J Smith

Nox2 NADPH Oxidase Has a Critical Role in Insulin Resistance–Related Endothelial Cell Dysfunction

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.

Novel Role of the IGF-1 Receptor in Endothelial Function and Repair: Studies in Endothelium-Targeted IGF-1 Receptor Transgenic Mice

We recently demonstrated that reducing IGF-1 receptor (IGF-1R) numbers in the endothelium enhances nitric oxide (NO) bioavailability and endothelial cell insulin sensitivity. In the present report, we aimed to examine the effect of increasing IGF-1R on endothelial cell function and repair. To examine the effect of increasing IGF-1R in the endothelium, we generated mice overexpressing human IGF-1R in the endothelium (human IGF-1R endothelium-overexpressing mice [hIGFREO]) under direction of the Tie2 promoter enhancer. hIGFREO aorta had reduced basal NO bioavailability (percent constriction to NG-monomethyl-l-arginine [mean (SEM) wild type 106% (30%); hIGFREO 48% (10%)]; P < 0.05). Endothelial cells from hIGFREO had reduced insulin-stimulated endothelial NO synthase activation (mean [SEM] wild type 170% [25%], hIGFREO 58% [3%]; P = 0.04) and insulin-stimulated NO release (mean [SEM] wild type 4,500 AU [1,000], hIGFREO 1,500 AU [700]; P < 0.05). hIGFREO mice had enhanced endothelium regeneration after denuding arterial injury (mean [SEM] percent recovered area, wild type 57% [2%], hIGFREO 47% [5%]; P < 0.05) and enhanced endothelial cell migration in vitro. The IGF-1R, although reducing NO bioavailability, enhances in situ endothelium regeneration. Manipulating IGF-1R in the endothelium may be a useful strategy to treat disorders of vascular growth and repair.

Insulinlike growth factor – binding protein-1 improves vascular endothelial repair in male mice in the setting of insulin resistance

Insulin resistance is associated with impaired endothelial regeneration in response to mechanical injury. We recently demonstrated that insulinlike growth factor-binding protein-1 (IGFBP1) ameliorated insulin resistance and increased nitric oxide generation in the endothelium. In this study, we hypothesized that IGFBP1 would improve endothelial regeneration and restore endothelial reparative functions in the setting of insulin resistance. In male mice heterozygous for deletion of insulin receptors, endothelial regeneration after femoral artery wire injury was enhanced by transgenic expression of human IGFBP1 (hIGFBP1). This was not explained by altered abundance of circulating myeloid angiogenic cells. Incubation of human endothelial cells with hIGFBP1 increased integrin expression and enhanced their ability to adhere to and repopulate denuded human saphenous vein ex vivo. In vitro, induction of insulin resistance by tumor necrosis factor α (TNFα) significantly inhibited endothelial cell migration and proliferation. Coincubation with hIGFBP1 restored endothelial migratory and proliferative capacity. At the molecular level, hIGFBP1 induced phosphorylation of focal adhesion kinase, activated RhoA and modulated TNFα-induced actin fiber anisotropy. Collectively, the effects of hIGFBP1 on endothelial cell responses and acceleration of endothelial regeneration in mice indicate that manipulating IGFBP1 could be exploited as a putative strategy to improve endothelial repair in the setting of insulin resistance.

139 Endothelial specific insulin resistance promotes the development of atherosclerosis

Background Global insulin resistance and endothelial dysfunction have been identified as predisposing factors for atherosclerosis. However, it is unclear whether selective insulin resistance in endothelial cells alone, is sufficient to promote atherosclerosis. Here we addressed this question by crossing Endothelial Specific Mutant Insulin Receptor Over-expressing (ESMIRO) mice with ApoE null mice. ESMIRO mice over-express a human insulin receptor with Ala-Thr1134 mutation in the tyrosine kinase domain (which disrupts insulin signalling) selectively in endothelial cells under the control of the tie-2 promoter/enhancer. Methods Male ApoE−/−ESMIRO mice were compared with sex-matched littermate ApoE−/− mice (both on a C57Bl6 background) after feeding a Western-style diet for 12u2005weeks. Results ApoE−/−ESMIRO mice were morphologically indistinguishable from ApoE−/− control littermates, with normal development and no difference between groups in body mass. Heart rate, systolic blood pressure, glucose tolerance, insulin sensitivity and fasting glucose levels were similar in ApoE-/-ESMIRO and ApoE−/− mice. Aortic lipid deposition, assessed by en-face oil red O staining, was similar in ApoE−/−ESMIRO and ApoE−/− mice (6.4%±0.5% vs 5.8%±0.5%; p=0.39). However, atherosclerotic lesion area in cross sections of aortic sinus was significantly increased in ApoE−/−ESMIRO mice compared to ApoE−/− controls (24.8%±2.4% vs 16.6%±2.4%; p=0.02). Absolute plaque size was also significantly increased in ApoE−/−ESMIRO mice compared to ApoE controls (226u2008448.9±16u2008154u2005μm2 vs 149u2008424.41±24u2008221u2005μm2; p=0.01). Conclusions Endothelial specific insulin resistance is sufficient to promote atherosclerosis and increase lesion area in ApoE null mice. This suggests that enhancing endothelial insulin sensitivity may be an appropriate target to prevent atherosclerosis in insulin-resistant conditions.

Preservation of vascular endothelial repair in mice with diet-induced obesity: Vascular repair in mice with diet-induced obesity

Preservation of structural integrity of the endothelial monolayer and maintenance of endothelial cell function are of critical importance in preventing arterial thrombosis, restenosis and atherosclerosis. Obesity has been intimately linked with endothelial dysfunction, and reports of reduced abundance and functional impairment of circulating progenitor cells in obesity have led to the suggestion that defective endothelial repair contributes to obesity‐related cardiovascular disease.

217 Insulin-like Growth Factor Binding Protein-1 Enhances Vascular Endothelial Repair in the Setting of Insulin Resistance

Introduction Insulin resistance predisposes to cardiovascular disease (CVD) by inducing endothelial cell (EC) dysfunction and impairs the capacity for endothelial repair. Additionally, we have discovered that a circulating protein, insulin-like growth factor binding protein-1 (IGFBP-1), is potentially protective in the vasculature by stimulating nitric oxide production and enhancing insulin sensitivity. In cross-sectional studies, low IGFBP-1 levels are associated with diabetes and CVD. In this project, we investigated whether IGFBP-1 can enhance vascular endothelial repair in insulin resistant mice in vivo and examined potential mechanisms in human EC and angiogenic progenitor cells (APCs) in vitro . Methods Endothelial regeneration following femoral artery wire-injury was quantified after 5 days in mice hemizygous for knockout of the insulin receptor (IRKO) with or without transgenic over-expression of human-IGFBP-1. We quantified APCs and assessed function in IRKO, IRKO*IGFBP-1, IGFBP-1 and Wild-type (WT) litter mate controls. Endothelial cell adhesion was assessed ex-vivo in human tissues by seeding segments of endothelium-denuded human saphenous vein with a sub-confluent density of human coronary artery EC, which were pre-incubated with or without IGFBP-1. The effects of IGFBP-1 on the functional properties of EC in vitro were examined using cell migration and proliferation assays. Mechanisms involved in endothelial repair were also investigated through Western blotting for focal adhesion kinase and RhoA activity and Integrin binding assays. Results Following wire injury, endothelial regeneration was enhanced in IRKO mice expressing IGFBP-1 compared to IRKO controls (47+/-3% vs. 54+/-2%; P < 0.05, Figure 1. A). This was not explained by altered abundance or function of APCs. Incubation of human EC with IGFBP-1 significantly increased cell-surface expression of α5β1 and αVβ3 integrins and enhanced the ability of EC to adhere and regenerate denuded human vein ex vivo (P < 0.001, Figure 1. B). Insulin resistance was induced in EC in vitro by the pro-inflammatory cytokine tumour necrosis factor-alpha (TNF-α) which significantly inhibited EC migration (P < 0.01) and proliferation (P < 0.01). Co-incubation with IGFBP-1 restored the migratory (P < 0.05) and proliferative (P < 0.05) capacity of EC to control levels (Figure 2.A and 2.B). IGFBP-1 induced rapid activation of RhoA in EC and significantly increased phosphorylation of focal adhesion kinase. Abstract 217 Figure 1 Abstract 217 Figure 2 Conclusions IGFBP-1 ameliorates insulin-resistance related defects in endothelial regeneration by enhancing endothelial cell migration, proliferation and adhesion through mechanisms involving RhoA, integrins and focal adhesion kinase phosphorylation. Our findings raise the possibility that manipulating IGFBP-1 could be a strategy to enhance endothelial repair in patients with insulin resistance.

TARGETING NOX2 NADPH OXIDASE IN INSULIN RESISTANCE RELATED ENDOTHELIAL DYSFUNCTION

Laboratory based cell culture studies have shown that reactive oxygen species (ROS) induced oxidative stress as an important molecular mechanism in the development and progression of cardiovascular diseases. Despite that, large scale clinical trials have shown that general antioxidants are not only ineffective in treating cardiovascular diseases but also, in some cases, can have harmful effects. In this context, the next logical step is to specifically targeting critical molecules in ROS generation rather than using non-specific antioxidants. Insulin resistance, the antecedent of type 2 diabetes mellitus, can itself initiate various vascular complications of diabetes before the development of clinically diagnosable diabetes. Insulin resistance is characterised by excessive endothelial cell generation of cytotoxic concentrations of ROS. NADPH oxidases (NOX), a group of ROS producing enzymes, have been recently shown as the major source of excessive ROS in insulin resistant endothelial cells. We hypothesised that specific targeting of NOX could be the way forward in developing antioxidant molecules as therapeutic agents for vascular diseases. In this study, we examined the role of NOX enzymes and specifically Nox2 isoform in superoxide generation in 2 complementary in vivo models (endothelial specific and whole body) of human insulin resistance. Using gp91-ds tat peptide, a non-specific NOX inhibitor, we have specifically targeted NOX generated superoxide production. For acute inhibition we have treated cells or tissues with 50u2005µM gp91ds-tat or control peptide for 30 minutes. For chronic inhibition, mice were implanted with osmotic mini-pumps which delivered 10u2005mg/kg/day of drug for 28 days. The excessive level of superoxide found in insulin resistant endothelial cells was significantly reduced by acute or chronic treatment with gp91ds-tat peptide. Also NO dependent vasorelaxation response, which was impaired in insulin resistance, was improved with gp91ds-tat peptide treatment. Since insulin resistant endothelial cells showed higher expression of Nox2 isoform, Nox2 gene was knocked down using siRNA which significantly reduced superoxide levels. Furthermore, to clearly examine the involvement of Nox2 isoform in this context, double transgenic mice with endothelial specific insulin resistance and Nox2 gene knockout were generated. These mice showed reduced superoxide production and improved vascular function. Thus Nox2 is the critical molecule in insulin resistance induced endothelial dysfunction. In conclusion, our study establishes that pharmacological inhibition of NOX as a novel way to treat insulin resistance related vascular disease.

171 NOX2-DERIVED REACTIVE OXYGEN SPECIES CAUSES VASCULAR DYSFUNCTION IN MURINE MODEL OF ENDOTHELIAL INSULIN SENSITIVITY AND ACTIVATION OF NRF2 TRANSCRIPTION FACTOR

Introduction The escalating number of individuals suffering from Type 2 Diabetes is a significant healthcare burden, globally. A critical pathophysiological feature of type 2 diabetes is insulin resistance. It is well-established that insulin stimulates generation of the endothelium-derived anti-inflammatory/anti-atherosclerotic signalling radical, nitric oxide (NO). We investigated the hypothesis that increasing insulin sensitivity specifically, in the endothelium will lead to beneficial effects on NO bioavailability and vascular endothelial function. A novel transgenic mouse over-expressing Type A human Insulin Receptor (HIRECO) in the endothelium, driven by the Tie-2 promoter-enhancer was generated in order to explore the effects of increasing insulin signalling in the vascular bed. Methods Various tissues and pulmonary endothelial cells from the HIRECO mice were analysed using RT-PCR to confirm significant levels of human insulin receptor mRNA, while protein expression was confirmed by western blotting. Lucigenin-enhanced chemiluminescence was used to measure superoxide anion levels while; vasomotor function was assessed in thoracic aortic rings mounted in an organ bath. Results HIRECO mice demonstrated no significant morphological, metabolic phenotype or blood pressure abnormality compared to wild type (WT) littermates. Plasma insulin levels were similar following an overnight fast, but were decreased in the HIRECO after a glucose challenge. HIRECO mice exhibited significant endothelial dysfunction with a blunted response to acetylcholine (Emax, WT vs. HIRECO: 84±3% vs. 68±3% respectively, p<0.05). The impaired aortic response to acetylcholine was normalized by the NADPH oxidase inhibitor peptide, gp91ds-tat, (Emax: 93±5%; p<0.05), as well as the superoxide dismutase mimetic, MnTmPyP. HIRECO mice had a 1.65-fold increase in the level of superoxide anion production compared to WT. Basal NO bioactivity was decreased in HIRECO compared to WT littermates (Emax upon exposure to eNOS inhibitor, L-NAME in phenylephrine-constricted aorta, WT vs. HIRECO: 144±27.9% vs. 32±33%, n=5, p<0.05). However, basal eNOS and Akt phosphorylation in isolated endothelial cells of HIRECO mice was enhanced 1.56 fold compared to WT littermates. Additionally, increased expressions of NADPH oxidase isoform, Nox2 and redox-sensitive transcription factor, Nrf2 were detected in HIRECO endothelial cells. Conclusions/Implications These data clearly suggest that enhanced oxidative stress in a novel murine model of increased endothelial insulin signalling, leads to reduced bioavailability of nitric oxide and vascular dysfunction. These data also demonstrate for the first time, that increased insulin sensitivity in the endothelium, increases the generation of free radical generation and reduces NO bioavailability.

Endothelial SHIP2 confers age-dependent contrasting affects on whole body glucose homeostasis and vascular function

Introduction Aging is an important risk factor for diabetes and cardiovascular disease and integrity of the endothelium plays a critical role in cardiovascular pathophysiology. Although the vascular implications of endothelial insulin resistance are well understood, the effect of enhanced endothelial insulin signaling on whole body glucose regulation and vascular function remains poorly characterized. We therefore generated mice with endothelial-specific downregulation of the lipid phosphatase SHIP2 (a negative regulator of insulin signaling) to investigate whether enhanced insulin signaling in the endothelium modulates vascular function and whole body glucose regulation. Methods Exons 18–19 of the ship 2 gene were deleted using Cre-Lox technology under control of the Tie2 promoter generating a catalytically inactivate protein. Male heterozygotes for the inactive protein (EC-SHIP2+/−) were compared to sex-matched littermate controls. Results EC-SHIP+/− mice were morphologically indistinguishable from controls, exhibiting normal development. At 8 weeks of age EC-SHIP2+/− mice displayed increased glucose tolerance after glucose challenge (P=0.03) and improved insulin sensitivity (P=0.02) after insulin challenge compared to controls. Surprisingly however, by 40 weeks of age this phenotype was reversed; EC-SHIP2+/− mice revealed significant insulin resistance 60u2005min after insulin challenge (P=<0.05). This phenotype was confirmed by euglycemic hyperinsulinemic clamping showing whole body insulin resistance in EC-SHIP+/− mice (decreased glucose infusion rate of 26% P<0.05). In young mice ex vivo aortic vasomotor studies in both controls and EC-SHIP+/− revealed similar contractile responses to phenylephrine and displayed decreased contraction after insulin incubation (Emax 0.88±0.05g vs 0.62±0.05g P=0.002 and 0.83±0.05g vs 0.69±0.05g P=0.025 respectively). Both groups displayed increased contraction after NO synthase inhibitor LNMMA incubation (Emax 0.88±0.05g vs 1.31±0.11g P=0.01 and 0.83±0.05g vs 1.28±0.02g P=<0.0001 respectively). However, at 40 weeks old in EC-SHIP2+/− mice the vasodilatory aortic ring response to insulin was abolished (Emax controls 0.59±0.04g vs 0.47±0.03g P=0.04, EC-SHIP2+/− 0.64±0.04g vs 0.63±0.06g P=0.9) and EC-SHIP2+/− displayed no increase in contraction to LNMMA incubation (Emax controls 0.59±0.04g vs 0.82±0.08g P=0.02, EC-SHIP2+/− 0.64±0.04g vs 0.69±0.07g P=0.5) indicating insulin resistance and lower basal NO production, suggesting the change in glucose homeostasis may be mediated by changes in NO bioavailability. Conclusion Endothelial functional downregulation of SHIP2 augments whole body glucose disposal in young mice but attenuates whole body glucose disposal in older mice. Although further studies are required to elucidate the molecular mechanisms our data suggest a previously unrecognised age dependent role for the vascular endothelium in whole body glucose regulation.

NOX2 NADPH-OXIDASE A NOVEL TARGET TO PREVENT INSULIN RESISTANCE RELATED ENDOTHELIAL CELL DYSFUNCTION

Introduction Insulin resistance, a central pathophysiological feature of type 2 diabetes is characterised by a deleterious change in endothelial cell phenotype, a hallmark of which is increased generation of reactive oxygen species. We examined the role of NADPH oxidase and specifically NOX2 NADPH oxidase in insulin resistance induced endothelial cell dysfunction. We studied mice with endothelium specific over expression of a dominant negative insulin receptor (ESMIRO) and mice with whole body haploinsufficiency of the insulin receptor (IR+/−). Methods ESMIRO mice, a model of endothelium specific insulin resistance, and IR+/− mice a model of whole body insulin resistance were used to examine the effect of acute and chronic pharmacological inhibition of NADPH oxidase on superoxide production (lucigenin enhanced chemiluminescence) and endothelial function (acetylcholine mediated aortic relaxation). To specifically investigate the role of NOX2, we crossed mice with holoinsufficiency of NOX2 with ESMIRO mice to generate ESMIRO/NOX2y/− mice. Data expressed as mean±SEM; male mice used for all experiments. Results Basal superoxide generation in isolated pulmonary endothelial cells (PEC) was increased in both models of insulin resistance (by 130% in ESMIRO and 220% in IR+/− compared to wild type, both p<0.01; n=3 for each group). Pre-treating PEC with gp91ds-tat, a cell permeable specific blocker of NOX subunit assembly and function, reduced the excessive superoxide generation in ESMIRO and IR+/−. Endothelial NO mediated vasorelaxation in aortic rings from ESMIRO and IR+/− was impaired (101%±11% relaxation to 1u2005μM acetylcholine in wild type, 61%±3% in ESMIRO (n=5, p<0.01); 91%±3% relaxation in wild type, 75%±6% in IR+/− (n=4, p=0.03)). This was restored by pre-incubating rings with gp91ds-tat (92%±6% relaxation in ESMIRO and 93%±6% in IR+/−). Chronic (4u2005weeks) administration of gp91-ds tat peptide (using osmotic mini-pump) to ESMIRO and IR+/− mice also restored endothelial dependent relaxation (from 83%±11% to 100%±9% in ESMIRO and to 136%±11% in IR+/−). NOX2 gene expression was significantly higher in ESMIRO mice. ESMIRO/NOX2y/− mice with complete deletion of NOX2 had significantly greater relaxation responses to acetylcholine than ESMIRO (77%±6% relaxation in ESMIRO and 100%±4% in ESMIRO/NOX2y/−; n=5, p=0.002). Neither pharmacological nor genetic inhibition of NADPH oxidase had any effect on glucose homeostasis. Discussion These data in complementary models of insulin resistance demonstrate that acute or chronic pharmacological inhibition of NADPH oxidase reduces superoxide generation and improves endothelial function. Specifically targeting NOX2 also restored endothelial function in ESMIRO mice.