Beta Blockade Prevents Cardiac Morphological and Molecular Remodelling in Experimental Uremia.

Heart failure and chronic kidney disease (CKD) share several mediators of cardiac pathological remodelling. Akin to heart failure, this remodelling sets in motion a vicious cycle of progressive pathological hypertrophy and myocardial dysfunction in CKD. Several decades of heart failure research have shown that beta blockade is a powerful tool in preventing cardiac remodelling and breaking this vicious cycle. This phenomenon remains hitherto untested in CKD. Therefore, we set out to test the hypothesis that beta blockade prevents cardiac pathological remodelling in experimental uremia. Wistar rats had subtotal nephrectomy or sham surgery and were followed up for 10 weeks. The animals were randomly allocated to the beta blocker metoprolol (10 mg/kg/day) or vehicle. In vivo and in vitro cardiac assessments were performed. Cardiac tissue was extracted, and protein expression was quantified using immunoblotting. Histological analyses were performed to quantify myocardial fibrosis. Beta blockade attenuated cardiac pathological remodelling in nephrectomised animals. The echocardiographic left ventricular mass and the heart weight to tibial length ratio were significantly lower in nephrectomised animals treated with metoprolol. Furthermore, beta blockade attenuated myocardial fibrosis associated with subtotal nephrectomy. In addition, the Ca++- calmodulin-dependent kinase II (CAMKII) pathway was shown to be activated in uremia and attenuated by beta blockade, offering a potential mechanism of action. In conclusion, beta blockade attenuated hypertrophic signalling pathways and ameliorated cardiac pathological remodelling in experimental uremia. The study provides a strong scientific rationale for repurposing beta blockers, a tried and tested treatment in heart failure, for the benefit of patients with CKD.

Disparity in association of obesity measures with ankle and brachial systolic blood pressures in Europeans and South Asians.

Obesity causes increases in brachial systolic-blood-pressures (SBP), risks of type 2 diabetes (T2DM) and cardiovascular diseases (CVD). Brachial and ankle SBPs have differential relationship with T2DM and CVD. Our objective was to study the relationship of obesity measures with brachial and ankle SBPs. A population of 1098 adults (South Asians n = 699; 41.70% male and 58.3% female) were recruited over 5 years from primary care practices in England. Their four limbs SBPs were measured using Doppler machine and body-mass-index (BMI) and waist-to-height-ratio (WHtR) calculated. Linear regressions were performed between SBPs and obesity measures, after adjustments for sex, age, ethnicity, T2DM and CVD. The mean age of all participants was 51.3 (SD = 17.2), European was 57.7 (SD 17.2) and South Asian was 47.8 (SD = 16.1). The left posterior tibial [Beta = 1.179, P = 4.559 × 10-15] and the right posterior tibial SBP [Beta = 1.178, P = 1.114 × 10-13] most significantly associated with the BMI. In South Asians, although the left brachial [Beta = 25.775, P = 0.032] and right brachial SBP [Beta = 22.792, P = 0.045] were associated to the WHtR, the left posterior tibial SBP [Beta = 39.894, P = 0.023], association was the strongest. For the first time, we have demonstrated that ankle SBPs had significant association with generalised obesity than brachial systolic blood pressures (SBP), irrespective of ethnicity. However, with respect to visceral obesity, the association with ankle SBP was more significant in South Asians compared to Europeans.

Cixutumumab reveals a critical role for IGF-1 in adipose and hepatic tissue remodelling during the development of diet-induced obesity.

High fat diet (HFD)-induced obesity leads to perturbation in the storage function of white adipose tissue (WAT) resulting in deposition of lipids in tissues ill-equipped to deal with this challenge. The role of insulin like growth factor-1 (IGF-1) in the systemic and organ-specific responses to HFD is unclear. Using cixutumumab, a monoclonal antibody that internalizes and degrades cell surface IGF-1 receptors (IGF-1 R), leaving insulin receptor expression unchanged we aimed to establish the role of IGF-1 R in the response to a HFD. Mice treated with cixutumumab fed standard chow developed mild hyperinsulinemia with no change in WAT. When challenged by HFD mice treated with cixutumumab had reduced weight gain, reduced WAT expansion, and reduced hepatic lipid vacuole formation. In HFD-fed mice, cixutumumab led to reduced levels of genes encoding proteins important in fatty acid metabolism in WAT and liver. Cixutumumab protected against blunting of insulin-stimulated phosphorylation of Akt in liver of HFD fed mice. These data reveal an important role for IGF-1 R in the WAT and hepatic response to short-term nutrient excess. IGF-1 R inhibition during HFD leads to a lipodystrophic phenotype with a failure of WAT lipid storage and protection from HFD-induced hepatic insulin resistance.

Inter-ankle Systolic Blood Pressure Difference Is a Marker of Increased Fasting Blood-Glucose in Asian Pregnant Women.

Objective: This cross-sectional study aimed to determine the relationship between clinical blood pressures and blood pressures measured using Doppler with blood glucose in pregnancy by ethnicity. Methods: We recruited 179 (52% White European, 48% Asian) pregnant women at 24-28 weeks of gestation who underwent a glucose tolerance test in an antenatal clinic in Bradford Royal Infirmary, the UK, from 2012 to 2013. Systolic blood pressures in the arm (left and right brachial) and ankle [left and right posterior tibial (PT) and dorsalis pedalis (DP)] blood pressures were measured using a Doppler probe. The inter-arm (brachial) and inter-ankle (PT and DP) systolic blood pressure differences were obtained. A multivariate linear regression model adjusted for age, body mass index, and diabetes risk was used to assess the relationship between blood pressures and blood glucose. Results: Asian pregnant women had higher blood glucose but lower ankle blood pressures than White Europeans. In White Europeans, brachial blood pressures and clinical blood pressures were positively associated with fasting blood glucose (FBG), but brachial blood pressures did not perform better as an indicator of FBG than clinical blood pressures. In Asians, increased inter-ankle blood pressure difference was associated with increased FBG. For each 10 mmHg increase in the inter-ankle blood pressure difference, FBG increased by 0.12 mmol/L (Beta=0.12, 95%CI: 0.01-0.23). Conclusion: The relationship between blood pressures with blood glucose differed by ethnicity. In Asians, inter-ankle systolic blood pressure difference was positively associated with blood glucose. This is first ever report on ankle blood pressures with blood glucose in pregnancy which suggests future potential as a non-invasive gestational diabetes risk screening tool.

Novel Paracrine Action of Endothelium Enhances Glucose Uptake in Muscle and Fat.

[Figure: see text].

Differential associations of ankle and brachial blood pressures with diabetes and cardiovascular diseases: cross-sectional study.

Increased brachial systolic blood-pressure (BP) predicts diabetes (T2DM) but is not fully effective. Value of absolute ankle systolic BP for T2DM compared to brachial systolic BP is not known. Our objectives were to assess independent relationships of ankle-systolic BP with T2DM and cardiovascular disease in Europeans and south Asians. Cross-sectional studies of anonymised data from registered adults (n = 1087) at inner city deprived primary care practices. Study includes 63.85% ethnic minority. Systolic BP of the left and right-brachial, posterior-tibial and dorsalis-pedis-arteries measured using a Doppler probe. Regression models' factors were age, sex, ethnicity, body mass index (BMI) and waist height ratio (WHtR). Both brachial and ankle systolic-BP increase with diabetes in Europeans and south Asians. We demonstrated that there was a significant positive independent association of ankle BP with diabetes, regardless of age and sex compared to Brachial. There was stronger negative association of ankle blood pressure with cardiovascular disease, after adjustment for BMI, WHtR and ethnicity. Additionally, we found that ankle BP were significantly associated with cardiovascular disease in south Asians more than the Europeans; right posterior tibial. Ankle systolic BPs are superior to brachial BPs to identify risks of Type 2DM and cardiovascular diseases for enhanced patient care.

Barium yttrium fluoride based upconversion nanoparticles as dual mode image contrast agents.

Dual labeled contrast agents could provide better complementary information for bioimaging than available solely from a single modality. In this paper we investigate the suitability of Yb3+ and Er3+-doped BaYF5 upconversion nanoparticles (UCNPs) as both optical and X-ray micro computed tomography (µCT) contrast agents. Stable, aqueous UCNP dispersions were synthesised using a hydrothermal method with the addition of polyethyleneimine (PEI). UCNPs were single crystal and had a truncated cuboidal and/or truncated octahedral morphology, with average particle size of 47 ±9 nm from transmission electron microscopy which was further used to characterize the structure and composition in detail. A zeta potential value of +51 mV was measured for the aqueous nanoparticle dispersions which is beneficial for cell permeability. The outer hydrated PEI layer is also advantageous for the attachment of proteins for targeted delivery in biological systems. The prepared UCNPs were proven to be non-toxic to endothelial cells up to a concentration of 3.5 mg/mL, when assessed using an MTT assay. The particles showed intense green upconversion photoluminescence when excited at a wavelength of 976 nm using a diode laser. Quantitative X-ray µCT contrast imaging confirmed the potential of these UCNPs as X-ray contrast agents and confirming their dual modality for bioimaging.

Divergent effects of genetic and pharmacological inhibition of Nox2 NADPH oxidase on insulin resistance-related vascular damage.

Insulin resistance leads to excessive endothelial cell (EC) superoxide generation and accelerated atherosclerosis. The principal source of superoxide from the insulin-resistant endothelium is the Nox2 isoform of NADPH oxidase. Here we examine the therapeutic potential of Nox2 inhibition on superoxide generation in saphenous vein ECs (SVECs) from patients with advanced atherosclerosis and type 2 diabetes and on vascular function, vascular damage, and lipid deposition in apolipoprotein E-deficient (ApoE-/-) mice with EC-specific insulin resistance (ESMIRO). To examine the effect of genetic inhibition of Nox2, ESMIRO mice deficient in ApoE-/- and Nox2 (ESMIRO/ApoE-/-/Nox2-/y) were generated and compared with ESMIRO/ApoE-/-/Nox2+/y littermates. To examine the effect of pharmacological inhibition of Nox2, we administered gp91dstat or scrambled peptide to ESMIRO/ApoE-/- mice. SVECs from diabetic patients had increased expression of Nox2 protein with concomitant increase in superoxide generation, which could be reduced by the Nox2 inhibitor gp91dstat. After 12 wk Western diet, ESMIRO/ApoE-/-/Nox2-/y mice had reduced EC superoxide generation and greater aortic relaxation to acetylcholine. ESMIRO/ApoE-/-/Nox2-/y mice developed more lipid deposition in the thoraco-abdominal aorta with multiple foci of elastin fragmentation at the level of the aortic sinus and greater expression of intercellular adhesion molecule-1 (ICAM-1). Gp91dstat reduced EC superoxide and lipid deposition in the thoraco-abdominal aorta of ESMIRO/ApoE-/- mice without causing elastin fragmentation or increased ICAM-1 expression. These results demonstrate that insulin resistance is characterized by increased Nox2-derived vascular superoxide. Complete deletion of Nox2 in mice with EC insulin resistance exacerbates, whereas partial pharmacological Nox2 inhibition protects against, insulin resistance-induced vascular damage.

Piezo1 channel activation mimics high glucose as a stimulator of insulin release.

Glucose and hypotonicity induced cell swelling stimulate insulin release from pancreatic ß-cells but the mechanisms are poorly understood. Recently, Piezo1 was identified as a mechanically-activated nonselective Ca2+ permeable cationic channel in a range of mammalian cells. As cell swelling induced insulin release could be through stimulation of Ca2+ permeable stretch activated channels, we hypothesised a role for Piezo1 in cell swelling induced insulin release. Two rat ß-cell lines (INS-1 and BRIN-BD11) and freshly-isolated mouse pancreatic islets were studied. Intracellular Ca2+ measurements were performed using the fura-2 Ca2+ indicator dye and ionic current was recorded by whole cell patch-clamp. Piezo1 agonist Yoda1, a competitive antagonist of Yoda1 (Dooku1) and an inactive analogue of Yoda1 (2e) were used as chemical probes. Piezo1 mRNA and insulin secretion were measured by RT-PCR and ELISA respectively. Piezo1 mRNA was detected in both ß-cell lines and mouse islets. Yoda1 evoked Ca2+ entry was inhibited by Yoda1 antagonist Dooku1 as well as other Piezo1 inhibitors gadolinium and ruthenium red, and not mimicked by 2e. Yoda1, but not 2e, stimulated Dooku1-sensitive insulin release from ß-cells and pancreatic islets. Hypotonicity and high glucose increased intracellular Ca2+ and enhanced Yoda1 Ca2+ influx responses. Yoda1 and hypotonicity induced insulin release were significantly inhibited by Piezo1 specific siRNA. Pancreatic islets from mice with haploinsufficiency of Piezo1 released less insulin upon exposure to Yoda1. The data show that Piezo1 channel agonist induces insulin release from ß-cell lines and mouse pancreatic islets suggesting a role for Piezo1 in cell swelling induced insulin release. Hence Piezo1 agonists have the potential to be used as enhancers of insulin release.

Endothelial Insulin Receptor Restoration Rescues Vascular Function in Male Insulin Receptor Haploinsufficient Mice.

Reduced systemic insulin signaling promotes endothelial dysfunction and diminished endogenous vascular repair. We investigated whether restoration of endothelial insulin receptor expression could rescue this phenotype. Insulin receptor knockout (IRKO) mice were crossed with mice expressing a human insulin receptor endothelial cell-specific overexpression (hIRECO) to produce IRKO-hIRECO progeny. No metabolic differences were noted between IRKO and IRKO-hIRECO mice in glucose and insulin tolerance tests. In contrast with control IRKO littermates, IRKO-hIRECO mice exhibited normal blood pressure and aortic vasodilatation in response to acetylcholine, comparable to parameters noted in wild type littermates. These phenotypic changes were associated with increased basal- and insulin-stimulated nitric oxide production. IRKO-hIRECO mice also demonstrated normalized endothelial repair after denuding arterial injury, which was associated with rescued endothelial cell migration in vitro but not with changes in circulating progenitor populations or culture-derived myeloid angiogenic cells. These data show that restoration of endothelial insulin receptor expression alone is sufficient to prevent the vascular dysfunction caused by systemically reduced insulin signaling.

Endothelial SHIP2 Suppresses Nox2 NADPH Oxidase-Dependent Vascular Oxidative Stress, Endothelial Dysfunction, and Systemic Insulin Resistance.

Shc homology 2-containing inositol 5' phosphatase-2 (SHIP2) is a lipid phosphatase that inhibits insulin signaling downstream of phosphatidylinositol 3-kinase (PI3K); its role in vascular function is poorly understood. To examine its role in endothelial cell (EC) biology, we generated mice with catalytic inactivation of one SHIP2 allele selectively in ECs (ECSHIP2Δ/+). Hyperinsulinemic-euglycemic clamping studies revealed that ECSHIP2Δ/+ was resistant to insulin-stimulated glucose uptake in adipose tissue and skeletal muscle compared with littermate controls. ECs from ECSHIP2Δ/+ mice had increased basal expression and activation of PI3K downstream targets, including Akt and endothelial nitric oxide synthase, although incremental activation by insulin and shear stress was impaired. Insulin-mediated vasodilation was blunted in ECSHIP2Δ/+ mice, as was aortic nitric oxide bioavailability. Acetylcholine-induced vasodilation was also impaired in ECSHIP2Δ/+ mice, which was exaggerated in the presence of a superoxide dismutase/catalase mimetic. Superoxide abundance was elevated in ECSHIP2Δ/+ ECs and was suppressed by PI3K and NADPH oxidase 2 inhibitors. These findings were phenocopied in healthy human ECs after SHIP2 silencing. Our data suggest that endothelial SHIP2 is required to maintain normal systemic glucose homeostasis and prevent oxidative stress-induced endothelial dysfunction.

Piezo1 channels sense whole body physical activity to reset cardiovascular homeostasis and enhance performance.

Mammalian biology adapts to physical activity but the molecular mechanisms sensing the activity remain enigmatic. Recent studies have revealed how Piezo1 protein senses mechanical force to enable vascular development. Here, we address Piezo1 in adult endothelium, the major control site in physical activity. Mice without endothelial Piezo1 lack obvious phenotype but close inspection reveals a specific effect on endothelium-dependent relaxation in mesenteric resistance artery. Strikingly, the Piezo1 is required for elevated blood pressure during whole body physical activity but not blood pressure during inactivity. Piezo1 is responsible for flow-sensitive non-inactivating non-selective cationic channels which depolarize the membrane potential. As fluid flow increases, depolarization increases to activate voltage-gated Ca2+ channels in the adjacent vascular smooth muscle cells, causing vasoconstriction. Physical performance is compromised in mice which lack endothelial Piezo1 and there is weight loss after sustained activity. The data suggest that Piezo1 channels sense physical activity to advantageously reset vascular control.The mechanisms that regulate the body's response to exercise are poorly understood. Here, Rode et al. show that the mechanically activated cation channel Piezo1 is a molecular sensor of physical exercise in the endothelium that triggers endothelial communication to mesenteric vessel muscle cells, leading to vasoconstriction.

Insulin-Like Growth Factor Binding Protein 1 Could Improve Glucose Regulation and Insulin Sensitivity Through Its RGD Domain.

Low circulating levels of insulin-like growth factor binding protein 1 (IGFBP-1) are associated with insulin resistance and predict the development of type 2 diabetes. IGFBP-1 can affect cellular functions independently of IGF binding through an Arg-Gly-Asp (RGD) integrin-binding motif. Whether causal mechanisms underlie the favorable association of high IGFBP-1 levels with insulin sensitivity and whether these could be exploited therapeutically remain unexplored. We used recombinant IGFBP-1 and a synthetic RGD-containing hexapeptide in complementary in vitro signaling assays and in vivo metabolic profiling in obese mice to investigate the effects of IGFBP-1 and its RGD domain on insulin sensitivity, insulin secretion, and whole-body glucose regulation. The RGD integrin-binding domain of IGFBP-1, through integrin engagement, focal adhesion kinase, and integrin-linked kinase, enhanced insulin sensitivity and insulin secretion in C2C12 myotubes and INS-1 832/13 pancreatic ß-cells. Both acute administration and chronic infusion of an RGD synthetic peptide to obese C57BL/6 mice improved glucose clearance and insulin sensitivity. These favorable effects on metabolic homeostasis suggest that the RGD integrin-binding domain of IGFBP-1 may be a promising candidate for therapeutic development in the field of insulin resistance.

Selective Enhancement of Insulin Sensitivity in the Endothelium In Vivo Reveals a Novel Proatherosclerotic Signaling Loop.

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.

Haploinsufficiency of the insulin-like growth factor-1 receptor enhances endothelial repair and favorably modifies angiogenic progenitor cell phenotype.

OBJECTIVES: Defective endothelial regeneration predisposes to adverse arterial remodeling and is thought to contribute to cardiovascular disease in type 2 diabetes mellitus. We recently demonstrated that the type 1 insulin-like growth factor receptor (IGF1R) is a negative regulator of insulin sensitivity and nitric oxide bioavailability. In this report, we examined partial deletion of the IGF1R as a potential strategy to enhance endothelial repair. APPROACH AND RESULTS: We assessed endothelial regeneration after wire injury in mice and abundance and function of angiogenic progenitor cells in mice with haploinsufficiency of the IGF1R (IGF1R(+/-)). Endothelial regeneration after arterial injury was accelerated in IGF1R(+/-) mice. Although the yield of angiogenic progenitor cells was lower in IGF1R(+/-) mice, these angiogenic progenitor cells displayed enhanced adhesion, increased secretion of insulin-like growth factor-1, and enhanced angiogenic capacity. To examine the relevance of IGF1R manipulation to cell-based therapy, we transfused IGF1R(+/-) bone marrow-derived CD117(+) cells into wild-type mice. IGF1R(+/-) cells accelerated endothelial regeneration after arterial injury compared with wild-type cells and did not alter atherosclerotic lesion formation. CONCLUSIONS: Haploinsufficiency of the IGF1R is associated with accelerated endothelial regeneration in vivo and enhanced tube forming and adhesive potential of angiogenic progenitor cells in vitro. Partial deletion of IGF1R in transfused bone marrow-derived CD117(+) cells enhanced their capacity to promote endothelial regeneration without altering atherosclerosis. Our data suggest that manipulation of the IGF1R could be exploited as novel therapeutic approach to enhance repair of the arterial wall after injury.

Restoring Akt1 activity in outgrowth endothelial cells from South Asian men rescues vascular reparative potential.

Recent data suggest reduced indices of vascular repair in South Asian men, a group at increased risk of cardiovascular events. Outgrowth endothelial cells (OEC) represent an attractive tool to study vascular repair in humans and may offer potential in cell-based repair therapies. We aimed to define and manipulate potential mechanisms of impaired vascular repair in South Asian (SA) men. In vitro and in vivo assays of vascular repair and angiogenesis were performed using OEC derived from SA men and matched European controls, prior defining potentially causal molecular mechanisms. SA OEC exhibited impaired colony formation, migration, and in vitro angiogenesis, associated with decreased expression of the proangiogenic molecules Akt1 and endothelial nitric oxide synthase (eNOS). Transfusion of European OEC into immunodeficient mice after wire-induced femoral artery injury augmented re-endothelialization, in contrast with SA OEC and vehicle; SA OEC also failed to promote angiogenesis after induction of hind limb ischemia. Expression of constitutively active Akt1 (E17KAkt), but not green fluorescent protein control, in SA OEC increased in vitro angiogenesis, which was abrogated by a NOS antagonist. Moreover, E17KAkt expressing SA OEC promoted re-endothelialization of wire-injured femoral arteries, and perfusion recovery of ischemic limbs, to a magnitude comparable with nonmanipulated European OEC. Silencing Akt1 in European OEC recapitulated the functional deficits noted in SA OEC. Reduced signaling via the Akt/eNOS axis is causally linked with impaired OEC-mediated vascular repair in South Asian men. These data prove the principle of rescuing marked reparative dysfunction in OEC derived from these men.

Endothelium-specific insulin resistance leads to accelerated atherosclerosis in areas with disturbed flow patterns: a role for reactive oxygen species.

OBJECTIVE: Systemic insulin resistance is associated with a portfolio of risk factors for atherosclerosis development. We sought to determine whether insulin resistance specifically at the level of the endothelium promotes atherosclerosis and to examine the potential involvement of reactive oxygen species. METHODS: We cross-bred mice expressing a dominant negative mutant human insulin receptor specifically in the endothelium (ESMIRO) with ApoE(-/-) mice to examine the effect of endothelium-specific insulin resistance on atherosclerosis. RESULTS: ApoE(-/-)/ESMIRO mice had similar blood pressure, plasma lipids and whole-body glucose tolerance, but blunted endothelial insulin signalling, in comparison to ApoE(-/-) mice. Atherosclerosis was significantly increased in ApoE(-/-)/ESMIRO mice at the aortic sinus (226 ± 16 versus 149 ± 24 × 10(3) µm(2), P = 0.01) and lesser curvature of the aortic arch (12.4 ± 1.2% versus 9.4 ± 0.9%, P = 0.035). Relaxation to acetylcholine was blunted in aorta from ApoE(-/-)/ESMIRO mice (Emax 65 ± 41% versus 103 ± 6%, P = 0.02) and was restored by the superoxide dismutase mimetic MnTMPyP (Emax 112 ± 15% versus 65 ± 41%, P = 0.048). Basal generation of superoxide was increased 1.55 fold (P = 0.01) in endothelial cells from ApoE(-/-)/ESMIRO mice and was inhibited by the NADPH oxidase inhibitor gp91ds-tat (-12 ± 0.04%, P = 0.04), the NO synthase inhibitor L-NMMA (-8 ± 0.02%, P = 0.001) and the mitochondrial specific inhibitor rotenone (-23 ± 0.04%, P = 0.006). CONCLUSIONS: Insulin resistance specifically at the level of the endothelium leads to acceleration of atherosclerosis in areas with disturbed flow patterns such as the aortic sinus and the lesser curvature of the aorta. We have identified a potential role for increased generation of reactive oxygen species from multiple enzymatic sources in promoting atherosclerosis in this setting.

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 N(G)-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.