A Skromna

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.

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.

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. Stem Cells 2014;32:2714–2723

173 Increasing Insulin Sensitivity in the Endothelium Leads to Reduced Nitric Oxide Bioavailability

Introduction Insulin resistance is known to precede Type 2 diabetes (T2DM). Insulin mediated release of the endothelial cell (EC) derived anti-atherosclerotic molecule, nitric oxide (NO) is blunted in patients suffering from insulin resistance T2DM. We examined the effects of enhancing EC insulin sensitivity in vivo , by generating a novel transgenic mouse, over-expressing Type A human Insulin Receptor (HIRECO) restricted to EC. Methods Western blotting and RT-PCR were carried out on tissues and isolated endothelial cells from lungs to measure protein levels and mRNA expression, respectively. NADPH-dependent lucigenin-enhanced chemiluminescence was used to measure superoxide anion levels. Isolated thoracic aortic rings suspended in an organ bath were used to determine vasomotor functions. eNOS activity was examined by citrulline assay with 14C-labelled L-arginine. HIRECO were compared to wild type littermates. Results Over-expressing human insulin receptors in EC had no significant effect on morphological features, metabolic phenotype or blood pressure of HIRECO. HIRECO demonstrated significant EC dysfunction measured by a blunted endothelium-dependent vasorelaxation to acetylcholine and reduced basal NO release. EC-independent response to sodium nitroprusside remained unchanged. EC dysfunction observed in the organ bath was normalised by a NADPH oxidase-specific inhibitor peptide, gp91ds-tat as well as the superoxide dismutase mimetic, MnTmPyp. HIRECO demonstrated significant increase in superoxide anion release compared to WT littermates. This data was supported by a concomitant increase in NADPH oxidase isoform, NOX2 protein expression. Basal eNOS and Akt phosphorylation levels in isolated EC of HIRECO mice were enhanced compared to WT mice. Interestingly, insulin-stimulated eNOS phosphorylation and activation was decreased, whereas Akt phosphorylation remained unchanged. eNOS tyrosine phosphorylation mediated by proline-rich tyrosine kinase (PYK2) was significantly enhanced in EC from HIRECO mice. In order to investigate, if the perturbations of insulin signalling in the EC have a pathological outcome, HIRECO mice were crossed with ApoE knockout mice. These mice demonstrated a significant enhancement of plaque formation in aorta. Conclusions/Implications These data show that increasing EC insulin sensitivity leads to reduced bioavailability of NO. These data demonstrate for the first time that increased insulin signalling in EC increases the generation of superoxide anion via activation of NOX2 NADPH oxidase and reduced NO production in response to insulin due at least in part to increased EC PYK2 activity leading to a pro-atherosclerotic state.

A Gene-based restoration of Akt activity in endothelial progenitor cells from human subjects at high cardiovascular risk rescues vascular reparative capacity

Introduction Late-outgrowth endothelial progenitor cells (LEPC) are putative mediators of endogenous vascular repair, and represent an attractive tool for future cell-based cardiovascular repair strategies. However, progenitor function may be impaired in high cardiovascular risk populations, such as those of South Asian (SA) ethnicity, in whom cell based therapies are likely to offer most benefit. Methods Detailed analysis of in vitro LEPC function (abundance, proliferation, migration, angiogenesis and senescence) was performed in 12 SA men and 12 matched White European (WE) controls. Molecular abnormalities within the Akt/eNOS signalling axis were analysed with PCR and western blotting. LEPC were transfused into immunodeficient mice subsequent to femoral artery injury to assess in vivo reparative function. In vitro and in vivo studies were repeated after lentiviral gene delivery of constitutively active Akt1 (E17K) or control (EGFP) to SA LEPC; augmented Akt activity was confirmed using a Glycogen Synthase Kinase phosphorylation assay. Data are expressed as mean [SEM] and compared with t tests as appropriate; statistical significance is defined as p<0.05 (denoted by *). Results The two groups were matched for age and cardiovascular risk factors, although the SA group was comparatively insulin resistant (HOMA-IR 1.2 [0.2] vs 0.5 [0.1] au*). SA LEPC exhibited impaired colony formation (0.06 [0.02] vs 0.19 [0.03] colonies/ml blood*), migration to vascular endothelial growth factor (5 [0.7] vs 10 [1.7] cells/microscopic field*) and in vitro angiogenesis (1.9 [0.6] vs 3.8 [0.5] tubular structures/microscopic field*), associated with markedly decreased abundance of the phosphorylated forms of the pro-angiogenic molecules S473-Akt (0.14 [0.05] vs 0.81 [0.2] au*) and S1177-eNOS (0.05 [0.02] vs 0.15 [0.01] au*). Transfusion of WE LEPC into immunodeficient mice after wire-induced femoral artery luminal injury augmented re-endothelialisation; however, neither SA LEPC, nor vehicle, augmented re-endothelialisation (WE: 54.2 [6.4], SA: 36.9 [3.4], vehicle: 31.1 [2.4] % re-endothelialised; WE vs SA*; SA vs vehicle p=0.2). Lentiviral gene delivery of a E17K, but not EGFP control, to SA LEPC was associated with augmented Akt1 activity and rescue of in vivo re-endothelialisation capacity (E17K: 55.2 [4.4] vs EGFP 24.1 [1.3] % re-endothelialised; E17K vs EGFP*; E17K vs WE non-transduced cells p=0.9). Conclusions These data provide proof of principle for human LEPC based vascular repair therapy, and demonstrate a mechanism by which to rescue marked progenitor dysfunction in a group at high risk of cardiovascular events, whom are likely to benefit from cardiovascular repair therapies.

Effects of obesity on insulin: insulin-like growth factor 1 hybrid receptor expression and Akt phosphorylation in conduit and resistance arteries

Insulin and insulin-like growth factor-1 stimulate specific responses in arteries, which may be disrupted by diet-induced obesity. We examined (1) temporal effects of high-fat diet compared to low-fat diet in mice on insulin receptor, insulin-like growth factor-1 receptor, insulin receptor/insulin-like growth factor-1 receptor hybrid receptor expression and insulin/insulin-like growth factor-1-mediated Akt phosphorylation in aorta; and (2) effects of high-fat diet on insulin and insulin-like growth factor-1-mediated Akt phosphorylation and vascular tone in resistance arteries. Medium-term high-fat diet (5 weeks) decreased insulin-like growth factor-1 receptor expression and increased hybrid expression (~30%) only. After long-term (16 weeks) high-fat diet, insulin receptor expression was reduced by ~30%, insulin-like growth factor-1 receptor expression decreased a further ~40% and hybrid expression increased a further ~60%. Independent correlates of hybrid receptor expression were high-fat diet, duration of high-fat diet and plasma insulin-like growth factor-1 (all p < 0.05). In aorta, insulin was a more potent activator of Akt than insulin-like growth factor-1, whereas in resistance arteries, insulin-like growth factor-1 was more potent than insulin. High-fat diet blunted insulin-mediated vasorelaxation (p < 0.01) but had no effect on insulin-like growth factor-1-mediated vasorelaxation in resistance arteries. Our findings support the possibility that hybrid receptor level is influenced by nutritional and metabolic cues. Moreover, vessel-dependent effects of insulin and insulin-like growth factor-1 on vascular tone and Akt activation may have implications in treating obesity-related vascular disease.

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.

169 Endothelial Insulin Sensitisation Enhances Vascular Repair and Aortic Vasomotor Function in Systemic Insulin Resistance

Introduction Insulin resistance is independently associated with cardiovascular events. We have previously shown that mice haploinsufficient for the insulin receptor (IRKO) exhibit hypertension, vasomotor dysfunction and impaired endothelial regeneration after denuding arterial injury, with concomitant reductions in endothelial progenitor cell (EPC) number and function. Importantly, these occur despite preserved glucocompetence. Murine models of endothelium-specific insulin resistance yield similar findings, implying that vascular insulin signalling plays a key role in maintaining functional vascular integrity. We hypothesise that selective restoration of endothelial insulin signalling can improve vascular function in the context of global insulin resistance. Methods We have generated transgenic mice in which the Tie-2 promoter targets human insulin receptor (IR) over-expression to endothelial cells (HIRECO). These mice were crossed with IRKO to derive HIRECOxIRKO offspring, which were compared with IRKO littermates. Metabolic phenotypes were assessed using glucose- and insulin-tolerance tests, and ELISA for plasma insulin concentrations. Femoral artery injury was performed using angioplasty guide wires; vessels were explanted 4 days later to quantify endothelial regeneration using Evans Blue dye. C-kit-expressing circulating progenitor cells (CPC) were enumerated using FACS, while cultured blood-derived EPCs were counted using DiI-ac-LDL/lectin staining. Finally, aortic rings were exposed to incremental acetylcholine (ACh) and phenylephrine (PE) doses in organ bath apparatus to assess endothelium-dependent vasorelaxation and constriction respectively. Data are expressed as mean (SE) and compared using t-tests; p < 0.05 is denoted with *. Results HIRECOxIRKO and IRKO had similar glucocompetence, insulin sensitivity, and fasting insulin concentrations [0.76 (0.14) ng/mL vs. 0.70 (0.18); p = 0.78]. Vascular regeneration was improved in HIRECOxIRKO [56.9 (4.2)% re-endothelialised vs. 46.0 (2.3)% in IRKO*]. This was not associated with improved numbers of peripheral blood CPC [212.3 (44.8) vs. 212.5 (43.9); p = 0.99], nor blood-derived EPCs in culture [0.5 (0.1)/HPF vs. 0.5 (0.2); p = 0.99]. In organ bath experiments, HIRECOxIRKO aortic rings demonstrated significantly greater ACh-induced relaxation than IRKO [Emax 99.8 (4.8)% of pre-constriction vs. 74.3 (9.3)%*] with a similar trend in PE constriction. Conclusions Endothelial insulin sensitisation in the context of global insulin resistance rescues vasomotor function and vascular repair, despite not altering metabolic function, CPC or EPC abundance. These data imply that endothelial insulin sensitisation may be a therapeutic target to augment vascular function and repair in insulin resistant subjects.