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Supplemental Digital Content is available in the text., Rationale: A hallmark of type 2 diabetes is insulin resistance, which leads to increased endothelial cell (EC) production of superoxide and a simultaneous reduction in the availability of the vasoprotective signaling radical NO. We recently demonstrated in preclinical models that type 2 diabetes simultaneously causes resistance to IGF-1 (insulin-like growth factor-1)–mediated glucose lowering and endothelial NO release. Objective: To examine the effect of insulin and IGF-1 resistance specifically in ECs in vivo. Methods and Results: We generated mice expressing mIGF-1Rs (mouse IGF-1 receptors), which form nonfunctioning hybrid receptors with native IRs (insulin receptors) and IGF-1R, directed to ECs under control of the Tie2 promoter-enhancer. Despite EC insulin and IGF-1 resistance, mIGFREO (mutant IGF-1R EC overexpressing) mice had enhanced insulin and IGF-1–mediated systemic glucose disposal, lower fasting free fatty acids, and triglycerides. In hyperinsulinemic-euglycemic clamp studies, mIGFREO had increased glucose disposal and increased glucose uptake into muscle and fat, in response to insulin. mIGFREO had increased Nox (NADPH oxidase)-4 expression due to reduced expression of the microRNA, miR-25. Consistent with increased Nox4, mIGFREO ECs generated increased hydrogen peroxide (H2O2), with no increase in superoxide. Treatment with catalase—a H2O2 dismutase—restored insulin tolerance to WT (wild type) levels in mIGFREO. Conclusions: Combined insulin and IGF-1 resistance restricted to the endothelium leads to a potentially favorable adaptation in contrast to pure insulin resistance, with increased Nox4-derived H2O2 generation mediating enhanced whole-body insulin sensitivity.

We have previously reported that overexpression of human insulin-like growth factor binding protein (IGFBP)-1 in mice leads to vascular insulin sensitization, increased nitric oxide bioavailability, reduced atherosclerosis, and enhanced vascular repair, and in the setting of obesity improves glucose tolerance. Human studies suggest that low levels of IGFBP-1 are permissive for the development of diabetes and cardiovascular disease. Here we seek to determine whether loss of IGFBP-1 plays a causal role in the predisposition to cardiometabolic disease. Metabolic phenotyping was performed in transgenic mice with homozygous knockout of IGFBP-1. This included glucose, insulin, and insulin-like growth factor I tolerance testing under normal diet and high-fat feeding conditions. Vascular phenotyping was then performed in the same mice using vasomotor aortic ring studies, flow cytometry, vascular wire injury, and angiogenesis assays. These were complemented with vascular phenotyping of IGFBP-1 overexpressing mice. Metabolic phenotype was similar in IGFBP-1 knockout and wild-type mice subjected to obesity. Deletion of IGFBP-1 inhibited endothelial regeneration following injury, suggesting that IGFBP-1 is required for effective vascular repair. Developmental angiogenesis was unaltered by deletion or overexpression of IGFBP-1. Recovery of perfusion following hind limb ischemia was unchanged in mice lacking or overexpressing IGFBP-1; however, overexpression of IGFBP-1 stimulated hindlimb perfusion and angiogenesis in insulin-resistant mice. These findings provide new insights into the role of IGFBP-1 in metabolic and vascular pathophysiology. Irrespective of whether loss of IGFBP-1 plays a causal role in the development of cardiometabolic disorders, increasing IGFBP-1 levels appears effective in promoting neovascularization in response to ischemia.

Transglutaminase activity has been widely implicated in bone deposition. A predominant role has been proposed for factor (F)XIII-A and a subsidiary role suggested for the homologous protein, transglutaminase 2. Full-length FXIII-A is an 83kDa protransglutaminase that is present both in plasma and also in haematopoietic and connective tissue lineages. Several studies have reported expression in murine cells, including osteocytes, of a 37 kDa protein that reacts with the monoclonal anti-FXIII-A antibody AC-1A1. This protein was presumed to be a catalytically active fragment of FXIII-A-83 and to play a major role in bone deposition. We detected a 37 kDa AC-1A1 reactive protein in FXIII-A mRNA negative cell lines and in tissues from FXIII-A(-/-) mice. By mass spectrometric sequencing of AC-1A1 immunoprecipitates, we identified this protein as transaldolase-1, and confirmed that recombinant transaldolase-1 is recognised by AC-1A1. We have also shown that bone deposition is normal in FXIII-A(-/-).TG2(-/-) double knockout mice, casting doubt on the role of transglutaminases in bone mineralisation. Various studies have used antibody AC-1A1 for immunohistochemistry or immunofluorescence. We observe strong FXIII-A dependent staining in paraffin embedded mouse heart sections, with relatively low background in non-expressing mouse cells. In contrast, FXIII-A independent staining predominates in cultured human cells using a standard immunofluorescence procedure. Immunofluorescence is present in membrane compartments that are expected to lack transaldolase, indicating that other off-target antigens are recognised by AC-1A1. This has significant implications for studies that have used this approach to define the subcellular trafficking of FXIII-A in osteocytes.

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.

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.

Objective Neprilysin (NEP), a zinc metallo-endopeptidase, has a role in blood pressure control and lipid metabolism. The present study tested the hypothesis that NEP is associated with insulin resistance and features of the metabolic syndrome (MetS) in a study of 318 healthy human subjects and in murine obesity and investigated NEP production by adipocytes in-vitro. Methods and Results In 318 white European males, plasma NEP was elevated in the MetS and increased progressively with increasing MetS components. Plasma NEP activity correlated with insulin, homeostasis model assessment and body mass index in all subjects (p

Background Transglutaminase 2 (TG2), a cross-linking enzyme that confers supra-molecular structures with extra rigidity and resistance against proteolytic degradation, is expressed in the shoulder regions of human atherosclerotic plaques. It has been proposed that TG2 prevents tearing and promotes plaque repair at these potential weak points, and also promotes ectopic calcification of arteries. TG2 is also expressed within plaques that develop within the brachiocephalic arteries of apolipoprotein E (apoE) deficient mice. Objectives To determine the role that TG2 plays in plaque development and calcification, mice were bred that were doubly deficient in apoE and TG2, and were maintained on a high-fat diet for 6 months. Results Lesion size and composition were not significantly altered in the apoE/TG2 double-knockout mice, with the exception of a 9.7% decrease in the proportion of the plaque occupied by lipid (p = 0.032). The frequency of buried fibrous caps within brachiocephalic plaques was significantly higher in male than in female mice, but TG2 deficiency had no effect on either gender. The extent of lesion calcification varied markedly between individual mice, but it was not decreased in the apoE/TG2 double-knockout mice. Conclusion These data indicate that, in the apoE knockout mouse model of atherosclerosis, TG2 does not influence plaque composition or calcification. The data further suggest that TG2 does not influence plaque stability or repair in these mice.

Factor XIII-A (FXIII-A) is present in the cytosol of platelets, megakaryocytes, monocytes, osteoblasts, and macrophages and may be released from cells by a nonclassical pathway. We observed that plasma FXIII-A levels were unchanged in thrombocytopenic mice (Bcl-xPlt20/Plt20 and Mpl−/−), which implicates nonclassical secretion from nucleated cells as the source of plasma FXIII-A. We, therefore, examined the intracellular targeting of FXIII-A in the THP-1 (monocyte/macrophage) cell line and in human monocyte–derived macrophages. Metabolic labeling of THP-1 cells did not show release of 35S-FXIII-A either under basal conditions or when interleukin 1-β was released in response to cell stress. However, immunofluorescence of THP-1 cells and primary macrophages showed that FXIII-A associated with podosomes and other structures adjacent to the plasma membrane, which also contain trans-Golgi network protein-46 and Golgi matrix protein-130 (GM130) but not the endoplasmic reticulum luminal protein, protein disulphide isomerase. Further, FXIII-A was present in GM130-positive intracellular vesicles that could mediate its transport, and in other contexts GM130 and its binding partner GRASP have been implicated in the delivery of nonclassically secreted proteins to the plasma membrane. Hence, this mechanism may precede FXIII-A release into the extracellular matrix from macrophages and its release into plasma from the cell type of origin.

Fibrin clot cross-linking FXIIIA has a plasma and cellular pool. It is present in platelets, macrophages and connective tissues. However, it is unknown how FXIIIA is secreted and which cell type maintains the plasma pool, although the platelet is considered the main candidate. To address this we have induced lineage specific deletion in mice floxed in coding exon 7 of the FXIIIA gene. Mice homozygous for the floxed gene and transgenic for Pf4cre-recombinase (thrombopoietic deletion), CD11bcre or LysMcre (myeloid deletions) were generated. A FXIIIA KO was obtained using a CMV-driven cre. Plasma and platelet FXIIIA protein & activity and FXIIIA mRNA in brain, heart and aorta were determined and compared to WT, FXIIIA floxed and thrombocytopenic Mpl (thrombopoietin receptor) KO mice. FXIIIA KO and Pf4FXIIIA KO mice lack platelet FXIIIA protein & activity, whereas both are normal in the Mpl KO. pFXIIIA is absent in the FXIIIA KO and decreased in Pf4FXIIIA KO (by 85%), CD11bFXIIIA KO (40%) and LysMFXIIIA KO (60%) whereas in WT, FXIIIA floxed and Mpl KO pFXIIIA was unaltered. Mpl KO platelet size as determined by FACS was also unaltered. These data confirm that platelets are not the source of pFXIIIA. These data also demonstrate that the pFXIIIA secreting cells are Pf4 dependent & Mpl independent: a combination not seen in BM-derived cells. Thus, we are looking for a macrophage-like cell outside of the BM. Resident macrophages in the brain are yolk sac-derived and uniquely, not replaced from BM under normal conditions. In young uninjured brain FXIIIA mRNA levels were knocked down by 96.8% in Pf4FXIIIA KO and by 37.8% in the Mpl KO, confirming, for the first time, the existence of a Pf4 dependent Mpl independent cell from the yolk sac. In heart cell fractions, the smallest Thy1+ fraction (3% of total) contained the largest FXIII-A mRNA pool, which decreases in Pf4cre & CD11bcre-Flox mice, further supporting involvement of yolk sac-derived macrophages. Further study of FXIIIA mRNA expression in Pf4 dependent, Mpl independent cells in the cardiovascular system demonstrated that the pFXIIIA profile was most closely mirrored by the reduction in arterial wall FXIIIA mRNA suggesting that yolk sac derived arterial wall resident macrophages are the cellular source of FXIIIA.

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.

Formation of a stable fibrin clot is dependent on interactions between factor XIII and fibrin. We have previously identified a key residue on the αC of fibrin(ogen) (Glu396) involved in binding activated factor XIII-A(2) (FXIII-A(2)*); however, the functional role of this interaction and binding site(s) on FXIII-A(2)* remains unknown. Here we (1) characterized the functional implications of this interaction; (2) identified by liquid-chromatography-tandem mass spectrometry the interacting residues on FXIII-A(2)* following chemical cross-linking of fibrin(ogen) αC389-402 peptides to FXIII-A(2)*; and (3) carried out molecular modeling of the FXIII-A(2)*/peptide complex to identify contact site(s) involved. Results demonstrated that inhibition of the FXIII-A(2)*/αC interaction using αC389-402 peptide (Pep1) significantly decreased incorporation of biotinamido-pentylamine and α2-antiplasmin to fibrin, and fibrin cross-linking, in contrast to Pep1-E396A and scrambled peptide controls. Pep1 did not inhibit transglutaminase-2 activity, and incorporation of biotinyl-TVQQEL to fibrin was only weakly inhibited. Molecular modeling predicted that Pep1 binds the activation peptide cleft (AP-cleft) within the β-sandwich domain of FXIII-A(2)* localizing αC cross-linking Q366 to the FXIII-A(2)* active site. Our findings demonstrate that binding of fibrin αC389-402 to the AP-cleft is fundamental to clot stabilization and presents this region of FXIII-A(2)* as a potential site involved in glutamine-donor substrate recognition.

FXIII (Factor XIII) is a Ca2+-dependent enzyme which forms covalent ϵ-(γ-glutamyl)lysine cross-links between the γ-carboxy-amine group of a glutamine residue and the ϵ-amino group of a lysine residue. FXIII was originally identified as a protein involved in fibrin clot stabilization; however, additional extracellular and intracellular roles for FXIII have been identified which influence thrombus resolution and tissue repair. The present review discusses the substrates of FXIIIa (activated FXIII) involved in thrombosis and wound healing with a particular focus on: (i) the influence of plasma FXIIIa on the formation of stable fibrin clots able to withstand mechanical and enzymatic breakdown through fibrin–fibrin cross-linking and cross-linking of fibrinolysis inhibitors, in particular α2-antiplasmin; (ii) the role of intracellular FXIIIa in clot retraction through cross-linking of platelet cytoskeleton proteins, including actin, myosin, filamin and vinculin; (iii) the role of intracellular FXIIIa in cross-linking the cytoplasmic tails of monocyte AT1Rs (angiotensin type 1 receptors) and potential effects on the development of atherosclerosis; and (iv) the role of FXIIIa on matrix deposition and tissue repair, including cross-linking of extracellular matrix proteins, such as fibronectin, collagen and von Willebrand factor, and the effects on matrix deposition and cell–matrix interactions. The review highlights the central role of FXIIIa in the regulation of thrombus stability, thrombus regulation, cell–matrix interactions and wound healing, which is supported by observations in FXIII-deficient humans and animals.

Fibrinogen αC residues 242-424 have been shown to have a major regulatory role in the activation of factor XIII-A2B2 (FXIII-A2B2); however, the interactions underpinning this enhancing effect have not been determined. Here, we have characterized the binding of recombinant (r)FXIII-A subunit and FXIII-A2B2 with fibrin(ogen) and fibrin αC residues 233-425. Using recombinant truncations of the fibrin αC region 233-425 and surface plasmon resonance, we found that activated rFXIII-A bound αC 233-425 (Kd of 2.35 ± 0.09μM) which was further localized to αC 389-403. Site-directed mutagenesis of this region highlighted Glu396 as a key residue for binding of activated rFXIII-A. The interaction was specific for activated rFXIII-A and depended on the calcium-induced conformational change known to occur in rFXIII-A during activation. Furthermore, nonactivated FXIII-A2B2, thrombin-cleaved FXIII-A2B2, and activated FXIII-A2B2 each bound fibrin(ogen) and specifically αC region 371-425 with high affinity (Kd < 35nM and Kd < 31nM, respectively), showing for the first time the potential involvement of the αC region in binding to FXIII-A2B2. These results suggest that in addition to fibrinogen γ′ chain binding, the fibrin αC region also provides a platform for the binding of FXIII-A2B2 and FXIII-A subunit.

In yeast and higher plants, separate genes encode the cytosolic and mitochondrial forms of glyoxalase II. In contrast, although glyoxalase II activity has been detected both in the cytosol and mitochondria of mammals, only a single gene encoding glyoxalase II has been identified. Previously it was thought that this gene (the hydroxyacylglutathione hydrolase gene), comprised 8 exons that are transcribed into mRNA and that the resulting mRNA species encoded a single cytosolic form of glyoxalase II. Here we show that this gene gives rise to two distinct mRNA species transcribed from 9 and 10 exons, respectively. The 9-exon-derived transcript encodes two protein species: mitochondrially targeted glyoxylase II, which is initiated from an AUG codon in a previously uncharacterized part of the mRNA sequence, and cytosolic glyoxalase II, which is initiated by internal ribosome entry at a downstream AUG codon. The transcript deriving from 10 exons has an in-frame termination codon between the two initiating AUG codons and hence only encodes the cytosolic form of the protein. Confocal fluorescence microscopy indicates that the mitochondrially targeted form of glyoxalase II is directed to the mitochondrial matrix. Analysis of glyoxalase II mRNA sequences from a number of species indicates that dual initiation from alternative AUG codons is conserved throughout vertebrates.

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 5 β 1 and α V β 3 integrins and enhanced the ability of EC to adhere and regenerate denuded human vein ex vivo (P in vitro by the pro-inflammatory cytokine tumour necrosis factor-alpha (TNF-α) which significantly inhibited EC migration (P 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.

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 (α 5 β 1 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 α 5 β 1 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.