S Galloway

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.

The Insulin-Like Growth Factor-1 Receptor Is a Negative Regulator of Nitric Oxide Bioavailability and Insulin Sensitivity in the Endothelium

OBJECTIVE In mice, haploinsufficiency of the IGF-1 receptor (IGF-1R+/−), at a whole-body level, increases resistance to inflammation and oxidative stress, but the underlying mechanisms are unclear. We hypothesized that by forming insulin-resistant heterodimers composed of one IGF-1Rαβ and one insulin receptor (IR), IRαβ complex in endothelial cells (ECs), IGF-1R reduces free IR, which reduces EC insulin sensitivity and generation of the antioxidant/anti-inflammatory signaling radical nitric oxide (NO). RESEARCH DESIGN AND METHODS Using a number of complementary gene-modified mice with reduced IGF-1R at a whole-body level and specifically in EC, and complementary studies in EC in vitro, we examined the effect of changing IGF-1R/IR stoichiometry on EC insulin sensitivity and NO bioavailability. RESULTS IGF-1R+/− mice had enhanced insulin-mediated glucose lowering. Aortas from these mice were hypocontractile to phenylephrine (PE) and had increased basal NO generation and augmented insulin-mediated NO release from EC. To dissect EC from whole-body effects we generated mice with EC-specific knockdown of IGF-1R. Aortas from these mice were also hypocontractile to PE and had increased basal NO generation. Whole-body and EC deletion of IGF-1R reduced hybrid receptor formation. By reducing IGF-1R in IR-haploinsufficient mice we reduced hybrid formation, restored insulin-mediated vasorelaxation in aorta, and insulin stimulated NO release in EC. Complementary studies in human umbilical vein EC in which IGF-1R was reduced using siRNA confirmed that reducing IGF-1R has favorable effects on NO bioavailability and EC insulin sensitivity. CONCLUSIONS These data demonstrate that IGF-1R is a critical negative regulator of insulin sensitivity and NO bioavailability in the endothelium.

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.

Polymorphisms of adrenoceptors are not associated with an increased risk of adverse event in heart failure: a MERIT-HF substudy.

BACKGROUND Enhanced sympathetic activation has a central role in the development of heart failure (HF). We assessed whether the alpha(2C)-adrenoceptor (Del322-325) polymorphism exclusively or in combination with a beta(1)-adrenoceptor (Arg389) polymorphism, each with known independent effects on sympathetic function, were associated with an increased risk of adverse events in HF. METHODS AND RESULTS A total of 526 patients enrolled in the Metoprolol CR/XL Randomized Intervention Trial in Congestive Heart Failure study were genotyped for both adrenoceptor polymorphisms. The distribution of alpha(2C) genotypes was similar between the event and nonevent groups. However, a reduced prevalence of the Del322-325 allele was found in individuals with ischemic congestive HF (P=.022). Patients possessing both the alpha(2C) Del322-325 and beta(1) Arg389 alleles had no increased risk of events. Adjusting for confounding variables and the beta(1) Arg389Gly polymorphism, the odds ratio of being ins/del + del/del for the alpha(2C) Del322-325 and having an event was 0.89 with 95% CI 0.49-1.63, P=.715. Similarly, adjusting for confounding variables and the alpha(2C) Del322-325 polymorphism the odds ratio of being Arg/Arg or Arg/Gly for the beta(1) Arg389Gly polymorphism and having an event was 1.13 with 95% CI 0.52-2.17, P=.864. CONCLUSIONS The alpha(2C) Del322-325 polymorphism exclusively or in combination with the beta(1)Arg389 allele is not associated with an increased risk of adverse events in HF.

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.

The −1562C/T MMP-9 promoter polymorphism does not predict MMP-9 expression levels or invasive capacity in saphenous vein smooth muscle cells cultured from different patients

Matrix metalloproteinase-9 (MMP-9) is an important regulator of vascular smooth muscle cell (SMC) invasion and proliferation. The T allele of the -1562C/T MMP-9 promoter polymorphism reportedly confers increased MMP-9 promoter activity, plasma MMP-9 levels and susceptibility to vascular pathologies. The aim of this study was to determine whether the MMP-9 -1562C/T polymorphism directly influences endogenous MMP-9 expression levels in saphenous vein (SV) SMC cultured from patients with different genotypes. Genotyping of 408 patients revealed -1562C/T genotype frequencies of 73.3% CC, 25.0% CT and 1.7% TT. Using a standardized, controlled protocol we investigated the effects of phorbol ester (TPA) and a physiological stimulus (PDGF+IL-1) on MMP-9 expression in cultured SV-SMC from 15 CC, 15 CT and 3 TT patients, and on PDGF+IL-1-induced SV-SMC invasion (Boyden chamber with Matrigel barrier). A strong correlation between MMP-9 mRNA levels (real-time RT-PCR) and MMP-9 protein secretion (gelatin zymography) was observed. However, no significant differences were observed in MMP-9 expression levels, or in SV-SMC invasion, between cells with different -1562C/T genotypes. Moreover, MMP-9 promoter activity of the C and T variants was similar. Our data challenge the functional nature of the -1562C/T polymorphism and its capacity to modulate MMP-9 expression levels and SV-SMC invasion, and hence susceptibility to vascular pathologies in vivo.

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

MMP-3 (5A/6A) Polymorphism Does Not Influence Human Smooth Muscle Cell Invasion

BACKGROUND Stromelysin (MMP-3) is an important regulator of vascular smooth muscle cell (SMC) invasion, a key contributor to saphenous vein (SV) bypass graft failure. The 5A allele of the common -1612 MMP-3 5A/6A promoter polymorphism reportedly confers increased promoter activity, MMP-3 tissue expression, and susceptibility to a number of vascular pathologies. The aim of this study was to determine whether the MMP-3 5A/6A polymorphism directly influences endogenous MMP-3 expression levels and, consequently, cell invasion, in SV-derived SMC cultured from patients with different genotypes. MATERIAL AND METHODS Genotyping of 226 patients revealed -1612 MMP-3 5A/6A genotype frequencies of 20.8% 5A/5A, 52.7% 5A/6A, and 26.5% 6A/6A. Using a standardized, controlled protocol, we investigated cytokine- and growth factor-induced MMP-3 expression (real-time polymerase chain reaction [RT-PCR], ELISA) and SV-SMC invasion (Boyden chamber with Matrigel barrier) using cultured SV-SMC from patients with different MMP-3 genotypes. RESULTS Despite observing a strong correlation between MMP-3 mRNA levels and MMP-3 protein secretion, no significant differences were apparent in MMP-3 expression levels or cell invasion between cells with different MMP-3 5A/6A genotypes. CONCLUSIONS Our data suggest that the MMP-3 5A/6A promoter polymorphism in isolation does not influence levels of MMP-3 secretion or cellular invasion in human SV-SMC.

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.