Activation of Nrf2 in Endothelial Cells Protects Arteries From Exhibiting a Proinflammatory State

Abstract

Objective-Proinflammatory mediators influence atherosclerosis by inducing adhesion molecules (eg, VCAM-1) on endothelial cells (ECs) via signaling intermediaries including p38 MAP kinase. Regions of arteries exposed to high shear stress are protected from inflammation and atherosclerosis, whereas low-shear regions are susceptible. Here we investigated whether the transcription factor Nrf2 regulates EC activation in arteries. Methods and Results-En face staining revealed that Nrf2 was activated in ECs at an atheroprotected region of the murine aorta where it negatively regulated p38 -VCAM-1 signaling, but was expressed in an inactive form in ECs at an atherosusceptible site. Treatment with sulforaphane, a dietary antioxidant, activated Nrf2 and suppressed p38 -VCAM-1 signaling at the susceptible site in wild-type but not Nrf2 Ϫ/Ϫ animals, indicating that it suppresses EC activation via Nrf2. Studies of cultured ECs revealed that Nrf2 inactivates p38 by suppressing an upstream activator MKK3/6 and by enhancing the activity of the negative regulator MKP-1. Key Words: Nrf2 Ⅲ arterial endothelium Ⅲ shear stress Ⅲ sulforaphane Ⅲ proinflammatory activation Ⅲ p38 Ⅲ MKK3/6 Ⅲ MKP-1 E arly atherosclerotic lesions contain monocytes and T-lymphocytes which are recruited from the circulation by adhesion to activated vascular endothelial cells (ECs). 1 This process is triggered by proinflammatory mediators (eg, TNF␣) which induce cellular adhesion molecules (eg, VCAM-1) via signaling intermediaries including p38 mitogen-activated protein (MAP) kinase, which is activated by phosphorylation by MAP kinase kinases 3 and 6 (MKK 3/6). 2,3 Vascular inflammation and atherosclerosis develop predominantly at distinct sites of the arterial tree located near branches and bends which are exposed to nonuniform blood flow, which exerts relatively low shear stress on vascular endothelium, whereas regions of arteries that are exposed to unidirectional high shear stress are protected. 4 -6 Proinflammatory activation of ECs is reduced at high-shear sites compared to low-shear regions, thus providing a potential explanation for the distinct spatial localization of vascular inflammation and lesion formation. 5,7-10 Similarly, the application of unidirectional high shear stress can suppress proinflammatory activation of cultured ECs, whereas low or oscillatory shear can act as a positive regulator of EC activation. 5,10 -15 The molecular mechanisms underlying the antiinflammatory effects of shear stress are uncertain, but previous studies of cultured cells have suggested a role for the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2). 16 -20 In unstimulated cells, Nrf2 is suppressed by kelch-like ECH-associated protein 1 (Keap1) which targets it for ubiquitination and proteasomal processing. Nrf2 can be activated by shear stress, dietary antioxidants (eg, sulforaphane) and other physiological stimuli which disrupt Keap1-Nrf2 interactions leading to stabilization and nuclear translocation of Nrf2. 16 -22 A previous study...

Nrf2 as an Endothelial Mechanosensitive Transcription Factor

“…[6][7][8][54][55][56][57] However, the involvement of Nrf2 as a shear-responsive transcription factor in EC has only been demonstrated in relatively few in vitro FSS culture models (Table) using primarily HUVEC which do not represent a cell type of relevance to the study of atherosclerosis, 3,11,12,47,[58][59][60] and in vivo mouse models of atherosclerosis. 13,14 As disturbed oscillatory FSS patterns have been established as modulators of EC redox status and inflammatory phenotype in regions of the vasculature prone to atherogenesis, 2,4 it is likely that augmented Nrf2 activity and ARE-linked gene expression in EC exposed to laminar FSS confers protection against vascular dysfunction and atherosclerosis. 3,28 Studies in cultured human EC have shown that the Nrf2 pathway is highly sensitive to laminar FSS and leads to induction of ARE-related genes such as HO-1, NQO1, sequestosome-1, glutamate-cysteine ligase modifier subunit, and ferritin heavy chain, an effect that is attenuated by siRNA knockdown of Nrf2.…”

“…The study provides mechanistic evidence that different classes of HDACs modulate EC responses to different flow patterns and shear stresses and implicate HDAC-3 as an epigenetic factor that may modulate endothelial oxidative and inflammatory responses to disturbed OS through deacetylation of Nrf2, thereby leading to decreases in Nrf2 ARE-binding activity and antioxidant gene induction. 65,66 Nuclear localization of Nrf2 in the endothelium of mouse aortae has been shown by enface immunofluorescence in atheroprotected regions of the descending aorta exposed to laminar FSS; however, its expression remains predominantly cytoplasmic in the inner curvature of the aortic arch where FSS patterns are oscillatory and disturbed 13,14 indicative of diminished ARE-mediated antioxidant gene induction in these atheroprone regions. In the atheroprone regions of the vasculature, sustained activation of the mitogen-activated protein kinase (MAPK), p38, and c-Jun N-terminal kinase-1/2 (JNK-1/2) results in enhanced NF-κB and activator protein-1 transcriptional activity, which subsequently promotes a proinflammatory EC phenotype through induction of adhesion molecules such as vascular cell adhesion molecule-1.…”

“…In the atheroprone regions of the vasculature, sustained activation of the mitogen-activated protein kinase (MAPK), p38, and c-Jun N-terminal kinase-1/2 (JNK-1/2) results in enhanced NF-κB and activator protein-1 transcriptional activity, which subsequently promotes a proinflammatory EC phenotype through induction of adhesion molecules such as vascular cell adhesion molecule-1. 14,67 . Activation of the Nrf2 pathway has been shown to negatively regulate the activity of MAPK signaling by enhancing the catalytic activity of MAPK kinase phosphatases by promoting its reduced form and suppressing the activation of the MAPK kinase 3/6, resulting in the reduced activation of p38 MAPK-mediated signaling.…”

“…[8][9][10] It has been reported that EC exposed to OS but not to US flow patterns in both in vivo and in vitro models exhibits greater nuclear factor-κB (NF-κB) activity and deficiencies in the Nrf2/ARE redox signaling pathway and are thus predisposed to oxidative stress and a proinflammatory phenotype because of the enhanced generation and reduced scavenging of reactive oxygen species (ROS). 3,[11][12][13][14] This review will highlight the role of the Nrf2 pathway in EC as a mechanosensitive transcriptional regulator of redox signaling in maintaining physiological endothelial function and summarize the mechanisms underlying the differential modulation of Nrf2 activity, ARE-dependent antioxidant gene expression and EC redox/inflammatory phenotype by laminar and disturbed FSS. Because therapeutic strategies for modulating Nrf2 activity have become an increasing area of basic and clinical research, 15 it is important to consider how these may also alter physiological responses of EC to FSS mediated by redox signaling.…”

Laminar Flow Activation of ERK5 Protein in Vascular Endothelium Leads to Atheroprotective Effect via NF-E2-related Factor 2 (Nrf2) Activation

“…Upon protein modification by electrophiles and kinase-activating signaling pathways, Nrf2 escapes from the Keap1-Cul3 complex and translocates into the nucleus (9,10). A recent study shows that Nrf2 activation ameliorates inflammatory responses at atherosusceptible sites in wild-type mice, but not in Nrf2 Ϫ/Ϫ mice, suggesting the protective role of Nrf2 in arterial inflammation in vivo (11). However, the underlying molecular mechanism by which laminar flow induces Nrf2 activation has not been fully addressed yet.…”