The nuclear factor (erythroid-derived 2)-like 2 (Nrf2) activator dh404 protects against diabetes-induced endothelial dysfunction

Arpeeta Sharma, Luddwi Rizky, Nada Stefanovic, Mitchel Tate, Rebecca H. Ritchie, Keith W. Ward, Judy B. de Haan

Vascular dysfunction is a pivotal event in the development of diabetes-associated vascular disease. Increased inflammation and oxidative stress are major contributors to vascular dysfunction. Nrf2, a master regulator of several anti-oxidant genes and a suppressor of inflammatory NF-κB, has potential as a target to combat oxidative stress and inflammation. The aim of this study was to investigate the effects of a novel Nrf2 activator, the bardoxolone methyl derivative dh404, on endothelial function in vitro and in vivo. dh404 at 3 mg/kg was administered to male Akita mice, an established diabetic mouse model of insulin insufficiency and hyperglycemia, from 6 weeks of age. At 26 weeks of age, vascular reactivity was assessed by wire myography, pro-inflammatory expression was assessed in the aortas by qRT-PCR and immunohistochemistry, and systemic and vascular oxidative stress measurements were determined. Additionally, studies in human aortic endothelial cells (HAECs) derived from normal and diabetic patients in the presence or absence of dh404 included assessment of pro-inflammatory genes by qRT-PCR and western blotting. Oxidative stress was assessed by three methods; L-012, DCFDA and amplex red. Static adhesion assays were performed to determine the leukocyte-endothelial interaction in the presence or absence of dh404. Dh404 significantly attenuated endothelial dysfunction in diabetic Akita mice characterized by reduced contraction in response to phenylephrine and the downregulation of inflammatory genes (VCAM-1, ICAM-1, p65, IL-1β) and pro-oxidant genes (Nox1 and Nox2). Furthermore, reduced systemic and vascular oxidative stress levels were observed in diabetic Akita mice. dh404 exhibited cytoprotective effects in diabetic HAECs in vitro, reflected by significant upregulation of Nrf2-responsive genes, NAD(P)H quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1), reduction of oxidative stress markers (O 2(·-) and H2O2), inhibition of inflammatory genes (VCAM-1 and the p65 subunit of NF-κB) and attenuation of leukocyte-endothelial interactions (P < 0.05 for all in vitro and in vivo parameters; one or two-way ANOVA as appropriate with post hoc testing). These studies demonstrate that upregulation of Nrf2 by dh404 represents a novel therapeutic strategy to limit diabetes-associated vascular injury.