Background-Increased production of reactive oxygen species (ROS) throughout the vascular wall is a feature of cardiovascular disease states, but therapeutic strategies remain limited by our incomplete understanding of the role and contribution of specific vascular cell ROS to disease pathogenesis. To investigate the specific role of endothelial cell (EC) ROS in the development of structural vascular disease, we generated a mouse model of endothelium-specific Nox2 overexpression and tested the susceptibility to aortic dissection after angiotensin II (Ang II) infusion. Methods and Results-A specific increase in endothelial ROS production in Nox2 transgenic mice was sufficient to cause Ang II-mediated aortic dissection, which was never observed in wild-type mice. Nox2 transgenic aortas had increased endothelial ROS production, endothelial vascular cell adhesion molecule-1 expression, matrix metalloproteinase activity, and CD45 + inflammatory cell infiltration. Conditioned media from Nox2 transgenic ECs induced greater Erk1/2 phosphorylation in vascular smooth muscle cells compared with wild-type controls through secreted cyclophilin A (CypA). Nox2 transgenic ECs (but not vascular smooth muscle cells) and aortas had greater secretion of CypA both at baseline and in response to Ang II stimulation. Knockdown of CypA in ECs abolished the increase in vascular smooth muscle cell Erk1/2 phosphorylation conferred by EC conditioned media, and preincubation with CypA augmented Ang II-induced vascular smooth muscle cell ROS production.Conclusions-These findings demonstrate a pivotal role for EC-derived ROS in the determination of the susceptibility of the aortic wall to Ang II-mediated aortic dissection. ROS-dependent CypA secretion by ECs is an important signaling mechanism through which EC ROS regulate susceptibility of structural components of the aortic wall to aortic dissection.

BACKGROUND AND PURPOSESystemic oxidative stress associated with dietary calorie overload plays an important role in the deterioration of vascular function in middle-aged patients suffering from obesity and insulin resistance. However, effective therapy is still lacking. EXPERIMENTAL APPROACHIn this study, we used a mouse model of middle-aged obesity to investigate the therapeutic potential of pharmaceutical inhibition (apocynin, 5 mM supplied in the drinking water) or knockout of Nox2, an enzyme generating reactive oxygen species (ROS), in high-fat diet (HFD)-induced obesity, oxidative stress, insulin resistance and endothelial dysfunction. Littermates of C57BL/6J wild-type (WT) and Nox2 knockout (KO) mice (7 months old) were fed with a HFD (45% kcal fat) or normal chow diet (NCD, 12% kcal fat) for 16 weeks and used at 11 months of age. KEY RESULTSCompared to NCD WT mice, HFD WT mice developed obesity, insulin resistance, dyslipidaemia and hypertension. Aortic vessels from these mice showed significantly increased Nox2 expression and ROS production, accompanied by significantly increased ERK1/2 activation, reduced insulin receptor expression, decreased Akt and eNOS phosphorylation and impaired endothelium-dependent vessel relaxation to acetylcholine. All these HFD-induced abnormalities (except the hyperinsulinaemia) were absent in apocynin-treated WT or Nox2 KO mice given the same HFD. CONCLUSIONS AND IMPLICATIONSIn conclusion, Nox2-derived ROS played a key role in damaging insulin receptor and endothelial function in dietary obesity after middle-age. Targeting Nox2 could represent a valuable therapeutic strategy in the metabolic syndrome.

Dietary obesity is a major risk factor for the development of cardiovascular diseases. However, the mechanism of high fat diet (HFD)-induced vascular dysfunction remains unclear. In this study we investigated the role of a Nox2-containing NADPH oxidase in high fat diet (HFD: 45% kcal fat)-induced metabolic disorders using Nox2/ApoE double knockout mice in comparison to age-matched ApoE−/− mice on the C57BL/6 background (n=9). ApoE−/− mice under 10 weeks of HFD had significant increases in body weight (NCD 27.9±1.5 vs HFD 31.3±1.1 g) and blood pressure (NCD 114±3.6 vs HFD 128.5±5.7 mm Hg) together with hyperglycaemia and hyperinsulinaemia as compared to age-matched littermates under a normal chow diet (NCD: 9.3% kcal fat). Aortas from HFD ApoE−/− mice had significant increases in O2.-production as detected by lucigenin-chemiluminescence and DHE fluorescence, and this was accompanied by increased endothelium Nox2 expression, Akt and ERK1/2 phosphorylation (immunofluorescence) and impaired endothelium function as assessed by an aorta organ bath. However, all these HFD-induced abnormalities were absent in Nox2/ApoE double knockout mice under the same HFD. Ex vivo organ culture (24 h) further confirmed that high levels of glucose (30 mM) plus insulin (1.2 nM) caused damages to ApoE−/− vessels (but not to vessels from Nox2/ApoE double knockout mice) characterised by ERK1/2 activation, reduced insulin receptor expression and deterioration of endothelial function. In conclusion, Nox2-derived oxidative stress plays an important role in the pathogenesis of dietary obesity-associated metabolic syndrome and endothelial dysfunction. Targeting Nox2-derived ROS represents a valuable therapeutic strategy to these diseases.

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NADPH oxidase 2 (Nox2) by generating reactive oxygen species (ROS) plays an important role in endothelial oxidative stress associated with obesity, insulin resistance and type-2 diabetes. In order to investigate the roles of Nox2 activation and ROS in high fat diet (HFD)-induced endothelial dysfunction and metabolic disorders, we crossed the Nox2-/- mice with the apolipoprotein E knockout (ApoE-/-) mice which is an established animal model to study dietary metabolic disorders. Littermates of ApoE-/- and Nox2/ApoE double knockout mice on the C57BL/6J background (6 weeks old, n = 9) were fed with HFD (45% kcal fat, 20% kcal protein and 35% kcal carbohydrate) or normal chow diet (NCD, 9.3% kcal fat, 25.9% kcal protein and 64.8% kcal carbohydrate) for 10 weeks. Body weights were measured weekly and mice were used at 16 weeks of age. Compared to age-matched NCD-fed mice, HFD ApoE-/- mice had hyperlipidaemia, hyperglycaemia and significant increases in body weight (NCD 27.9 ± 1.5 vs HFD 31.3 ± 1.1 g; p < 0.05). These metabolic changes were accompanied by significant increases in aorta ROS production as measured by lucigenin (5 µM)-chemiluminescence and DHE fluorescence, and a decrease in endothelium-dependent vessel relaxation to acetylcholine (Emax NCD 82.2 ± 5.5% vs HFD 68.7 ± 10.7%; p < 0.05) as assessed by an organ bath. HFD ApoE-/- mice had high blood pressure as compared to NCD controls (p < 0.05). Compared to ApoE-/- mice, Nox2/ApoE double knockout mice under the same HFD had no significant increases in levels of fasting LDL, triglyceride and glucose and their body weights were maintained at the NCD levels (NCD 29.1 ± 0.8 vs HFD 29.0 ± 0.7 g). The aorta ROS production and the endothelium-dependent vessel relaxation to acetylcholine were well preserved (Emax NCD 75.1 ± 11.0% vs HFD 78.1 ± 3.6%). HFD Nox2/ApoE double knockout mice had normal blood pressure. In conclusion, Nox2-derived oxidative stress plays an important role in the pathogenesis of dietary obesity-associated metabolic syndrome and endothelial dysfunction. Targeting Nox2-derived ROS represents a valuable therapeutic strategy to these diseases.