Affimers and nanobodies as molecular probes and their applications in imaging.

Antibodies are the most widely used, traditional tool for labelling molecules in cells. In the past five to ten years, many new labelling tools have been developed with significant advantages over the traditional antibody. Here, we focus on nanobodies and the non-antibody binding scaffold proteins called Affimers. We explain how they are generated, selected and produced, and we describe how their small size, high binding affinity and specificity provides them with many advantages compared to antibodies. Of particular importance, their small size enables them to better penetrate dense cytoskeletal regions within cells, as well as tissues, providing them with specific advantage for super-resolution imaging, as they place the fluorophore with a few nanometres of the target protein being imaged. We expect these novel tools to be of broad interest to many cell biologists and anticipate them becoming the tools of choice for super-resolution imaging.

Novel Paracrine Action of Endothelium Enhances Glucose Uptake in Muscle and Fat.

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IGFBP-1 in Cardiometabolic Pathophysiology-Insights From Loss-of-Function and Gain-of-Function Studies in Male Mice.

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.

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.

Cre/lox Studies Identify Resident Macrophages as the Major Source of Circulating Coagulation Factor XIII-A.

OBJECTIVE: To establish the cellular source of plasma factor (F)XIII-A. APPROACH AND RESULTS: A novel mouse floxed for the F13a1 gene, FXIII-Aflox/flox (Flox), was crossed with myeloid- and platelet-cre-expressing mice, and cellular FXIII-A mRNA expression and plasma and platelet FXIII-A levels were measured. The platelet factor 4-cre.Flox cross abolished platelet FXIII-A and reduced plasma FXIII-A to 23±3% (P<0.001). However, the effect of platelet factor 4-cre on plasma FXIII-A was exerted outside of the megakaryocyte lineage because plasma FXIII-A was not reduced in the Mpl-/- mouse, despite marked thrombocytopenia. In support of this, platelet factor 4-cre depleted FXIII-A mRNA in brain, aorta, and heart of floxed mice, where FXIII-Apos cells were identified as macrophages as they costained with CD163. In the integrin αM-cre.Flox and the double copy lysozyme 2-cre.cre.Flox crosses, plasma FXIII-A was reduced to, respectively, 75±5% (P=0.003) and 30±7% (P<0.001), with no change in FXIII-A content per platelet, further consistent with a macrophage origin of plasma FXIII-A. The change in plasma FXIII-A levels across the various mouse genotypes mirrored the change in FXIII-A mRNA expression in aorta. Bone marrow transplantation of FXIII-A+/+ bone marrow into FXIII-A-/- mice both restored plasma FXIII-A to normal levels and replaced aortic and cardiac FXIII-A mRNA, while its transplantation into FXIII-A+/+ mice did not increase plasma FXIII-A levels, suggesting that a limited population of niches exists that support FXIII-A-releasing cells. CONCLUSIONS: This work suggests that resident macrophages maintain plasma FXIII-A and exclude the platelet lineage as a major contributor.

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 ß-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.

Normal Bone Deposition Occurs in Mice Deficient in Factor XIII-A and Transglutaminase 2.

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.

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 activation peptide cleft exposed by thrombin cleavage of FXIII-A(2) contains a recognition site for the fibrinogen α chain.

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 ß-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.

Substrates of Factor XIII-A: roles in thrombosis and wound healing.

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.

Interactions between factor XIII and the alphaC region of fibrinogen.

Fibrinogen αC residues 242-424 have been shown to have a major regulatory role in the activation of factor XIII-A(2)B(2) (FXIII-A(2)B(2)); 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-A(2)B(2) 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 (K(d) of 2.35 ± 0.09 µM) 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-A(2)B(2), thrombin-cleaved FXIII-A(2)B(2), and activated FXIII-A(2)B(2) each bound fibrin(ogen) and specifically αC region 371-425 with high affinity (K(d) < 35 nM and K(d) < 31 nM, respectively), showing for the first time the potential involvement of the αC region in binding to FXIII-A(2)B(2). These results suggest that in addition to fibrinogen γ' chain binding, the fibrin αC region also provides a platform for the binding of FXIII-A(2)B(2) and FXIII-A subunit.

Association of coagulation factor XIII-A with Golgi proteins within monocyte-macrophages: implications for subcellular trafficking and secretion.

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-x(Plt20/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 (35)S-FXIII-A either under basal conditions or when interleukin 1-beta 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.

Effect of transglutaminase 2 (TG2) deficiency on atherosclerotic plaque stability in the apolipoprotein E deficient mouse.

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.

The Human hydroxyacylglutathione hydrolase (HAGH) gene encodes both cytosolic and mitochondrial forms of glyoxalase II.

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.