associated with stunted endothelial cell growth, migration, capillary tube formation, and angiogenesis. This work sheds new light on the pathophysiologic basis of diabetic wounds and thereby helps to direct future study at potential therapeutic targets.
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