Publications by authors named "Maria Swiatkowska"

21 Publications

  • Page 1 of 1

Effects of Dual Purinoceptor-dependent Approach on Release of Vascular Endothelial Growth Factor From Human Microvascular Endothelial Cell (HMEC-1) and Endothelial Cell Condition.

J Cardiovasc Pharmacol 2020 Sep;76(3):349-359

Department of Cytobiology and Proteomics, Medical University of Lodz, Lodz, Poland.

In the recent years, the awareness of the role purinergic signaling plays as a therapeutic target has increased considerably. The purinoceptor allows the action of extracellular nucleotides (P2 receptors) and intermediary products of their metabolism, such as adenosine (P1 receptors), regulating pivotal processes occurring in the cardiovascular system. This study focuses on a dual purinoreceptor-dependent approach, based on the activation of adenosine P1 receptors with the simultaneous inhibition of P2Y12 receptors that can be used as novel platelet inhibitors in antithrombotic therapy. Endothelial cells are directly exposed to the drugs circulating in the bloodstream. That is why effects of our concept on human microvascular endothelial cells (HMEC-1) were examined in in vitro studies, such as enzyme-linked immunosorbent assay and scratch assays. In response to adenosine receptor agonists, levels of secreted vascular endothelial growth factor varied. Two of them, 5'-N-ethylcarboxamidoadenosine and MRE0094 remarkably increased vascular endothelial growth factor release. The elevated levels were reduced when used together with the P2Y12 receptor antagonist. Also, rates of wound closure in a scratch assay were significantly reduced in these cases. The results suggest that the proposed treatment does not impair endothelial cell condition. In addition, it is suggested as a collateral benefit, namely solving the problem of excessive activation of endothelial cells during antiplatelet therapy.
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http://dx.doi.org/10.1097/FJC.0000000000000866DOI Listing
September 2020

data: treatment with the F11R/JAM-A peptide 4D decreases mortality and reduces the generation of atherosclerotic plaques in ApoE-deficient mice.

Data Brief 2020 Jun 23;30:105516. Epub 2020 Apr 23.

Department of Medicine, State University of New York, Downstate Medical Center, Brooklyn, New York 11203, USA.

The data in this article focus on the F11 Receptor (F11R/JAM-A; Junctional Adhesion Molecule-A; JAM-A, F11R), a cell adhesion protein constitutively expressed on the membrane surface of circulating platelets and localized within the tight junctions of healthy endothelial cells (ECs). Previous reports have shown that F11R/JAM-A plays a critical role in the adhesion of platelets to an inflamed endothelium due to its' pathological expression on the luminal surface of the cytokine-inflamed endothelium. Since platelet adhesion to an inflamed endothelium is an early step in the development of atherosclerotic plaque formation, and with time, resulting in heart attacks and stroke, we conducted a long-term, study utilizing the atherosclerosis-prone ApoE mice to attempt a blockade of the formation of atherosclerotic plaques by preventing the adhesion of platelets to the inflamed vasculature . Utilizing a nonhydrolyzable peptide derived from an amino acid sequence of F11R/JAM-A, peptide 4D, we have shown that the adhesion of platelets to the inflamed endothelial cells could be blocked by peptide 4D. The present data demonstrate the positive health benefits of chronic peptide 4D administration to the atherosclerosis-prone ApoE mice, and provides new information for potential use of this F11R derived peptide in the prevention of atherosclerosis. The data presented in this article provide further experimental support for the study presented in Babinska et al., Atherosclerosis 284 (2019) 92-101.
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http://dx.doi.org/10.1016/j.dib.2020.105516DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7206208PMC
June 2020

P2Y receptor antagonists and AR receptor agonists regulates Protein Disulfide Isomerase secretion from platelets and endothelial cells.

Biochem Biophys Res Commun 2020 06 4;526(3):756-763. Epub 2020 Apr 4.

Department of Cytobiology and Proteomics, Medical University of Lodz, 6/8 Mazowiecka St., 92-215, Lodz, Poland.

Secretion of PDI from platelets and endothelial cells is an important step of all thrombotic events. In the absence of extracellular PDI thrombus formation and fibrin generation may be impaired. Thrombin-mediated PDI secretion is regulated by the stimulation of P2Y receptors. This paper provides evidences that P2Y antagonists or AR agonists may modulate release of PDI molecules from platelets and with less efficiency from endothelial cells. Moreover P2Y antagonization or AR agonization modulates platelet-endothelial interaction. We prove that combinations of P2Y antagonists and AR agonists inhibit platelet-dependent adhesion of cancer cells to endothelium and attenuate cancer cell invasiveness, but longer exposition to AR agonists may stimulate migration of invasive breast cancer cells through endothelium thus leading to increased metastasis.
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http://dx.doi.org/10.1016/j.bbrc.2020.03.143DOI Listing
June 2020

Functional inhibition of F11 receptor (F11R/junctional adhesion molecule-A/JAM-A) activity by a F11R-derived peptide in breast cancer and its microenvironment.

Breast Cancer Res Treat 2020 Jan 24;179(2):325-335. Epub 2019 Oct 24.

Department of Cytobiology and Proteomics, Medical University of Lodz, Lodz, Poland.

Purpose: To examine the involvement of the F11R/JAM-A protein in breast cancer metastasis, we utilized the F11R/JAM-A antagonistic peptide 4D (P4D) in experiments of transendothelial migration (TEM) of breast cancer cells.

Methods: Experiments were conducted in the mouse 4T1 breast cancer model utilizing the human mammary epithelial cell and endothelial cell lines. The levels of soluble F11R/JAM-A (sJAM-A) in the murine plasmas were measured by ELISA. Levels of F11R/JAM-A mRNA and protein in cell lines were assessed by qRT-PCR and Western blot, respectively. Cell surface expression of F11R/JAM-A was demonstrated by flow cytometry. Functional tests included the TEM of breast cancer cells and adhesion of breast cancer cells to the endothelium. The endothelial permeability was studied by fluorescent tracer assay and by the Real-Time Cell Analysis (RTCA).

Results: The tumor inducers Tβ4 and TGF-β1 reduced the levels of sJAM-A in murine plasma, and reduced the F11R/JAM-A protein levels in the human microvascular endothelial cell line HMEC-1. The adhesion and TEM measured between breast cancer cells and inflamed or Tβ4-treated endothelium were inhibited by P4D. The presence of P4D did not destabilize the pre-existing tight junctions in the endothelial monolayer. The barrier-protecting effect of P4D was stronger than that of forskolin, when a booster dose of P4D was applied to the inflamed endothelium.

Conclusions: F11R/JAM-A protein can be considered as a novel target in the treatment of breast cancer metastasis. In vivo and clinical studies are needed to further investigate the effectiveness of F11R/JAM-A-derived peptide as a possible anti-metastatic drug.
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http://dx.doi.org/10.1007/s10549-019-05471-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6987052PMC
January 2020

Modifications of disulfide bonds in breast cancer cell migration and invasiveness.

Am J Cancer Res 2019 1;9(8):1554-1582. Epub 2019 Aug 1.

Department of Cytobiology and Proteomics, Medical University of Lodz 6/8 Mazowiecka St., 92-215 Lodz, Poland.

Cancer metastasis involves the adhesion of cancer cells to the endothelium. This process can be mediated by integrins which are surface receptors responsible for interactions with ECM proteins. Integrins β and αβ represent factors are involved in cancer progression and metastasis. Activation of integrins can be promoted by thiol-disulfide exchanges initiated by Protein Disulfide Isomerase (PDI). The purpose of this study was to prove the involvement of disulfide rearrangements in the molecules of integrins in the course of cancer cell adhesion and migration through the endothelium. We present the evidence which proves that highly metastatic MDA-MB-231 breast cancer cell lines adhere to endothelial cells are more effective than non-invasive MCF-10A and MCF-7 cell lines and that the attachment of MDA-MB-231 to the endothelium can be attenuated either by the agents blocking free thiol groups (DTNB, cystamine or PCMBS) or by PDI inhibitors (Q3Rut, 16F16 or PACMA-31). Furthermore, we prove that the transendothelial migration of MDA-MB-231 cells and contraction of collagen can be blocked by thiol blockers or PDI inhibitors and that these factors affect exposition of free thiols on integrin molecules.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6727000PMC
August 2019

A peptide antagonist of F11R/JAM-A reduces plaque formation and prolongs survival in an animal model of atherosclerosis.

Atherosclerosis 2019 05 22;284:92-101. Epub 2019 Feb 22.

Department of Medicine, State University of New York, Downstate Medical Center, Brooklyn, NY, 11203, USA.

Background And Aims: The F11 Receptor (F11R), AKA Junctional Adhesion Molecule-A (JAM-A) (F11R/JAM-A), is an adhesion protein constitutively expressed on the membrane surface of circulating platelets and the luminal surface of inflamed endothelial cells (EC). Platelet adhesion to an inflamed endothelium is one of the early steps of atherosclerotic plaque formation. Our previous studies, conducted with cultured EC in vitro, have demonstrated the expression of F11R/JAM-A on the luminal surface of inflamed EC, platelet adhesion to inflamed EC through F11R/JAM-A interactions, and inhibition of this interaction by the presence of F11R/JAM-A antagonistic peptide (F11Rpeptide 4D). In the present study, we examined in vivo the overall health-benefits and cardiovascular effects of long-term treatment of animals prone to atherosclerosis, ApoE mice, with F11R-peptide 4D.

Methods: Twenty ApoE mice were assigned to daily treatment with peptide 4D and compared to their counterparts control untreated mice. Mice were observed for wellness and survival. Plaque size in the aorta and heart was measured using histological analysis. Effects of peptide 4D (or scramble control) on platelet adhesion to inflamed endothelium were measured using intravital microscopy.

Results: Significant reductions in atherosclerotic plaques number and size, an overall robust health with longer survival were found in the peptide 4D treated group of ApoE mice. Intravital microscopic studies conducted in exposed vessels of ApoE mice demonstrated significant inhibition by peptide 4D of platelet adhesion to the cytokine-inflamed endothelium.

Conclusions: Our results demonstrate that peptide 4D significantly reduces atherosclerotic plaque formation in ApoE mice and inhibits platelet adhesion to the inflamed arterial endothelium.
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http://dx.doi.org/10.1016/j.atherosclerosis.2019.02.014DOI Listing
May 2019

Contribution of activated beta3 integrin in the PDI release from endothelial cells.

Front Biosci (Landmark Ed) 2018 03 1;23:1612-1627. Epub 2018 Mar 1.

Department of Cytobiology and Proteomics, Medical University of Lodz, Poland,

Protein disulfide isomerase (PDI) is an abundant reticulum endoplasmic protein but also acts as an important functional regulator of some extracellular surface proteins. Recent studies suggest that PDI plays a role in integrin activation and thrombus formation. The aim of this study was to examine whether activation of integrin is the first stage leading to release of PDI from the subcellular compartments of endothelial cells to extracellular space. Our results show that endothelial cells which adhere to fibronectin or fibrinogen release significantly more PDI than those which adhere to poly-L-lysine. Cells treated with RGD peptide, Src and FAK kinase inhibitors and anti alphaVbeta3 antibody display lower PDI secretion. The destruction of the actin cytoskeleton of endothelial cells by cytochalasin D inhibits PDI release. When the endothelial cells adhere to fibrinogen or fibronectin, PDI and alphaVbeta3 gain free thiol groups. Our data suggest that upon activation of integrins, PDI is released from endothelial cells and forms a disulfide bond complex with alphaVbeta3 integrin.
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http://dx.doi.org/10.2741/4663DOI Listing
March 2018

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

Biochem Biophys Res Commun 2018 01 2;495(2):1635-1641. Epub 2017 Dec 2.

Department of Cytobiology and Proteomics, Medical University of Lodz, 6/8 Mazowiecka St., 92-215 Lodz, Poland.

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.
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http://dx.doi.org/10.1016/j.bbrc.2017.11.186DOI Listing
January 2018

Virus-directed enzyme prodrug therapy and the assessment of the cytotoxic impact of some benzimidazole derivatives.

Tumour Biol 2017 Jul;39(7):1010428317713675

1 Department of Applied Pharmacy, Medical University of Lodz, Lodz, Poland.

Virus-directed enzyme prodrug therapy is one of the major strategy of increasing cytotoxicity of bioreductive agents. This research intended to examine new selected benzimidazole derivatives as a substrate for nitroreductase, the enzyme involved in nitroreduction which is responsible to the production of cytotoxic metabolites. In this way, the selectivity and strength of cytotoxicity can be raised. The effect of benzimidazoles on virus transfected cells and non-virus transfected cells A549 cell line was established by Annexin V + propidium iodide test, western blot, and polymerase chain reaction analysis of specific pro- and anti-apoptotic proteins in the corresponding gene expression and additionally nitroreductase gene expression. Our results proved the pro-apoptotic properties of all tested compounds in normoxia and hypoxia, especially according to virused A549 cells where the time of exposition was reduced from 48 to 4 h. In this shorten period of time, the strongest activity was shown by N-oxide compounds with nitro-groups. The apoptosis was confirmed by generation of BAX gene and protein and reduction of BCL2 gene and protein.
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http://dx.doi.org/10.1177/1010428317713675DOI Listing
July 2017

[Role of protein disulfide isomerase in activation of integrins].

Postepy Hig Med Dosw (Online) 2014 May 30;68:666-83. Epub 2014 May 30.

Katedra i Zakład Biofizyki Molekularnej i Medycznej Uniwersytet Medyczny w Łodzi.

Integrins belong to a large family of transmembrane cell adhesion receptors that communicate biochemical and mechanical signals in a bidirectional manner across the plasma membrane. Integrins and their ligands play a crucial role in a number of physiological and pathological processes, including cell migration, cell differentiation, hemostasis, adhesion, angiogenesis, cancer, cell invasiveness and wound healing. Intracellular signals switch integrins into a ligand-competent state as a result of conformational changes within the integrin molecule. Binding of extracellular ligands induces structural changes that can transmit signals to the cell interior. Transition of integrins from an inactive to a ligand binding state involves rearrangement of the disulfide bonding pattern. The rearrangement of disulfide bonds is modulated by protein disulfide isomerase (PDI). PDI has been found on the surface of several types of cells, including endothelial cells, hepatocytes, cancer cells, pancreatic cells and B cells. PDI was identified on the platelet surface, where it plays an important role in platelet reactions such as adhesion, aggregation and secretion. PDI was found to directly interact with integrins. Disulfide-thiol exchange mediated by PDI appears to be involved in the conformational changes in integrin activation. In this report we describe the structure of integrin and the role of disulfide bond rearrangement in its activation.
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http://dx.doi.org/10.5604/17322693.1105766DOI Listing
May 2014

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

J Biol Chem 2014 Feb 10;289(9):5758-73. Epub 2014 Jan 10.

From the Department of Molecular and Medical Biophysics, Medical University of Lodz, 92-215 Lodz, Poland and.

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 Cys(374) 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.
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http://dx.doi.org/10.1074/jbc.M113.479477DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3937648PMC
February 2014

Proteomic analysis of plasma profiles in children with recurrent bone fractures.

Acta Biochim Pol 2011 12;58(4):553-61. Epub 2011 Dec 12.

Department of Paediatric Propedeutics and Bone Metabolic Diseases, Medical University of Lodz, Lodz, Poland.

Unlabelled: The aim of the study is proteomic analysis of the plasma profile in children with recurrent bone fractures. The study involved 16 children: 6 patients with recurrent low-energy fractures and normal bone mass and 10 with osteogenesis imperfecta. In the analysis of the protein profile, the two-dimensional protein electrophoresis was used (Ettan DALT II, Amersham Bioscience). The images of protein gels were compared with controls. The protein spots with changed expression were cut from the gel and the amino acid sequence was analyzed with the mass spectrometry method (Q-Tof Premier(TM) API MASS SPECTROMETR, Waters) for protein identification. The most prevalent protein with changed expression, with respect to controls, was haptoglobin observed in 6 patients with a severe form of osteogenesis imperfecta. Increased haptoglobin concentration in these patients was confirmed by the ELISA method. Peptides corresponding to alpha-1 acid glycoprotein and serum amyloid P-component, apolipoprotein A-I, and transthyretin were detected in one, two and three children, respectively.

Conclusions: 1) The results show increased haptoglobin which may be suggestive of an inflammatory component taking part in the course of osteogenesis imperfecta. 2) Further studies to explain the possible relationship of this protein with increased bone fragility are necessary.
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May 2012

Ero1alpha is expressed on blood platelets in association with protein-disulfide isomerase and contributes to redox-controlled remodeling of alphaIIbbeta3.

J Biol Chem 2010 Sep 18;285(39):29874-83. Epub 2010 Jun 18.

Department of Molecular and Medical Biophysics, Medical University of Lodz, 92-215 Lodz, Poland.

Recent evidence supports a role of protein-disulfide isomerase (PDI) in redox-controlled remodeling of the exofacial domains of α(IIb)β(3) 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 α(IIb)β(3), as suggested by colocalization analysis in confocal microscopy and confirmed by immunoprecipitation experiments. Apart from monomeric oxidized Ero1α, anti-α(IIb)β(3) immunoprecipitates showed the presence of several Ero1α-positive bands that corresponded to the complexes α(IIb)β(3)-PDI-Ero1α, PDI-Ero1α, and Ero1α-Ero1α dimers. It binds more efficiently to the activated α(IIb)β(3) conformer, and its interaction is inhibited by RGD peptides. Ero1α appears to be involved in the regulation of α(IIb)β(3) 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 α(IIb)β(3) receptor activity, as indicated by increased binding of fibrinogen.
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http://dx.doi.org/10.1074/jbc.M109.092486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2943306PMC
September 2010

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

Biochem Cell Biol 2008 Dec;86(6):477-86

Department of Medical Biochemistry Medical University of Lodz, 6/8 Mazowiecka Street, 92-215 Lodz, Poland.

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.
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http://dx.doi.org/10.1139/O08-137DOI Listing
December 2008

Interaction and functional association of protein disulfide isomerase with alphaVbeta3 integrin on endothelial cells.

FEBS J 2008 Apr 7;275(8):1813-23. Epub 2008 Mar 7.

Department of Molecular and Medical Biophysics, Medical University in Lodz, 6/8 Mazowiecka Street, Lodz, Poland.

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 alpha(V)beta(3), in Mn(2+)-treated endothelial cells. By employing confocal microscopy, flow cytometry and coimmunoprecipitation experiments, we showed that exposure of endothelial cells to Mn(2+) resulted in: (a) the appearance of surface protein thiol groups, which can be found in PDI and alpha(V)beta(3), and both proteins colocalizing on the cellular surface; and (b) the formation of the PDI-alpha(V)beta(3) complex, which dissociates upon reduction. In addition, PDI in a complex with alpha(V)beta(3) 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 Mn(2+). Consistently, LM609 almost completely blocked binding of vitronectin to such cells. The formation of the PDI-alpha(V)beta(3) 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 Mn(2+), probably mediated by disulfide bonds. Thus, we show that Mn(2+) simultaneously modulates the thiol isomerase activity of PDI that is bound to alpha(V)beta(3) and induces its transition to the ligand-competent state, suggesting an alternative mechanism of integrin regulation.
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http://dx.doi.org/10.1111/j.1742-4658.2008.06339.xDOI Listing
April 2008

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

FEBS J 2005 Nov;272(22):5821-31

Department of Molecular and Medical Biophysics, Medical University in Lodz, Poland.

Plasminogen activator inhibitor type 1 (PAI-1) is induced by many proinflammatory and pro-oxidant factors. Among them, tumor necrosis factor alpha (TNFalpha), a pivotal early mediator that regulates and amplifies the development of inflammation, is one of the strongest PAI-1 synthesis activators. Location of the TNFalpha 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 TNFalpha 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-kappaB 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 IkappaBalpha. We have shown that TNFalpha-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 TNFalpha-induced generation of superoxide leading to activation of NF-kappaB, and can be abolished by antioxidants and by overexpression of a super-suppressor phosphorylation-resistant IkappaBalpha. 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-kappaB. We show that activation of PAI-1 gene by TNFalpha and reactive oxygen species is mediated by interaction of NF-kappaB with the cis-acting element located in the -675 4G/5G insertion/deletion in the PAI-1 promoter.
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http://dx.doi.org/10.1111/j.1742-4658.2005.04979.xDOI Listing
November 2005

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.

Platelets 2005 Mar;16(2):99-109

Department of Anatomy and Cell Biology, State University of New York, Downstate Medical Center at Brooklyn, Brooklyn, NY 11203, USA.

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 IIb/3 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 K(a) of 5.47 x 10(6) and K(d) of 1.83 x 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.
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http://dx.doi.org/10.1080/09537100400010329DOI Listing
March 2005

Thymosin beta 4 induces the synthesis of plasminogen activator inhibitor 1 in cultured endothelial cells and increases its extracellular expression.

Blood 2004 Feb 30;103(4):1319-24. Epub 2003 Oct 30.

Center for Medical Biology and Microbiology, Polish Academy of Sciences, Lodz, Poland.

Thymosin beta 4(T beta 4), a 4.9-kDa polypeptide primarily known as a main G-actin-sequestering peptide, is present in high concentrations in various cells and in the circulation. We have found that T beta 4 upregulates the expression of plasminogen activator inhibitor 1 (PAI-1) in endothelial cells measured both at the level of mRNA and protein synthesis. This effect seems to be cell specific and was not observed when other cells such as human fibroblasts, PC3, and U937 were tested. T beta 4 significantly activated the PAI-1 promoter in EA.hy 926 cells transiently transfected either with plasmid p800LUC containing PAI-1 promoter fragment (-800 to +71) or the PAI-1 promoter linked with green fluorescent protein. T beta 4 mediated up-regulation of PAI-1 involved activation of the mitogen-activated protein kinase cascade. Furthermore, T beta 4 enhanced c-Fos/c-Jun DNA-binding activity to the activator protein 1 (AP-1)-like element (-59 to -52). The specificity of this binding activity was demonstrated by competition electrophoretic mobility shift assay and after transfection of EA.hy 926 cells with the mutated PAI-1 promoter. Taken together, these data indicate that, in response to T beta 4 stimulation, AP-1 activity increases to enhance PAI-1 transcription through its unique AP-1-like element at -59 to -52 in the PAI-1 promoter.
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http://dx.doi.org/10.1182/blood-2003-04-1015DOI Listing
February 2004

Natriuretic peptides reduce plasminogen activator inhibitor-1 expression in human endothelial cells.

Cell Mol Biol Lett 2002 ;7(4):1153-7

Department of Molecular and Medical Biophysics, Medical University of Łódź, Łódź, Poland.

Plasma concentrations of natriuretic peptides increase in some pathological conditions, but very little is known about the effect of these vasodilator peptides on the regulation of the blood coagulation system. The fundamental role in the regulation of fibrinolysis is played by plasminogen activator inhibitor type 1 (PAI-1). Recent studies demonstrate that natriuretic peptides can modulate PAI-1 expression in bovine aortic smooth muscle cells and rat aortic endothelial cells. In this report, we tested the effect of natriuretic peptides on PAI-1 expression in the human endothelial cell line (EA.hy 926). For this purpose, we treated the cell cultures with ANP, BNP and CNP, and modulation of PAI-1 synthesis was evaluated. We compared the effect of natriuretic peptides on synthesis and release of PAI-1 in unstimulated cells, and after activation with tumour necrosis factor alpha (TNFalpha). Natriuretic peptides abolished TNFalpha - induced upregulation of PAI-1 expression at both the PAI-1 mRNA and the antigen levels. The inhibitory efficiency was higher in the case of CNP when compared to that produced by ANP and BNP, particularly when TNFalpha-stimulated cells were used. We observed an inhibition of stimulatory effect of TNFalpha on PAI-1 expression also at the level of the PAI-1 promoter in cells transfected with a PAI-1 promoter fragment (+71 to -800). The PAI-1 promoter activity was markedly inhibited by C-type natriuretic peptide, already at a very low (0.001 micro M) concentration of the peptide.
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July 2003

Reactive oxygen species upregulate expression of PAI-1 in endothelial cells.

Cell Mol Biol Lett 2002 ;7(4):1065-71

Department of Molecular and Medical Biophysics, Medical University of Łódź, Łódź, Poland.

Second messengers involved in the signal transduction pathway leading to induction of the plasminogen activator inhibitor (PAI-1) have not yet been well characterized. This study focuses on the mechanisms of regulation of PAI-1 expression by reactive oxygen species (ROS) in human endothelial cells. Inhibition of the tumor necrosis factor alpha (TNFalpha?-induced expression of PAI-1 by antioxidant N-acetyl-L-cysteine (NAC) indicated redox-sensitive mechanisms involved in the signaling pathway. Because TNFalpha induces PAI-1 production in endothelial cells, and NAC attenuated this response, we attempted to investigate the possible involvement of ROS in the activation of PAI-1 by TNFalpha. Upregulation of PAI-1 expression in endothelial cells by the stimulation with TNFalpha (50 ng/ml) or H2O2 (10-200 micro M), observed by measurement of the antigen and mRNA levels, was reversed in the presence of NAC (20mM). The stimulatory effect of ROS was detected also at the level of the PAI-1 promoter in endothelial cells transfected with plasmid p800 LUC containing a PAI-1 promoter fragment (+71 to -800). The PAI-1 promoter activity was increased in the presence of ROS, and was suppressed by up to 75% in the presence of antioxidants. On the basis of this study we can conclude that reactive oxygen species play an important role in a cytokine-induced activation of PAI-1 expression, and may act as a signal transduction messenger.
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July 2003

Cerivastatin, a HMG-CoA reductase inhibitor, reduces plasminogen activator inhibitor-1 (PAI-1) expression in endothelial cells by down-regulation of cellular signaling and the inhibition of PAI-1 promoter activity.

Jpn J Pharmacol 2002 Dec;90(4):337-44

Department of Molecular and Medical Biophysics, Medical University of Lodz, Poland.

Statins, which competitively inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity and reduce mevalonate synthesis, are believed to exert a plethora of pleiotropic effects. In this report, molecular mechanisms of the inhibitory effect on plasminogen activator inhibitor type 1 (PAI-1) expression produced by cerivastatin (CRV), the most active compound in this class, were studied using monocultures of human endothelial cell line (EA.hy 926). CRV similar to another statin, lovastatin (LOV), significantly inhibited PAI-1 expression and its release from endothelial cells, nonstimulated and stimulated with TNF-alpha. The inhibitory effect of CRV could be detected at the level of PAI-1 promoter in EA.hy 926 cells transfected with plasmid p800 LUC containing PAI-1 promoter fragment (+71 to -800), as well as at the level of PAI-1 mRNA. The PAI-1 promoter activity was markedly suppressed in the nonstimulated cells and almost completely inhibited in TNF-alpha-stimulated cells. In addition, CRV at low doses (IC(50) of 4 - 6 microM) significantly inhibited mitogen-activated protein kinases (MAPKs) phosphorylation. The majority of inhibitory effects occurred at significantly lower concentrations for CRV compared to LOV. The mechanism by which CRV inhibits PAI-1 expression appears to be directly associated with geranylgeranylation of some cell proteins, since the inhibitory effect on PAI-1 expression can be reversed by geranylgeranyl-pyrophosphate but not by farnesyl-pyrophosphate.
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http://dx.doi.org/10.1254/jjp.90.337DOI Listing
December 2002