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Adhesive properties of endothelial cells are influenced by the thioldisulfide balance. However, the molecular mechanism of this effect is unclear, although recent observations indicate that integrin receptors may be direct targets for redox modulation. The purpose of this study was to examine whether protein disulfide isomerase (PDI) is directly involved in this process. As manganese ions are known to affect the thioldisulfide balance and activate integrins to maximal affinity, we searched for PDI interactions with integrins, particularly with αVβ3, in Mn2+‐treated endothelial cells. By employing confocal microscopy, flow cytometry and coimmunoprecipitation experiments, we showed that exposure of endothelial cells to Mn2+ resulted in: (a) the appearance of surface protein thiol groups, which can be found in PDI and αVβ3, and both proteins colocalizing on the cellular surface; and (b) the formation of the PDI–αVβ3 complex, which dissociates upon reduction. In addition, PDI in a complex with αVβ3 induces conversion of the integrin to the ligand‐competent high‐affinity state, as evidenced by increased binding of vitronectin. The membrane‐impermeable sulfhydryl blockers 3‐N‐maleimidylpropionyl biocytin 3‐N‐maleimidylpropionyl biocytin and p‐chloromercuriphenyl sulfonate, as well as the PDI inhibitors bacitracin, MA3 018, and MA3 019, abolished the binding of vitronectin and LM609 to endothelial cells that is activated by Mn2+. Consistently, LM609 almost completely blocked binding of vitronectin to such cells. The formation of the PDI–αVβ3 stoichiometric complex was further demonstrated by surface plasmon resonance analysis, which showed that the initial reversible binding of PDI becomes irreversible in the presence of Mn2+, probably mediated by disulfide bonds. Thus, we show that Mn2+ simultaneously modulates the thiol isomerase activity of PDI that is bound to αVβ3 and induces its transition to the ligand‐competent state, suggesting an alternative mechanism of integrin regulation.

The intracellular ability of the "10 -23" DNAzyme to efficiently inhibit expression of targeted proteins has been evidenced by in vitro and in vivo studies. However, standard conditions for kinetic measurements of the DNAzyme catalytic activity in vitro include 25 mM Mg 2؉ , a concentration that is very unlikely to be achieved intracellularly. To study this discrepancy, we analyzed the folding transitions of the 10 -23 DNAzyme induced by Mg 2؉ . For this purpose, spectroscopic analyzes such as fluorescence resonance energy transfer, fluorescence anisotropy, circular dichroism, and surface plasmon resonance measurements were performed. The global geometry of the DNAzyme in the absence of added Mg 2؉ seems to be essentially extended, has no catalytic activity, and shows a very low binding affinity to its RNA substrate. The folding of the DNAzyme induced by binding of Mg 2؉ may occur in several distinct stages. The first stage, observed at 0.5 mM Mg 2؉ , corresponds to the formation of a compact structure with limited binding properties and without catalytic activity. Then, at 5 mM Mg 2؉ , flanking arms are projected at right position and angles to bind RNA. In such a state, DNAzyme shows substantial binding to its substrate and significant catalytic activity. Finally, the transition occurring at 15 mM Mg 2؉ leads to the formation of the catalytic domain, and DNAzyme shows high binding affinity toward substrate and efficient catalytic activity. Under conditions simulating intracellular conditions, the DNAzyme was only partially folded, did not bind to its substrate, and showed only residual catalytic activity, suggesting that it may be inactive in the transfected cells and behave like antisense oligodeoxynucleotide.The typical DNAzyme, 1 known as the "10 -23" model, has tremendous potential in gene suppression for both target validation and therapeutic applications (1). It is capable of cleaving single-stranded RNA at specific sites under simulated physiological conditions and can be used to control even complex biological processes such as tumor angiogenesis. For example, DNAzymes to ␤1 and ␤3 mRNA reduced expression of targeted integrin subunits in endothelial cells and blocked proliferation, migration, and network formation in a fibrin and Matrigel™ matrix (2). In a cell culture system, a 10 -23 deoxyribozyme designed against 12-lipoxygenase mRNA specifically down-regulated expression of this protein and its metabolites, which are known to play a crucial role in tumor angiogenesis (3). Similarly, the DNAzyme to VEGFR2 mRNA cleaved its substrate efficiently and inhibited the proliferation of endothelial cells with a concomitant reduction of VEGFR2 mRNA and blocked tumor growth in vivo (4).The origins of the DNAzyme catalytic activity are not yet fully understood, but the observed rate enhancements probably are generated by a number of factors, including metal ion and nucleobase catalysis and local stereochemical effects. The 10 -23 DNAzyme has been developed using an in vitro selection strategy on the basis of it...

Objective-Previous studies supported the contribution of exosomes to an acellular mode of communication, leading to intercellular transfer of molecules. In this study we provide evidence that mast cell-derived exosomes induce plasminogen activator inhibitor type 1 (PAI-1) expression in endothelial cells, detectable at the level of PAI-1 mRNA and protein synthesis. The stimulating effect was also measured at the level of PAI-1 promoter activity. Methods and Results-To identify components responsible for this activity, exosome proteins were separated by 2-dimensional PAGE, and protein spots were identified by microsequencing using electrospray (ISI-Q-TOF-Micromass) spectrometer. Components of 3 independent systems that can be involved in activation of endothelial cells, namely the prothrombinase complex, tumor necrosis factor-␣, and angiotensinogen precursors were identified. Procoagulant activity of exosomes was confirmed by a thrombin generation assay using a specific chromogenic substrate. Because the potential of mast cell-derived exosomes to induce PAI-1 expression was completely abolished by hirudin, thrombin generated on exosomes seems to be responsible for this activity. Conclusions-It can be concluded that mast cell-derived exosomes via significant upregulation of PAI-1 secretion from endothelial cells may provide feedback between the characteristically increased PAI-1 levels and procoagulant states, both observed in diverse syndromes manifesting as endothelial cell dysfunction. 5,6 or diabetes mellitus, 7 and contributes to procoagulant state in these and other conditions. Mast cells exert profound pleiotropic effects on immune cell reactions at inflammatory sites, where they are most likely influenced not only by the extracellular matrix and inflammatory mediators but also by the proximity of activated T lymphocytes. These cells have been implicated in 2 contrasting types of immune responses, the immediate hypersensitivity reactions associated with allergic phenomena and their acute activation by bacterial products during infection. 8 Mast cells are localized near blood vessels and are involved in the activation of the clotting system during inflammation to contain the injury and initiate tissue repair. This concept is supported by studies of the reverse passive Arthus reaction in mast cell-deficient mice cells, which showed that these cells contributed to the exudation of clotting factors resulting in fibrin deposition and enhancement of fibrin cross-linkage. 9,10 In view of the role of mast cells in deposition of fibrin during inflammation near the site of injury, the aim of the present study was to examine the effect of mast cells-derived exosomes on expression and secretion of plasminogen activator inhibitor type 1 (PAI-1) from endothelial cells. Because exocytosis is the process by which stimulation of plasma membrane receptors on secretory cells results in the release of proteins and/or peptides from the intracellular stores into the extracellular space, 11 we attempted to identify active compone...

The spatial relationship between the binding sites for two cyclic peptides, cyclo(S,S)KYGCRGDWPC (cRGD) and cyclo(S,S)KYGCHarGDWPC (cHarGD), high affinity analogs for the RGD and HLGGAKQAGDV peptide ligands, in integrin ␣ IIb ␤ 3 (GPIIb-IIIa) has been characterized. For this purpose, cRGD and cHarGD were labeled with fluorescein isothiocyanate and tetramethylrhodamine 5-isothiocyanate, respectively. Both cyclic peptides were potent inhibitors of fibrinogen binding to ␣ IIb ␤ 3 , particularly in the presence of Mn 2؉; IC 50 values for cRGD and cHarGD were 1 and <0.1 nM in the presence of Mn 2؉. Direct binding experiments and fluorescence resonance energy transfer analysis using the purified receptor showed that both peptides interacted simultaneously with distinct sites in ␣ IIb ␤ 3 . The distance between these sites was estimated to be 6.1 ؎ 0.5 nm. Although cRGD bound preferentially to one site and cHarGD to the other, the sites were not fully specific, and each cyclic peptide or its linear counterpart could displace the other to some extent. The binding affinity of the cHarGD site was dramatically affected by Mn 2؉. cRGD, but not cHarGD, bound to recombinant ␤ 3 -(95-373) in a cation-dependent manner, indicating that the cRGD site is located entirely within this fragment. With intact platelets, binding of c-RGD and cHarGD to ␣ IIb ␤ 3 resulted in distinct conformational alterations in the receptor as indicated by the differential exposure of ligand-induced binding site epitopes and also induced the opposite on membrane fluidity as shown by electron paramagnetic resonance analyses using 5-doxylstearic acid as a spin probe. These data support the concept the two peptide ligands bind to distinct sites in ␣ IIb ␤ 3 and initiate different functional consequences within the receptor itself and within platelets. ␣ IIb ␤ 3 (GPIIb-IIIa) is a member of integrin family of cell adhesion receptors (1-3) and is the most abundant membrane protein on the platelet surface (4). On nonstimulated platelets, ␣ IIb ␤ 3 is incapable of binding most of its soluble macromolecular ligands (5), but after exposure of the cells to appropriate agonists, the receptor undergoes a conformational change (6) as a consequence of signal transmission from inside the cell to the extracellular domain of the receptor (inside-out signaling (7, 8)) and becomes competent to interact with several plasma protein ligands, including fibrinogen, fibronectin, and von Willebrand factor (9 -11). Fibrinogen inhibits the binding of other two ligands to ␣ IIb ␤ 3 (10 -13), and a common set of monoclonal antibodies (mAbs) 1 to the receptor blocks the interaction of these adhesive ligands with ␣ IIb ␤ 3 (9). Two sets of ligand peptides, ␥ chain peptides, which correspond to the sequence at the carboxyl-terminal sequence of the fibrinogen ␥ chain, and RGD(X) peptides, which correspond to sequences present in all three macromolecular ligands, define the recognition specificity of ␣ IIb ␤ 3 for its macromolecular ligands (reviewed in Ref. 14). Both peptide sets inhib...

Gradual occlusion of coronary arteries may result in reversible loss of cardiomyocyte function (hibernating myocardium), which is amenable to therapeutic neovascularization. The role of myocardin-related transcription factors (MRTFs) co-activating serum response factor (SRF) in this process is largely unknown. Here we show that forced MRTF-A expression induces CCN1 and CCN2 to promote capillary proliferation and pericyte recruitment, respectively. We demonstrate that, upon G-actin binding, thymosin 4 (T 4), induces MRTF translocation to the nucleus, SRF-activation and CCN1/2 transcription. In a murine ischaemic hindlimb model, MRTF-A or T 4 promotes neovascularization, whereas loss of MRTF-A/B or CCN1-function abrogates the T 4 effect. We further show that, in ischaemic rabbit hindlimbs, MRTF-A as well as T 4 induce functional neovascularization, and that this process is inhibited by angiopoietin-2, which antagonizes pericyte recruitment. Moreover, MRTF-A improves contractile function of chronic hibernating myocardium of pigs to a level comparable to that of transgenic pigs overexpressing T 4 (T 4tg). We conclude that MRTF-A promotes microvessel growth (via CCN1) and maturation (via CCN2), thereby enabling functional improvement of ischaemic muscle tissue.

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Background: Cancer cell invasion requires integrins for adhesion/de-adhesion and MMPs for focalized proteolysis. Results: MMP-2 is up-regulated in invasive colorectal tumors and degrades ␤1 integrins. Conclusion: Shedding of the I-like domain from ␤1 integrins results in decreased adhesion and enhanced cell motility. Significance: MMP-2 amplifies the motility of cancer cells, not only degrading extracellular matrix but also reducing the ␤1 integrin expression.