Maria Swiatkowska

Interaction and functional association of protein disulfide isomerase with αVβ3 integrin on endothelial cells

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.

Induction of PAI‐1 expression by tumor necrosis factor α in endothelial cells is mediated by its responsive element located in the 4G/5G site

Plasminogen activator inhibitor type 1 (PAI‐1) is induced by many proinflammatory and pro‐oxidant factors. Among them, tumor necrosis factor α (TNFα), a pivotal early mediator that regulates and amplifies the development of inflammation, is one of the strongest PAI‐1 synthesis activators. Location of the TNFα response element in the PAI‐1 promoter is still ambiguous. In this study, we attempted to evaluate the significance of the element located in the 4G/5G site of the PAI‐1 promoter in the TNFα stimulation of PAI‐1 expression in endothelial cells. PAI‐1 expression was monitored at: (a) the level of mRNA using real‐time PCR, (b) PAI‐1 gene transcription by transfection reporter assays, and (c) protein synthesis using the enzyme immunoassay. NF‐κB activity was monitored using the electrophoretic mobility shift assay. Its activity was modified by either antisense oligonucleotides or transfection of endothelial cells with the wild‐type or mutated IκBα. We have shown that TNFα‐induced expression and gene transcription of PAI‐1 involves a regulatory region present in segment −664/−680 of the PAI‐1 promoter. This reaction involves the TNFα‐induced generation of superoxide leading to activation of NF‐κB, and can be abolished by antioxidants and by overexpression of a super‐suppressor phosphorylation‐resistant IκBα. Stimulation of PAI‐1 under these conditions involves the motif of the PAI‐1 promoter adjacent to the 4G/5G site, which can directly interact with NF‐κB. We show that activation of PAI‐1 gene by TNFα and reactive oxygen species is mediated by interaction of NF‐κB with the cis‐acting element located in the −675 4G/5G insertion/deletion in the PAI‐1 promoter.

Ero1α Is Expressed on Blood Platelets in Association with Protein-disulfide Isomerase and Contributes to Redox-controlled Remodeling of αIIbβ3

Recent evidence supports a role of protein-disulfide isomerase (PDI) in redox-controlled remodeling of the exofacial domains of αIIbβ3 in blood platelets. The aim of this study was to explain whether Ero1α can be responsible for extracellular reoxidation of the PDI active site. We showed that Ero1α can be found on platelets and is rapidly recruited to the cell surface in response to platelet agonists. It is physically associated with PDI and αIIbβ3, as suggested by colocalization analysis in confocal microscopy and confirmed by immunoprecipitation experiments. Apart from monomeric oxidized Ero1α, anti-αIIbβ3 immunoprecipitates showed the presence of several Ero1α-positive bands that corresponded to the complexes αIIbβ3-PDI-Ero1α, PDI-Ero1α, and Ero1α-Ero1α dimers. It binds more efficiently to the activated αIIbβ3 conformer, and its interaction is inhibited by RGD peptides. Ero1α appears to be involved in the regulation of αIIbβ3 receptor activity because of the following: (a) blocking the cell surface Ero1α by antibodies leads to a decrease in platelet aggregation in response to agonists and a decrease in fibrinogen and PAC-1 binding, and (b) transfection of MEG01 with Ero1α increases αIIbβ3 receptor activity, as indicated by increased binding of fibrinogen.

Effect of oxidative stress on the expression of t-PA, u-PA, u-PAR, and PAI-1 in endothelial cells.

In this study we examined the effects of exogenous nitric oxide (sodium nitroprusside, SNP) and hydrogen peroxide (H2O2) on the expression level of tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), urokinase-type plasminogen activator receptor (u-PAR), and plasminogen activator inhibitor type 1 (PAI-1) in human umbilical vein endothelial cells (HUVEC). The expression of selected genes involved in fibrynolysis under the influence of oxidative stress was analyzed at the levels of mRNA, protein, and promoter activity. The results of the conducted studies revealed that oxidative stress in endothelial cells causes a significant increase in PAI-1 and u-PAR expression and a moderate increase in t-PA and u-PA expression at all of the investigated levels. We attempted to elucidate the molecular signaling mechanisms by which SNP and H2O2 regulate expression of the respective fibrinolytic factors. Therefore, we tested the protein levels of AP-1, NF-kappaB, and HIF-1 and their DNA-binding activity in endothelial cells subjected to oxidative stress. We found strong correlation between AP-1, NF-kappaB, and HIF-1 in the contribution of regulation of selected genes. In addition, we also found that the inhibition of PAI-1 synthesis by antisense oligonucleotide to PAI-1 mRNA results in markedly increased u-PAR expression and that NF-kappaB and AP-1 are involved in this regulation.

Protein disulfide isomerase directly interacts with β-actin Cys374 and regulates cytoskeleton reorganization.

Background: PDI regulates cytoskeleton reorganization by the thiol-disulfide exchange in β-actin. Results: PDI directly binds to Cys374 of β-actin during cell adhesion and spreading. Conclusion: Interaction of PDI with β-actin is induced by integrin-mediated cell adhesion and promotes cytoskeleton reorganization. Significance: PDI is a new regulator of the intramolecular disulfide-thiol rearrangement of β-actin in response to αIIbβ3 integrin engagement. Recent studies support the role of cysteine oxidation in actin cytoskeleton reorganization during cell adhesion. The aim of this study was to explain whether protein disulfide isomerase (PDI) is responsible for the thiol-disulfide rearrangement in the β-actin molecule of adhering cells. First, we showed that PDI forms a disulfide-bonded complex with β-actin with a molecular mass of 110 kDa. Specific interaction of both proteins was demonstrated by a solid phase binding assay, surface plasmon resonance analysis, and immunoprecipitation experiments. Second, using confocal microscopy, we found that both proteins colocalized when spreading MEG-01 cells on fibronectin. Colocalization of PDI and β-actin could be abolished by the membrane-permeable sulfhydryl blocker, N-ethylmaleimide, by the RGD peptide, and by anti-αIIbβ3 antibodies. Consequently, down-regulation of PDI expression by antisense oligonucleotides impaired the spreading of cells and initiated reorganization of the cytoskeleton. Third, because of transfection experiments followed by immunoprecipitation and confocal analysis, we provided evidence that PDI binds to the β-actin Cys374 thiol. Formation of the β-actin-PDI complex was mediated by integrin-dependent signaling in response to the adhesion of cells to the extracellular matrix. Our data suggest that PDI is released from subcellular compartments to the cytosol and translocated toward the periphery of the cell, where it forms a disulfide bond with β-actin when MEG-01 cells adhere via the αIIbβ3 integrin to fibronectin. Thus, PDI appears to regulate cytoskeletal reorganization by the thiol-disulfide exchange in β-actin via a redox-dependent mechanism.

Increased platelet-fibrinogen interaction in patients with hypercholesterolemia and hypertriglyceridemia

Binding of fibrinogen to platelets washed from the blood of patients with hypercholesterolemia and hypertriglyceridemia (n = 25) and control donors (n = 12) was compared. In addition, the content of platelet glycoprotein IIb was determined by radioimmunoassay. Fibrinogen was bound in significantly higher amounts (P < 0.02) to hyperlipidaemic platelets activated by ADP than to control ones (107,112 +/- 16,371 and 45,612 +/- 6495 molecules per platelet, respectively). The mean content of GPIIb was the same in hyperlipidaemic and in control platelets (2.06 +/- 0.16 and 1.94 +/- 0.21 micrograms/10(8) platelets, respectively). The amount of fibrinogen bound to the activated hyperlipidaemic platelets showed a positive correlation with total plasma cholesterol and LDL (r = 0.45 and 0.47, respectively) whereas a negative correlation with plasma HDL was found (r = -0.50). The increased expression of fibrinogen binding sites similar to that of hyperlipidaemic platelets could be produced by preincubation of normal platelets with palmitic acid. This was evidenced by a significant increase of fibrinogen binding sites in control platelets. This suggests that either palmitoylation of the receptor or microenvironment changes in the membrane lipid bilayer may be responsible for the enhanced platelet receptor capacity to bind fibrinogen.

Expression of a recombinant protein of the platelet F11 receptor (F11R) (JAM-1/JAM-A) in insect cells: F11R is naturally phosphorylated in the extracellular domain

The F11 receptor (F11R/JAM) is a member of the immunoglobulin superfamily localized on the membrane surface of human platelets and a component of tight junctions of endothelial and epithelial cells. F11R was demonstrated to participate in the adhesion of human platelets to cytokine-inflamed endothelial cells (EC), indicating an important role for F11R in inflammatory thrombosis and atherosclerosis. Domains responsible for the formation of tight junctions, the adhesion of platelets to EC, activation of platelets resulting in granule release, the activation of αIIb/β3 integrin and platelet aggregation, were identified in the external portion of F11R. To further examine critical sites of F11R, we utilized the baculovirus system to generate the F11R recombinant protein with the sequence of the extracellular domain, in two types of insect cells, Sf9 and H5. The F11R recombinant protein was detected in the cytoplasm of both infected Sf9 and H5 insect cells, but only infected H5 cells secreted a soluble F11R protein. The purified recombinant F11R proteins, obtained from both types of insect cells, were recognizeable by a conformation-dependent monoclonal antibody, M.Ab.F11, directed against domains within the N-terminus and the first Ig-like fold of F11R. Assessment of the phosphorylation state in the recombinant F11R protein revealed phosphorylation of serine, threonine and tyrosine amino acid residues within the external domain. Real-time biomolecular interaction analysis, performed to assess kinetic constants associated with the binding of active molecules to the purified recombinant F11R protein revealed high affinity binding of the phosphorylated recombinant protein by M.Ab.F11 with Ka of 5.47 × 106 and Kd of 1.83 × 10−7, comparable to values measured with intact human platelets. The findings reported here provide new information on specific domains of F11R that can lead to the generation of therapeutic agents expected to be useful in the treatment of cardiovascular diseases.

The role of Protein Disulfide Isomerase and thiol bonds modifications in activation of integrin subunit alpha11

Integrins belong to a family of transmembrane receptors that mediate cell migration and adhesion to ECM. Extracellular domains of integrin heterodimers contain cysteine-rich regions, which are potential sites of thiol-disulfide exchanges. Rearrangements of extracellular disulfide bonds regulate activation of integrin receptors by promoting transition from an inactive state into a ligand-binding competent state. Modifications of integrin disulfide bonds dependent on oxidation-reduction can be mediated by Protein Disulfide Isomerse (PDI). This paper provides evidences that binding to integrin ligands initiate changes in free thiol pattern on cell surface and that thiol-disulfide exchange mediated by PDI leads to activation of integrin subunit α11. By employing co-immunoprecipitation and confocal microscopy analysis we showed that α11β1 and PDI create complexes bounded by disulfide bonds. Using surface plasmon resonance we provide biochemical evidence that PDI can interact directly with integrin subunit α11.

Differential Quantitative Proteomics of Human Microvascular Endothelial Cells 1 by iTRAQ Reveals Palladin to be a New Biomarker During TGF-?1 Induced Endothelial Mesenchymal Transition

The study uses global quantitative proteomics to investigate the molecular mechanisms behind the induction of endothelial-mesenchymal transition (EndMT) by transforming growth factor–β (TGF-β). Orbitrap Velos mass spectrometers and iTRAQ – a labeling-based analysis were used to perform a global and quantitative comparison of two proteomes of Human Microvascular Endothelial Cells-1 (HMEC-1) treated or not treated by TGF-β1. iTRAQ analysis identified 43 differentially-expressed proteins in the early stages of EndMT induced by TGF-β1. From 5522 identified proteins, 26 were downregulated and 17 were upregulated, including proteins such as palladin, POTE I, torsin A and nucleoporin (NDC1). Further analysis of palladin revealed its increased mRNA and protein expression in response to TGF-β and Snail transcription factor. Our findings demonstrate that the newly- identified proteins may be involved in early stages of biological processes leading to EndMT. Biological Significance: Endothelial to mesenchymal transition is a possible source of myofibroblasts, which play a crucial role in the pathogenesis of fibrosis. EndMT participate in tissue fibrotic processes in various organs. TGF-β family growth factors are involved in the initiation of EndMT. The intracellular cascades activated by TGF-β that result in the remarkable phenotypic change of endothelial cells to mesenchymal cells have not been entirely elucidated. The downstream signaling pathway initiated by TGF-β resulted in a strong upregulation of the Snail1 transcriptional repressor. Our proteomics data demonstrated that TGF-β -induced EndMT leads to alterations in protein profiles, more specifically, the upregulation of palladin. This upregulation is mediated by Snail transcription factor and GSK-3 β signaling kinase. Our results also suggest that palladin could be considered a new biomarker in the early stages of cellular transdifferentiation, eventually leading to endothelial-mesenchymal transition.

Structure-Based Design, Synthesis and Evaluation of Novel Peptidic Inhibitors of Thrombin-Induced Activation of Platelets Aggregation

Janet Gonzalez, Anna Babinska, Ebenezer L.V. Ewul, Edem Timpo, Alhassan Jallow, Zhiyong Qiu, Radoslaw Bednarek, Maria Swiatkowska, Moro O. Salifu, Manfred Philipp, and Cristina C. Clement Department of Natural Sciences, LaGuardia Community College, New York, NY, 11104, USA; Division of Nephrology, Department of Medicine, State University of New York, Downstate Medical Center, Brooklyn, NY, 11203, USA; Department of Chemistry, Lehman College of the City University of New York, Bronx, NY, 10468, USA; Medical University of Lodz Department of Cytobiology and Proteomics, Lodz, Poland