Mark T. Kearney

A Endothelial cell nitric oxide bioavailability and insulin sensitivity are regulated by IGF-1 and insulin receptor levels

Background In a similar manner to insulin, the growth promoting hormone Insulin-like Growth Factor-1 (IGF-1), may be an important regulator of endothelial nitric oxide (NO) bioavailability. We have previously reported evidence of increased basal NO production in the vasculature in two murine models of reduced IGF-1 receptor (global hemizygous knockout (IGFRKO) and endothelial cell specific IGF-1R knockout (ECIGFRKO)). Augmentation of this increase in NO is relative to progressive decrease in IGF-1R number (WT vs ECIGFRKO hemizygotes p=0.01, WT vs ECIGFRKO homozygotes p=0.001). Furthermore, by decreasing IGF-1R numbers in the insulin resistant hemizygous insulin receptor knockout (IRKO) model (IRKO × IGFRKO) we have shown insulin sensitivity in the vasculature can be restored. In this study, we have investigated further these receptor interactions with the generation of a mouse overexpressing the human IGF-1R specifically in the endothelium under control of the Tie-2 promoter-enhancer (hIGFREO), and by targeted knockdown of the IGF-1R in human umbilical vein endothelial cells (HUVECs). Methods Metabolic function was assessed in mice by tolerance tests using whole-blood micro-sampling after insulin or glucose intraperitoneal injection. Cardiovascular function was assessed by thoracic aortic vasomotion ex vivo in the organbath. Complimentary in vitro studies were conducted by siRNA mediated downregulation of the IGF-1 receptor in HUVECs with and wihout insulin stimulation. Nitric oxide synthase activity was measured using an assay measuring conversion of [14C]-L-arginine to [14 C]-L-citrulline. Results Glucose and insulin tolerance testing showed no difference between hIGFREO mice and wild-type (WT) littermates. Murine thoracic aorta from hIGFREO mice were hypercontractile to phenylepherine (PE) compared to WT (Emax hIGFREO=0.91±0.045u2005g; WT=0.62±0.045u2005g, p=0.0036) with decreased response to LNMMA (Emax hIGFREO=47.70±9.87u2005g; WT=106.1±30.10u2005g, p=0.048). These data indicate reduced endothelial NO bioavailability in hIGFREO mice compared to WT. HUVECs transfected with IGF1R-siRNA showed increased basal and insulin mediated eNOS phosphorylation in the presence of insulin (Ins: 164±4.9% vs siRNA+Ins: 192±0.7%, p<0.05). eNOS activity (L-arginine, L-citrulline assay) was enhanced upon transfection with IGF1R-siRNA (Scrambled siRNA: 95.7±13.7% vs IGF1R-siRNA: 188.7±48.3%, p<0.05). Implications These data demonstrate that increasing numbers of IGF-1R specifically in murine endothelium leads to reduced NO bioavailability. Complementary siRNA studies confirm results of previous murine studies that reducing IGF-1R numbers enhance NO bioavailability. Therefore this raises the intriguing possibility that manipulation of IGF-1R numbers may represent a novel therapeutic strategy by which to modify vascular NO bioavailability and endothelial cell insulin sensitivity.Abstract A Figure 1Abstract A Figure 2Abstract A Figure 3Abstract A Figure 4Abstract A Figure 5Abstract A Figure 6

Effects of Ivabradine on Hemodynamic and Functional Parameters in Left Ventricular Systolic Dysfunction: a Systematic Review and Meta-analysis

BackgroundIvabradine is licensed as add-on therapy in patients with severe left ventricular systolic dysfunction (LVSD), normal sinus rhythm, and suboptimal heart rate (HR) control, but effects are not fully established. This study sought to assess the impact of ivabradine therapy on hemodynamic and functional outcome measures in all patients with LVSD.MethodsMEDLINE (1996–2017), Embase (1996–2017), Cochrane Central Register of Controlled Trials (CENTRAL), Cochrane Database of Systematic Reviews, ClinicalTrials.gov, and ISI Web of Science were searched for randomized clinical trials (RCTs) comparing standard medical therapy (SMT) plus ivabradine to SMT alone for patients with LVSD of any severity. Each trial was assessed using the Cochrane Collaborations Risk of Bias tool.ResultsEight RCTs with 17,823 patients were included. Add-on use of ivabradine reduced resting HR (mean difference [MD] 10.3xa0bpm; pu2009<u20090.001), improved ejection fraction (EF) (MD 3.6%, pu2009<u20090.001), and preserved systolic blood pressure (MD 3.4xa0mmHg; pu2009=u20090.09). Stratified analyses according to severity of LVSD did not influence conferred benefits on HR and EF. Small improvements were noted in exercise tolerance (standardized MD 5.9xa0s; pu2009=u20090.004) and peak oxygen consumption (MD 2.9xa0ml/kg/min; pu2009=u20090.02).DiscussionAdjunct therapy with ivabradine in patients with LVSD results in a favorable hemodynamic profile and correlates with improved functional capacity. Benefits appear to be broadly preserved irrespective of baseline EF. This was a meta-analysis of RCTs, though limited by exclusion of post hoc analyses, lack of access to patient level data, and inter-study variability in some baseline characteristics. Further, large-scale RCTs are warranted to evaluate effectiveness of ivabradine in cohorts with non-severe LVSD.

56 The Influence of Chronotropic Incompetence on Exercise Capacity in Chronic Heart Failure

Introduction Increased cardiac output during exercise is the result of greater stroke volume and an increase in heart rate. It is therefore assumed that a limitation to heart rate rise (HRR) in response to activity, chronotropic incompetence (CI), could contribute to the exercise intolerance that is characteristic of chronic heart failure (CHF). However, HRR is closely related to workload and subjects with impaired exercise capacity have a lower peak heart rate (PHR) and a lower HRR. Hence, whether a limited increase in heart rate (HR) during exercise in patients with CHF is aetiological in their exercise limitation or merely a bystander remains unproven. The aim of this study was to examine the effects of correcting CI in patients with CHF on peak and submaximal exercise capacity. Methods We enrolled 50 subjects with CHF due to left ventricular systolic dysfunction (LVSD), (left ventricular ejection fraction <45%, and symptoms of breathlessness or fatigue), on optimal medical therapy with cardiac pacing devices, into a randomised double blind cross-over study of rate adaptive versus fixed rate pacing. At baseline, each participant underwent a full echocardiographic assessment, and performed a peak, symptom-limited treadmill-based familiarisation cardiopulmonary exercise test with breath-by-breath metabolic gas analysis. After at least one week, subjects were invited back for two further exercise tests (at least one week apart) immediately prior to which, the pacemaker was either programmed to rate-adaptive or fixed rate pacing, with the maximum paced HR determined using the age-predicted peak HR equation (220-age). Results Rate-adaptive pacing led to higher mean (SE) peak HR (128 (21) vs 107 (28) bpm; p < 0.0001, Figure 1) but no difference in mean peak oxygen consumption (16.6(4.7) v 15.9(4.5) ml/kg/min, p = 0.24), anaerobic threshold (11.7 (2.7) vs 11.3 (2.6) ml/kg/min; p = 0.24), exercise time (470 (239) v 451 (222)s; p = 0.33, Figure 2), stage-by-stage HR or perceived exertion levels. Abstract 56 Figure 1 Rate-adaptive (RR on) versus fixed-rate (RR off) pacing in CHF patients showing increased heart rates (HR) at submaximal and peak exercise Abstract 56 Figure 2 Rate-adaptive (RR on) versus fixed-rate (RR off) pacing in CHF patients showing no benefit on oxygen consumption with increased heart rate Conclusions Exercise intolerance due to breathlessness and fatigue is a cardinal feature of CHF and remains a problem for many patients despite optimal medical and device therapy. Agents that lower HR and induce CI are proven to improve outcomes for patients with CHF due to LVSD yet traditional models of heart failure suggest that a lower cardiac output, potentially the consequence of CI, reduces blood supply to exercising muscles thereby limiting work. By demonstrating that higher HR throughout exercise is not associated with greater exercise capacity in CHF patients, we propose that CI does not play a major role in exercise intolerance in CHF.

104 Enhancing endothelial insulin sensitivity reduces nitric oxide bioavailability: a role for NADPH oxidase-derived reactive oxygen species

Introduction Insulin resistance at a whole body level and in the endothelium precedes type 2 diabetes. Insulin resistance at the level of the endothelium reduces bioavailability of the anti-atherosclerotic signalling radical nitric oxide (NO). We explored the effects of increasing insulin signalling in the endothelium, using novel transgenic mice, over-expressing Type A human Insulin Receptor (HIRECO) in the endothelium, driven by the Tie-2 promoter-enhancer. Methods Semi-quantitative RT-PCT was carried out on various tissues and isolated endothelial cells from lungs to confirm significant levels of human insulin receptor mRNA while the protein expression was confirmed by western blotting on aortic sections or endothelial cells. Lucigenin-enhanced chemiluminescence was exploited to measure superoxide anion levels while; vasomotor functions were assessed in thoracic aortic rings mounted in organ baths. Results No significant changes in morphological features, metabolic phenotypes or blood pressure regulation were observed between the HIRECO and wild type (WT) littermates. However, plasma insulin levels were similar following an overnight fast, but were decreased in the HIRECO after glucose challenge. HIRECO mice demonstrated significant endothelial dysfunction measured by a blunted response to acetylcholine (Emax, WT vs HIRECO: 84±3% vs 68±3% respectively; n=5, p>0.05). Endothelium-independent response to sodium nitroprusside remained unchanged. The impaired aortic response to acetylcholine was normalised by the specific NADPH oxidase inhibitor peptide, gp91ds-tat, (Emax: 93±5%; n=6, p<0.05), as well as the superoxide dismutase mimetic, Mn(III) tetrakis (1-methyl-4-pyrydil) porphyrin pentachloride. Isolated aortic rings of HIRECO exhibited a hypercontractile response to phenylephrine compared to wild type mice (log EC50, WT vs HIRECO: 6.96±0.03 vs 7.24±0.08, n=6, p<0.01). Indeed, HIRECO mice elicited 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 phosphorylation levels in isolated endothelial cells of HIRECO mice was enhanced 1.56-fold compared to WT littermates. Conclusions/Implications These data demonstrate enhanced oxidative stress in a novel murine model of increased insulin signalling in the endothelium, leading to reduced bioavailability of nitric oxide and atherosclerosis. These data also demonstrate for the first time, that increased insulin sensitivity in the endothelium, increases the generation of superoxide anion and reduces NO bioavailability.

109 Overexpression of endothelial insulin-like growth factor-1 receptors (IGF-1R): a novel role for IGF-1R in endothelial function and repair

Introduction Endothelium-derived nitric oxide (NO) is a critical regulator of vascular homeostasis, repair and regeneration. We recently demonstrated that reducing insulin-like growth factor-1 receptor (IGF-1R) numbers in the endothelium, thereby decreasing the proportion of insulin resistant hybrid receptors, enhances NO bioavailability and increases endothelial cell (EC) insulin sensitivity (Abbas*, Imrie*, Viswambharan* et al. Diabetes 2011;60:2169–78). Methods To examine the effect of increasing IGF-1R in EC we generated transgenic mice overexpressing the human IGF-1R in EC (human IGF-1R endothelium overexpressing mice (hIGFREO)). Glucose and insulin sensitivity were measured by tolerance testing and plasma insulin and IGF-1 levels were analysed by ELISA. The response of aortic rings to increasing doses of phenylepherine (PE), with and without L-NMMA (a NO inhibitor), were measured ex vivo in an organ bath. NO release, eNOS activity and phosphorylation of eNOS (all in response to insulin) were measured by DAF fluorescence, the conversion of L-arginine to L-citrulline, and western blotting respectively. Endothelial regeneration was investigated by guide-wire injury of the femoral artery with quantification of Evans-blue dyed denuded area and migration assays were performed in response to VEGF by H&E staining. Results Increased endothelial IGF-1R numbers had no effect on glucose tolerance or insulin sensitivity in hIGFREO mice compared to wild-type (wt) littermates and fasting plasma glucose, insulin and IGF-1 levels were similar. Aortae from hIGFREO mice were hypercontractile to phenylephrine (PE) (Emax wt =0.62±0.045 vs hIGFREO =0.91±0.045, p=0.036) and had blunted constrictor responses to LNMMA (Emax wt =106.1±30.10 vs hIGFREO =47.7±9.87, p=0.048) indicating reduced basal NO bioavailability. In response to insulin EC from hIGFREO had: reduced NO release (wt =4500±1000 vs hIGFREO =1500±700, p<0.05); reduced eNOS activation (wt =170%±25 vs hIGFREO 58%±3, p<0.04); and decreased phosphorylation of eNOS (p=0.027). After endothelium denuding arterial injury hIGFREO mice demonstrated accelerated endothelium regeneration (recovered area: wt =40.27%±5.7 vs hIGFREO =57.25%±2.3, p=0.003) and enhanced endothelial cell migration under control conditions and in response to VEGF (p<0.001). Conclusions Manipulation of IGF1-receptor numbers may represent a novel strategy for altering insulin sensitivity and vascular NO production. This data demonstrates uncoupling of endothelial NO bioavailability and vascular repair.

D Endothelium specific insulin resistance leads to accelerated atherosclerosis: a role for reactive oxygen species

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. We addressed this question by crossing Endothelial Specific Mutant Insulin Receptor Over-expressing (ESMIRO) mice with ApoE−/− mice. ESMIRO mice over-express a human insulin receptor with an 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 and showed normal development with no differences 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. ApoE−/−/ESMIRO cultured endothelial cells demonstrated insulin resistance through significantly reduced insulin mediated eNOS activity (p=0.003). Aortic lipid deposition along the whole aorta, 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). Analysis of lipid deposition along the lesser curvature of the aortic arch revealed a significant increase in ApoE−/−ESMIRO when compared to controls (9.4±0.89 vs 12.43±1.19% p=0.035). Atherosclerotic lesion area in cross sections of aortic sinus was also significantly increased in ApoE−/−/ESMIRO mice compared to ApoE−/− controls (24.8%±2.4% vs 16.6%±2.4%; p=0.02). Vascular function assessed through relaxation responses of aortic rings in response to the endothelial specific vasodilator acetylcholine revealed that aortic rings from ApoE−/−/ESMIRO mice had blunted relaxation responses to acetylcholine (Emax ApoE−/− 102.88±6, Emax ApoE−/−/ESMIRO 65±41%, p=0.02), which was restored by the superoxide dismutase mimetic and antioxidant MnTMPyP (Emax ApoE−/−/ESMIRO without MnTMPyP 65±41%, with MnTMPyP 112±15% p=0.048). Endothelial cells from ApoE−/−/ESMIRO mice had significantly increased basal generation of superoxide (1.87-fold increase compared to ApoE−/− p<0.05) which was blunted by the selective NADPH oxidase inhibitor gp91ds-tat (11% reduction ±0.02, p=0.03) and the non-selective NO synthase inhibitor L-NMMA (6% reduction ±0.01, p=0.03). Conclusions Endothelial specific insulin resistance is sufficient to promote atherosclerosis and increase lesion area in ApoE null mice potentially via the increased ROS displayed in this model. This suggests that enhancing endothelial insulin sensitivity may be an appropriate target to prevent atherosclerosis in insulin-resistant conditions.

C Insulin resistance impairs angiogenic progenitor cell function and delays endothelial repair following vascular injury

Introduction Insulin-resistance, the primary metabolic abnormality underpinning type-2-diabetes mellitus (T2DM) and obesity, is an important risk factor for the development of atherosclerotic cardiovascular disease. Circulating-angiogenic-progenitor-cells (APCs) participate in endothelial-repair following arterial injury. Type-2 diabetes is associated with fewer circulating APCs, APC dysfunction and impaired endothelial-repair. We set out to determine whether insulin-resistance per se adversely affects APCs and endothelial-regeneration. Research Design and Methods We quantified APCs and assessed APC-mobilisation and function in mice hemizygous for knockout of the insulin receptor (IRKO) and wild-type (WT) littermate controls. Endothelial-regeneration following femoral artery wire-injury was also quantified at time intervals after denudation and following APC transfusion. Results The metabolic phenotype of IRKO mice was consistent with compensated insulin resistance, with hyperinsulinaemia after a glucose challenge but a normal blood glucose response to a glucose tolerance test. IRKO mice had fewer circulating Sca-1+/Flk-1+ APCs than WT mice at baseline. Culture of mononuclear-cells demonstrated that IRKO mice had fewer APCs in peripheral-blood, but not in bone-marrow or spleen, suggestive of a mobilisation defect. Defective VEGF-stimulated APC mobilisation was confirmed in IRKO mice, consistent with reduced eNOS expression in bone marrow and impaired vascular eNOS activity. Paracrine-angiogenic-activity of APCs from IRKO mice was impaired compared to those from WT animals. Endothelial-regeneration of the femoral artery following denuding wire-injury was delayed in IRKO mice compared to WT (re-endothelialised area 35.8±4.8% vs 66.6±5.2% at day 5 following injury and 35.6±4.8% vs 59.8±6.6% at day 7; P<0.05) (Abstract C Figure 1A). Transfusion of mononuclear-cells from WT mice normalised the impaired endothelial-regeneration in IRKO mice (57±4% vs 25±5%; p<0.002). Transfusion of c-kit+ bone-marrow cells from WT mice also restored endothelial-regeneration in IRKO mice (62±2% vs 25±5%; p<0.002). However, transfusion of c-kit+ cells from IRKO mice was less effective at improving endothelial-repair (62±2% vs 45±4%; p<0.02) (Abstract C Figure 1B).Abstract C Figure 1 (A) Time-dependent endothelial regeneration following vascular injury (n=5 mice per group; *denotes p<0.05). (B) Effects on endothelial regeneration 5 days after wire-injury of transfusion of spleen-derived MNCs or BM-derived c-kit (CD117)+ve cells from WT or IRKO mice (n=4 mice per group). Conclusions Insulin-resistance impairs APC function and delays endothelial-regeneration following arterial injury. These findings support the hypothesis that insulin-resistance per se is sufficient to jeopardise endogenous vascular repair. Defective endothelial-repair may be normalised by transfusion of APCs from insulin-sensitive animals but not from insulin-resistant animals. These data may have important implications for the development of therapeutic strategies for insulin-resistance associated cardiovascular disease.