Natalie Haywood

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.

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 β-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.

Hypertrophic cardiomyopathy mutations in the calponin-homology domain of ACTN2 affect actin binding and cardiomyocyte Z-disc incorporation

We have discovered that two mutations at the actin binding domain (ABD) of α-actinin-2 (ACTN2), which cause hypertrophic cardiomyopathy (HCM), have minor effects on its structure and ability to bind actin and integrate into Z-discs, providing a potential disease mechanism.

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.

Preservation of vascular endothelial repair in mice with diet-induced obesity: Vascular repair in mice with diet-induced obesity

Preservation of structural integrity of the endothelial monolayer and maintenance of endothelial cell function are of critical importance in preventing arterial thrombosis, restenosis and atherosclerosis. Obesity has been intimately linked with endothelial dysfunction, and reports of reduced abundance and functional impairment of circulating progenitor cells in obesity have led to the suggestion that defective endothelial repair contributes to obesity‐related cardiovascular disease.

The Insulin like growth factor and binding protein family: novel therapeutic targets in obesity & diabetes

Background Recent changes in nutrition and lifestyle have provoked an unprecedented increase in the prevalence of obesity and metabolic disorders. Recognition of the adverse effects on health has prompted intense efforts to understand the molecular determinants of insulin sensitivity and dysglycemia. In many respects, actions of insulin-like growth factors (IGFs) mirror those of insulin in metabolic regulation. Unlike insulin, however, the bioactivity of IGFs is regulated by a family of seven high-affinity binding proteins (IGFBPs) which confer temporospatial modulation with implications for metabolic homeostasis. In addition, evidence is accumulating that IGF-independent actions of certain of the IGFBPs can directly modulate insulin sensitivity. Scope of review In this review, we discuss the experimental data indicating a critical role for IGF/IGFBP axis in metabolic regulation. We highlight key discoveries through which IGFBPs have emerged as biomarkers or putative therapeutic targets in obesity and diabetes. Major conclusions Growing evidence suggests that several components of the IGF-IGFBP system could be explored for therapeutic potential in metabolic disorders. Both IGFBP-1 and IGFBP-2 have been favorably linked with insulin sensitivity in humans and preclinical data implicate direct involvement in the molecular regulation of insulin signaling and adiposity respectively. Further studies are warranted to evaluate clinical translation of these findings.

P4 Insulin-like growth factor binding protein-2 (IGFBP-2) and its role in angiogenesis

Ischaemic heart disease (IHD) presents a significant burden on global morbidity, healthcare costs and patient wellbeing, therefore novel therapeutic methods for IHD are urgently needed. A family of proteins demonstrating potential in therapeutic angiogenesis is the insulin like growth factor binding proteins (IGFBPs). The family of IGFBPs (IGFBP-1-7) modulate IGF bioactivity and bioavailability. Several members of the IGFBP family exert IGF-independent cellular effects and are signalling molecules implicated in many physiological processes, including angiogenesis, which may be exploited therapeutically. IGFBP-2 possesses IGF binding domains, an Arg-Gly-Asp integrin-recognition motif and heparin-binding domains (HBDs), therefore promoting high affinity IGF binding, integrin recruitment and glycosaminoglycan interactions (via the HBD). IGFBP-2 possesses a nuclear localisation sequence, enabling its entry into the nucleus. The effect of IGFBP-2 on angiogenic signalling pathways was investigated using Human Umbilical Vein Endothelial Cells (HUVECs). In vitro angiogenic assays were employed to study the angiogenic properties of IGFBP-2 in HUVECs. HUVECs possessed highest endogenous expression levels of IGFBP-2 across a range of different cell types. IGFBP-2 stimulated HUVECs exhibited dose- and time-dependent enhancement in AKT activation. Maximum Akt phosphorylation was achieved with 200 ng/ml IGFBP-2. HUVECs stimulated with IGFBP-2 at different time intervals demonstrated immediate Akt activation at 15 minutes. Addition of IGFBP-2 to HUVECs significantly enhanced cell adhesion, migration and tube formation. Future work involves investigating the mechanisms involved within the role of IGFBP-2 in angiogenesis by exploring each domain individually. In vivo studies are planned to confirm in vitro findings, supporting IGFBP-2 as a potential novel therapeutic for angiogenesis.

197 Alpha-5 Beta-1 Integrin: a Promising Therapeutic Target in the Field of Insulin Resistance and Cardiovascular Disease

Obesity is a key factor in the development of insulin resistance. Resistance to the vascular effects of insulin plays a central role in the initiation and progression of cardiovascular disease. Developing novel therapeutic strategies to prevent and treat insulin resistance is therefore important. IGFBP1 (Insulin like growth factor binding protein 1) is a 30kDa protein, derived mainly from the liver. We have previously shown that in vivo over expression of IGFBP1 improves insulin sensitivity, promotes nitric oxide production, lowers blood pressure and protects against atherosclerosis. IGFBP1 can impact on cellular functions via an RGD (α5β1 integrin binding) motif independent of IGF binding. However, whether the integrin binding motif of IGFBP1 could be exploited therapeutically remained unexplored. The cell line C2C12, an insulin responsive skeletal muscle cell line, was used to investigate the effects with acute treatment of GRGDTP synthetic hexapeptide (which binds α5β1 integrin) on the insulin signalling pathway, through immunoblotting of key insulin signalling proteins – IR (Insulin receptor), IRS1 (Insulin receptor substrate 1) and AKT (Protein kinase B). To investigate the possible role of integrin activation, the effects of acute treatment of GRGDTP on FAK (Focal adhesion kinase) phosphorylation were also investigated. C2C12 cells were also used in a glucose uptake assay, to determine the effects of GRGDTP on insulin stimulated glucose uptake. To examine whether the RGD motif of IGFBP1 could be exploited therapeutically synthetic hexapeptide GRGDTP was administered to C57BL/6 mice with diet-induced obesity. Treatment with GRGDTP prior to insulin stimulation of C2C12 cells enhances FAK, IRS1 and AKT phosphorylation (P ≤ 0.05). Treatment with GRGDTP prior to insulin stimulation of C2C12 cells increased insulin stimulated glucose uptake (P ≤ 0.01). Acute administration of GRGDTP (40 μg IP) significantly improved glucose tolerance (P ≤ 0.05) and insulin sensitivity in C57BL/6 mice with diet-induced obesity (P ≤ 0.05). For the first time, we have shown that the integrin binding domain of IGFBP1 may play an important role in insulin sensitivity and glucose regulation and represents a promising therapeutic agent in the field of insulin resistance and cardiovascular disease.

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.