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Rationale: In the endothelium, insulin stimulates endothelial NO synthase (eNOS) to generate the antiatherosclerotic signaling radical NO. Insulin-resistant type 2 diabetes mellitus is associated with reduced NO availability and accelerated atherosclerosis. The effect of enhancing endothelial insulin sensitivity on NO availability is unclear., Objective: To answer this question, we generated a mouse with endothelial cell (EC)-specific overexpression of the human insulin receptor (hIRECO) using the Tie2 promoter-enhancer., Methods and Results: hIRECO demonstrated significant endothelial dysfunction measured by blunted endothelium-dependent vasorelaxation to acetylcholine, which was normalized by a specific Nox2 NADPH oxidase inhibitor. Insulin-stimulated phosphorylation of protein kinase B was increased in hIRECO EC as was Nox2 NADPH oxidase-dependent generation of superoxide, whereas insulin-stimulated and shear stress-stimulated eNOS activations were blunted. Phosphorylation at the inhibitory residue Y657 of eNOS and expression of proline-rich tyrosine kinase 2 that phosphorylates this residue were significantly higher in hIRECO EC. Inhibition of proline-rich tyrosine kinase 2 improved insulin-induced and shear stress-induced eNOS activation in hIRECO EC., Conclusions: Enhancing insulin sensitivity specifically in EC leads to a paradoxical decline in endothelial function, mediated by increased tyrosine phosphorylation of eNOS and excess Nox2-derived superoxide. Increased EC insulin sensitivity leads to a proatherosclerotic imbalance between NO and superoxide. Inhibition of proline-rich tyrosine kinase 2 restores insulin-induced and shear stress-induced NO production. This study demonstrates for the first time that increased endothelial insulin sensitivity leads to a proatherosclerotic imbalance between NO and superoxide., (© 2016 American Heart Association, Inc.)

Insulin resistance is characterized by excessive endothelial cell generation of potentially cytotoxic concentrations of reactive oxygen species. We examined the role of NADPH oxidase (Nox) and specifically Nox2 isoform in superoxide generation in two complementary in vivo models of human insulin resistance (endothelial specific and whole body). Using three complementary methods to measure superoxide, we demonstrated higher levels of superoxide in insulin-resistant endothelial cells, which could be pharmacologically inhibited both acutely and chronically, using the Nox inhibitor gp91ds-tat. Similarly, insulin resistance-induced impairment of endothelial-mediated vasorelaxation could also be reversed using gp91ds-tat. siRNA-mediated knockdown of Nox2, which was specifically elevated in insulin-resistant endothelial cells, significantly reduced superoxide levels. Double transgenic mice with endothelial-specific insulin resistance and deletion of Nox2 showed reduced superoxide production and improved vascular function. This study identifies Nox2 as the central molecule in insulin resistance-mediated oxidative stress and vascular dysfunction. It also establishes pharmacological inhibition of Nox2 as a novel therapeutic target in insulin resistance-related vascular disease.

Purpose: To investigate the role of endothelial Nox2 in sepsis-induced systemic inflammatory response and hypotension. Methods: All experiments were conduced in accordance with the Scientific Procedures Act. 1986 (UK Home Office). Mice with endothelial-target deletion of Nox2 were generated by crossing Nox2fl/fl mice with Tie2Cre transgenic animals. We compared Nox2fl/fl Tie2Cre+/− mice (Knockout,KO) with matched littermate Nox2fl/fl Tie2Cre-/- (Control) animals on a C57BL/6 background. Mice received an iv injection of lipopolysaccharide (LPS, 10mg/kg) or saline. Systolic and diastolic blood pressures were determined by tail-cuff plethysmography. A clinical severity score comprising signs of lethargy, piloerection, tremor, periorbital exudates, respiratory distress and diarrhoea was determined 2-hourly after LPS injection. After 12 hours sepsis, animals were euthanized and neutrophil sequestration in lungs was evaluated by myeloperoxidase assay. ROS production in mesenteric vessels was assessed by dihydroethidium- image assay and 3-nitrotyrosine staining was used as a readout of nitrosative stress. Levels of tumor necrosis factor alpha (TNF-alpha) were determined by enzymatic linked immunoabsorbent assay. Results: KO mice had lower ROS production in mesenteric vessels than controls after LPS injection. Despite similar basal values (94.46±2.1 vs 96.43±2.50mmHg for systolic blood pressure and 68.19±1.74 vs 69.26±2.56mmHg for diastolic pressure), KO mice had lower blood pressure than control mice, 6 hours after LPS injection (63.04±2.56 vs 76.70±1.38mmHg for systolic blood pressure and 48.24±2.77 vs 59.14±1.89 mmHg for diastolic pressure; p<0.05; n=9). The clinical severity scores were also more aggravate in KO mice compared to controls (p=0.0003, n=9). Mice with endothelial-target Nox2 deletion had higher number of neutrophils trapped in lungs (12480±207.4 vs 6489±75.09 cells/mg lung, p=0.0004, n=6), and increased levels of TNF-alpha in plasma (95.70±13.51 vs 33.69±11.59 pg/ml, p=0.0207, n=5) compared to controls. Interestingly, 3-nitrotyrosine staining in mesenteric vessels was higher in KO mice compared to controls. Conclusions: Endothelial cell Nox2 appears to be protective during sepsis by reducing the extent of hypotension, neutrophil sequestration in lungs and levels of TNF-alpha in plasma. The severe hypotension in KO mice during sepsis could be associated with evidence of greater nitrosative stress in mesenteric vessels than controls. The cellular mechanisms underlying the Nox2 protective effect are currently being investigated. [ABSTRACT FROM AUTHOR]