Background— Recent clinical studies have suggested a major protective role for the antioxidant enzyme glutathione peroxidase-1 (GPx1) in diabetes-associated atherosclerosis. We induced diabetes in mice deficient for both GPx1 and apolipoprotein E (ApoE) to determine whether this is merely an association or whether GPx1 has a direct effect on diabetes-associated atherosclerosis. Methods and Results— ApoE-deficient (ApoE −/− ) and ApoE/GPx1 double-knockout (ApoE −/− GPx1 −/− ) mice were made diabetic with streptozotocin and aortic lesion formation, and atherogenic pathways were assessed after 10 and 20 weeks of diabetes. Aortic proinflammatory and profibrotic markers were determined by both quantitative reverse-transcription polymerase chain reaction analysis after 10 weeks of diabetes and immunohistochemical analysis after 10 and 20 weeks of diabetes. Sham-injected nondiabetic counterparts served as controls. Atherosclerotic lesions within the aortic sinus region, as well as arch, thoracic, and abdominal lesions, were significantly increased in diabetic ApoE −/− GPx1 −/− aortas compared with diabetic ApoE −/− aortas. This increase was accompanied by increased macrophages, α-smooth muscle actin, receptors for advanced glycation end products, and various proinflammatory (vascular cell adhesion molecule-1) and profibrotic (vascular endothelial growth factor and connective tissue growth factor) markers. Quantitative reverse-transcription polymerase chain reaction analysis showed increased expression of receptors for advanced glycation end products (RAGE), vascular cell adhesion molecule-1, vascular endothelial growth factor, and connective tissue growth factor. Nitrotyrosine levels were significantly increased in diabetic ApoE −/− GPx1 −/− mouse aortas. These findings were observed despite upregulation of other antioxidants. Conclusions— Lack of functional GPx1 accelerates diabetes-associated atherosclerosis via upregulation of proinflammatory and profibrotic pathways in ApoE −/− mice. Our study provides evidence of a protective role for GPx1 and establishes GPx1 as an important antiatherogenic therapeutic target in patients with or at risk of diabetic macrovascular disease.

OBJECTIVETo investigate the effect of the GPx1-mimetic ebselen on diabetes-associated atherosclerosis and renal injury in a model of increased oxidative stress.RESEARCH DESIGN AND METHODSThe study was performed using diabetic apolipoprotein E/GPx1 (ApoE−/−GPx1−/−)-double knockout (dKO) mice, a model combining hyperlipidemia and hyperglycemia with increased oxidative stress. Mice were randomized into two groups, one injected with streptozotocin, the other with vehicle, at 8 weeks of age. Groups were further randomized to receive either ebselen or no treatment for 20 weeks.RESULTSEbselen reduced diabetes-associated atherosclerosis in most aortic regions, with the exception of the aortic sinus, and protected dKO mice from renal structural and functional injury. The protective effects of ebselen were associated with a reduction in oxidative stress (hydroperoxides in plasma, 8-isoprostane in urine, nitrotyrosine in the kidney, and 4-hydroxynonenal in the aorta) as well as a reduction in VEGF, CTGF, VCAM-1, MCP-1, and Nox2 after 10 weeks of diabetes in the dKO aorta. Ebselen also significantly reduced the expression of proteins implicated in fibrosis and inflammation in the kidney as well as reducing related key intracellular signaling pathways.CONCLUSIONSEbselen has an antiatherosclerotic and renoprotective effect in a model of accelerated diabetic complications in the setting of enhanced oxidative stress. Our data suggest that ebselen effectively repletes the lack of GPx1, and indicate that ebselen may be an effective therapeutic for the treatment of diabetes-related atherosclerosis and nephropathy. Furthermore, this study highlights the feasibility of addressing two diabetic complications with one treatment regimen through the unifying approach of targeted antioxidant therapy.

Oxidative stress and inflammation are inextricably linked and play essential roles in the initiation and progression of diabetes complications such as diabetes-associated atherosclerosis and nephropathy. Bolstering antioxidant defenses is an important mechanism to lessen oxidative stress and inflammation. In this study, we have used a novel analog of the NFE2-related factor 2 (Nrf2) agonist bardoxolone methyl, dh404, to investigate its effects on diabetic macrovascular and renal injury in streptozotocininduced diabetic apolipoprotein E 2/2 mice. We show that dh404, at lower but not higher doses, significantly lessens diabetes-associated atherosclerosis with reductions in oxidative stress (in plasma, urine, and vascular tissue) and proinflammatory mediators tumor necrosis factor-a, intracellular adhesion molecule-1, vascular cell adhesion molecule-1, and monocyte chemotactic protein-1 (MCP-1). We demonstrate that dh404 attenuates functional (urinary albumin-to-creatinine ratio) and structural (mesangial expansion) glomerular injury and improves renal tubular injury. Liver functional and structural studies showed that dh404 is well tolerated. Complementary in vitro studies in normal rat kidney cells showed that dh404 significantly upregulates Nrf2-responsive genes, heme oxygenase-1, NAD(P)H quinone oxidoreductase 1, and glutathione-S transferase, with inhibition of transforming growth factor-b-mediated profibrotic fibronectin, collagen I, and proinflammatory interleukin-6. Higher doses of dh404 were associated with increased expression of proinflammatory mediators MCP-1 and nuclear factor-kB. These findings suggest that this class of compound is worthy of further study to lessen diabetes complications but that dosage needs consideration.

Objective-Recently we showed that lack of the antioxidant enzyme glutathione peroxidase-1 (GPx1) accelerates atherosclerosis and upregulates proatherogenic pathways in diabetic apoE/GPx1-deficient double-knockout mice, thereby establishing GPx1 as an important therapeutic target. In vivo studies now investigate ebselen, a seleno-organic GPx1-mimetic, for its potential to reduce diabetes-associated atherosclerosis. Methods and Results-Lesions were significantly increased in diabetic apoEϪ/Ϫ aortas (PϽ0.001) compared with nondiabetic controls after 20 weeks of diabetes. Ebselen-gavage significantly reduced total aortic lesions (PϽ0.001), with significant regional reductions in the arch (PϽ0.001), thoracic (PϽ0.001), and abdominal regions (PϽ0.05), but not within the aortic sinus of diabetic apoE Ϫ/Ϫ mice. These reductions were accompanied by significantly lower nitrotyrosine and Nox2 levels, reduced proatherogenic cellularity (macrophages and SMCs), and reduced expression of the proatherogenic mediator RAGE. Within the aortic sinus, ebselen reduced nitrotyrosine, Nox2, and VEGF levels but had no effect on RAGE. Studies in HAECs show that ebselen abrogates H 2 O 2 -induced increases in P-IKK, P-JNK, TNF-␣, and Nox2. Conclusions-Ebselen reduces atherosclerotic lesions in most regions of diabetic apoEϪ/Ϫ aorta, except within the aortic sinus, suggesting its effectiveness as a potential antiatherogenic therapy in diabetic-macrovascular disease.

BackgroundVascular 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.Methodsdh404 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.ResultsDh404 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 (O2·− 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).ConclusionThese studies demonstrate that upregulation of Nrf2 by dh404 represents a novel therapeutic strategy to limit diabetes-associated vascular injury.Electronic supplementary materialThe online version of this article (doi:10.1186/s12933-017-0513-y) contains supplementary material, which is available to authorized users.

With animal models, death as an intentional end point is ethically unacceptable. However, in the study of septic shock, death is still considered the only relevant end point. We defined eight humane end points into four stages of severity (from healthy to moribund) and used to design a clinically relevant scoring tool, termed "the mouse clinical assessment score for sepsis" (M-CASS). The M-CASS was used to enable a consistent approach to the assessment of disease severity. This allowed an ethical and objective assessment of disease after which euthanasia was performed, instead of worsening suffering. The M-CASS displayed a high internal consistency (Cronbach α = 0.97) with a high level of agreement and an intraclass correlation coefficient equal to 0.91. The plasma levels of cytokines and markers of oxidative stress were all associated with the M-CASS score (Kruskal-Wallis test, P < 0.05). The M-CASS allows tracking of disease progression and animal welfare requirements.

Patients with diabetes have an increased risk of developing atherosclerosis. Endothelial dysfunction, characterized by the lowered bioavailability of endothelial NO synthase (eNOS)-derived NO, is a critical inducer of atherosclerosis. However, the protective aspect of eNOS in diabetes-associated atherosclerosis remains controversial, a likely consequence of its capacity to release both protective NO or deleterious oxygen radicals in normal and disease settings, respectively. Harnessing the atheroprotective activity of eNOS in diabetic settings remains elusive, in part due to the lack of endogenous eNOS-specific NO release activators. We have recently shown in vitro that eNOS-derived NO release can be increased by blocking its binding to Caveolin-1, the main coat protein of caveolae, using a highly specific peptide, CavNOxin. However, whether targeting eNOS using this peptide can attenuate diabetes-associated atherosclerosis is unknown. In this study, we show that CavNOxin can attenuate atherosclerotic burden by ∼84% in vivo. In contrast, mice lacking eNOS show resistance to CavNOxin treatment, indicating eNOS specificity. Mechanistically, CavNOxin lowered oxidative stress markers, inhibited the expression of proatherogenic mediators, and blocked leukocyte-endothelial interactions. These data are the first to show that endogenous eNOS activation can provide atheroprotection in diabetes and suggest that CavNOxin is a viable strategy for the development of antiatherosclerotic compounds.Diabetes is widely regarded as an independent risk factor for the development of cardiovascular diseases, with ;80% of cardiovascular mortality and morbidity being linked to macrovascular complications, such as atherosclerosis (1-3). The increased risk of development of vascular complications in individuals with type 1 (T1D) or type 2 diabetes (T2D) occurs despite intensive glycemic control, stressing the need for novel approaches to lessen the burden of diabetes-mediated macrovascular injury (4).The vascular endothelium plays a crucial role in diabetesassociated atherosclerosis through the regulation of vessel permeability, inflammation, coordination of leukocyte trafficking, and thrombosis (1,5). Indeed, the function of the vascular endothelium is significantly impaired during diabetes, a phenomena termed endothelial dysfunction and characterized by the reduced bioavailability of an important endothelial cell mediator, nitric oxide (NO). Such chronic attenuation of endothelial-derived NO release promotes platelet and leukocyte activation and adhesion, compromises endothelial cell barrier integrity, and causes the upregulation of proinflammatory genes (6-8). Moreover, reduced NO-dependent vasodilation and increased leukocyte adhesion to the endothelium, both of which are hallmarks of endothelial dysfunction, have been observed in patients with diabetes and diabetic animal models (9-12). Similarly, in cultured endothelial cells exposed to high glucose, lowered endothelial NO synthase (eNOS)-derived NO release was observed (13-15). In...

Oxidative stress is thought to contribute to the initiation and progression of atherosclerosis. As glutathione peroxidase-1 (Gpx1) is an antioxidant enzyme that detoxifies lipid hydroperoxides, we tested the impact of Gpx1 deficiency on atherosclerotic processes and antioxidant enzyme expression in mice fed a high-fat diet (HFD). After 12 weeks of HFD, atherosclerotic lesions at the aortic sinus were of similar size in control and Gpx1-deficient mice. However, after 20 weeks of HFD, lesion size increased further in control but not in Gpx1-deficient mice, even though plasma and aortic wall markers of oxidative damage did not differ between groups. In control mice, the expression of Gpx1 increased and that of Gpx3 decreased at the aortic sinus after 20 weeks of HFD, with no change in the expression of Gpx2, Gpx4, catalase, peroxiredoxin-6, glutaredoxin-1 and -2, or thioredoxin-1 and -2. By comparison, in Gpx1-deficient mice, the expression of antioxidant genes was unaltered except for a decrease in glutaredoxin-1 and an increase in glutaredoxin-2. These changes were associated with increased expression of the proinflammatory marker monocyte chemoattractant protein-1 in control mice but not in Gpx1-deficient mice. In summary, a specific deficiency in Gpx1 was not accompanied by an increase in markers of oxidative damage or increased atherosclerosis in a murine model of HFD-induced atherogenesis.-de Haan,