Anshuman Sengupta

Importance of insulin resistance to vascular repair and regeneration.

Metabolic insulin resistance is apparent across a spectrum of clinical disorders, including obesity and diabetes, and is characterized by an adverse clustering of cardiovascular risk factors related to abnormal cellular responses to insulin. These disorders are becoming increasingly prevalent and represent a major global public health concern because of their association with significant increases in atherosclerosis-related mortality. Endogenous repair mechanisms are thought to retard the development of vascular disease, and a growing evidence base supports the adverse impact of the insulin-resistant phenotype upon indices of vascular repair. Beyond the impact of systemic metabolic changes, emerging data from murine studies also provide support for abnormal insulin signaling at the level of vascular cells in retarding vascular repair. Interrelated pathophysiological factors, including reduced nitric oxide bioavailability, oxidative stress, altered growth factor activity, and abnormal intracellular signaling, are likely to act in conjunction to impede vascular repair while also driving vascular damage. Understanding of these processes is shaping novel therapeutic paradigms that aim to promote vascular repair and regeneration, either by recruiting endogenous mechanisms or by the administration of cell-based therapies.

Socioeconomic deprivation and mode-specific outcomes in patients with chronic heart failure

Objective To characterise the association between socioeconomic deprivation and adverse outcomes in patients with chronic heart failure (CHF). Methods We prospectively observed 1802 patients with CHF and left ventricular ejection fraction (LVEF) ≤45%, recruited in four UK hospitals between 2006 and 2014. We assessed the association between deprivation defined by the UK Index of Multiple Deprivation (IMD) and: mode-specific mortality (mean follow-up 4 years); mode-specific hospitalisation; and the cumulative duration of hospitalisation (after 1 year). Results A 45-point difference in mean IMD score was noted between patients residing in the least and most deprived quintiles of geographical regions. Deprivation was associated with age, sex and comorbidity, but not CHF symptoms, LVEF or prescribed drug therapy. IMD score was associated with the risk of age-sex adjusted all-cause mortality (6% higher risk per 10-unit increase in IMD score; 95% CI 2% to 10%; P=0.004), and non-cardiovascular mortality (9% higher risk per 10-unit increase in IMD score; 95% CI 3% to 16%; P=0.003), but not cardiovascular mortality. All-cause, but not heart failure-specific, hospitalisation was also more common in the most deprived patients. Overall, patients spent a cumulative 3.3 days in hospital during 1 year of follow-up, with IMD score being associated with the age-sex adjusted cumulative duration of hospitalisations (4% increase in duration per 10-unit increase in IMD score; 95% CI 3% to 6%; P<0.0005). Conclusions Socioeconomic deprivation in people with CHF is linked to increased risk of death and hospitalisation due to an excess of non-cardiovascular events.

Performance of 2014 NICE defibrillator implantation guidelines in heart failure risk stratification

Objective Define the real-world performance of recently updated National Institute for Health and Care Excellence guidelines (TA314) on implantable cardioverter-defibrillator (ICD) use in people with chronic heart failure. Methods Multicentre prospective cohort study of 1026 patients with stable chronic heart failure, associated with left ventricular ejection fraction (LVEF) ≤45% recruited in cardiology outpatient departments of four UK hospitals. We assessed the capacity of TA314 to identify patients at increased risk of sudden cardiac death (SCD) or appropriate ICD shock. Results The overall risk of SCD or appropriate ICD shock was 2.1 events per 100 patient-years (95% CI 1.7 to 2.6). Patients meeting TA314 ICD criteria (31.1%) were 2.5-fold (95% CI 1.6 to 3.9) more likely to suffer SCD or appropriate ICD shock; they were also 1.5-fold (95% CI 1.1 to 2.2) more likely to die from non-cardiovascular causes and 1.6-fold (95% CI 1.1 to 2.3) more likely to die from progressive heart failure. Patients with diabetes not meeting TA314 criteria experienced comparable absolute risk of SCD or appropriate ICD shock to patients without diabetes who met TA314 criteria. Patients with ischaemic cardiomyopathy not meeting TA314 criteria experienced comparable absolute risk of SCD or appropriate ICD shock to patients with non-ischaemic cardiomyopathy who met TA314 criteria. Conclusions TA314 can identify patients with reduced LVEF who are at increased relative risk of sudden death. Clinicians should also consider clinical context and the absolute risk of SCD when advising patients about the potential risks and benefits of ICD therapy.

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.

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.

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

Abstract 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.

Role of vascular endothelial insulin sensitisation in vascular repair in systemic insulin resistance

Abstract Background Insulin resistance is an independent risk factor for cardiovascular disease. Murine models with global insulin receptor haploinsufficiency (IRKO) show endothelial dysfunction and impaired vascular repair, potentially related to reduced number and function of endothelial progenitor cells and bioavailability of nitric oxide. We hypothesise that endothelium-specific insulin sensitisation, in the context of global insulin resistance, could reverse these vascular abnormalities. Therefore, we have bred IRKO mice with HIRECO mice, which overexpress a human insulin receptor in endothelial cells under the transcriptional regulation of the Tie-2 promoter-enhancer. Methods Double-transgenic offspring from HIRECO mice crossed with IRKO mice (HIRECOxIRKO) were compared with IRKO mice after confirming genotype with PCR on DNA extracted from ear samples. Metabolic assessment was done with glucose-tolerance and insulin-tolerance tests, and ELISA was used to measure plasma insulin concentrations. Blood pressure (BP) was measured with tail cuff plethysmography. Femoral artery denuding injury was induced with an angioplasty guide-wire; 4 days later, vessels were explanted after perfusion with Evans blue dye to quantify endothelial denudation. Circulating progenitor cells (CPCs, positive for Sca-1/Flk-1) were counted in peripheral venous blood by fluorescence-activated cell sorting. Continuous data were expressed as means (SD) and analysed with paired and unpaired Students t tests as appropriate (significance at p Findings HIRECOxIRKO and IRKO mice had similar growth profiles (weight at 4 months 28·3 g [3·0] vs 27·4 [0·9], p=0·51) and systolic BP (98 mm Hg [12·4] vs 106 [10·4], p=0·28), with comparable insulin sensitivity on tolerance tests. Basal plasma insulin concentrations were also similar (0·70 ng/mL [0·50] vs 0·76 [0·47], p=0·78; n=7), though HIRECOxIRKO did not display the post prandial hyperinsulinaemia seen in IRKO mice previously and in this study. Vascular repair was significantly enhanced in HIRECOxIRKO after femoral artery wire injury (HIRECOxIRKO 56·9% regeneration [11·1] vs IRKO 46·0% [5·1], p=0·048). This repair was not associated with any improvement in numbers of CPCs, which were similar in the two groups of mice (HIRECOxIRKO 90 Sca-1/Flk-1 positive cells [27] vs IRKO 105 [42], p=0·99]. Interpretation In HIRECOxIRKO mice, restoration of vascular repair occurred after endothelial wire injury, which has been previously demonstrated as impaired in IRKO mice. This restoration happened even with comparable glucoregulation and BP, suggesting that the effect is related specifically to endothelial insulin sensitisation despite insulin resistance in traditional target tissues. These data are early and exploratory, and sample size was small and from murine colonies; nonetheless, they seem to support the possibility of vascular endothelial insulin sensitisation as a potential therapeutic target in insulin resistant states. A mechanism remains to be established and we are planning assays of endothelial function, angiogenesis, and downstream insulin molecular signalling. Funding British Heart Foundation.

The investigation and management of broad complex tachycardia and ventricular standstill presenting in pregnancy: A case report

A 23 year old pregnant lady at 35 weeks gestation presented to accident and emergency with worsening dyspnoea, palpitations and dizziness. Twelve lead electrocardiogram, routine bloods and echocardiography were normal. Ambulatory monitoring previously had shown an episode of monomorphic broad complex tachycardia (BCT) and a short episode of ventricular standstill. She was admitted for cardiac monitoring until delivery. Several episodes of ventricular standstill and self-terminating BCT were recorded, which were not associated with symptoms. The patients symptoms either corresponded with sinus rhythm or supraventricular tachycardia. She underwent elective caesarean section at 37 weeks with no complications. The patients symptoms reduced considerably post delivery, and she was discharged three days later. Unfortunately she then had a presyncopal episode whilst holding her baby. Due to concern regarding the safety of her baby she had a permanent pacemaker implanted to allow safe beta-blockade. She remains asymptomatic six months later.

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.

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.