scholarly journals 300 PCSK9 induces a prothrombotic response by activation of NFKB signalling pathway in mononuclear cells

2021 ◽  
Vol 23 (Supplement_G) ◽  
Author(s):  
Valentina Scalise ◽  
Chiara Sanguinetti ◽  
Tommaso Neri ◽  
Alessandro Celi ◽  
Roberto Pedrinelli
Keyword(s):  
Transcription Factor ◽  
Mononuclear Cells ◽  
Deductive Reasoning ◽  
Membrane Surface ◽  
Specific Inhibitor ◽  
Cell Types ◽  
Signalling Pathway ◽  
Procoagulant Activity ◽  
Direct Stimulation ◽  
Important Player

Abstract Aims Strong evidence both experimental and clinical studies point out to an involvement of proprotein convertase subtilisin/kexin 9 (PCSK9), as an important player in hypercholesterolemia and atherosclerosis pathophysiology, identifying it, as a key molecule in the development of new cholesterol-lowering drugs and therapeutic target for atherosclerosis and related diseases. Emerging evidence shown that PCSK9 is straight implicated in inflammatory process where it may directly influence the activity of various cell types through NFkB activation, a key transcription factor involved in the induction of both pro-inflammatory cytokines and Tissue Factor (TF) expression, a major regulator of haemostasis and thrombosis in monocytes. Deductive reasoning makes therefore plausible to investigate a mechanistic link between circulation PCSK9 levels and TF expression, both in monocytes as well as on the surface of microparticles (MPs) generated from the same cells, with NFkB acting as the chain in the link. To investigate the involvement of NFkB signalling pathway in PCSK9-induced TF expression. Methods THP-1 cell line were stimulated with human (h) PCSK9 (5 μg/mL) or pre-incubated with BAY-117082 (BAY, 10−5M) an NFκB inhibitor, CLI-095 (3 × 10−6M), a highly TLR-4 signalling specific inhibitor and LPS-RS(5 μg/mL) a TLR-4 antagonist. TF procoagulant activity (PCA) was assessed by one-stage clotting assay using a STart Max semi-automated coagulation analyser. Concentration of TF-bearing MPs was determined using the Zymuphen MP-activity kit. Results hPCSK9 stimulated TF activity in THP-1 (PCA: from 50 ± 20 to 120 ± 20 ρg/mL, n = 10, P < 0.01). BAY, an NFkB inhibitor (PCA: −71 ± 23%, n = 5, P < 0.01) and CLI-095, a TLR-4 signalling inhibitor, (PCA: −86 ± 26%, n = 3, P < 0.05) and LPS-RS (PCA: −71 ± 23%, n = 5, P < 0.01) down-regulated PCSK9-induced TF activity completely. Furthermore THP-1stimulation with hPCSK9 causes the release of prothrombotic MPs-TF+ (MPs release from: 0.13 ± 0.07 to 0.42 ± 0.1 nM PS, n = 7, P < 0.05; PCA-MP+ from: 14 ± 3 to 44 ± 28 ρg/mL, n = 5, P < 0.05). Conclusions These data confirm the pivotal role of monocytes in the response PCSK9-induced TF-expression as well as the involvement of PCSK9 in inflammatory-thrombotic diseases through a direct stimulation of TF procoagulant activity and by the release of TF-bearing MPs. The possible mechanism of action involves recognition of PCSK9 by TLRs 2 and/or 4, on monocytes membrane surface, leading to activation of the transcription factor NFκB via an intracellular signalling cascade. Further studies will be needed to better understand the regulatory mechanisms underlying this complex set of biological responses that bind PCSK9, and coagulation events.

Elevated Procoagulant Activity Due to Phosphatidylserine Exposure on Red Blood Cells during Storage.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1996-1996
Author(s):  
Jin Zhou ◽  
Chengfang Lu ◽  
Hongjuan Yu ◽  
Jinxiao Hou ◽  
Shuchuan Liu ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Mononuclear Cells ◽  
Cell Types ◽  
Procoagulant Activity ◽  
Organ Injury ◽  
Enzymatic Assays ◽  
Healthy Donors ◽  
Coagulant Activity ◽  
Pathogenetic Factor ◽  
Relationship Of

Abstract Serious complications and mortality after cardiac surgery are increased when transfused red cells are stored for more than 2 weeks (Koch, N Engl J Med. 2008). However, the mechanism remains unclear and the relationship of procoagulant activity to PS exposure on banked RBCs has not been clarified, although prior studies suggest that banked cells went to apoptosis. Procoagulant activity of RBCs was measured in a modified prothrombin time in which RBCs replaced thromboplastin. The presence of PS in neutrophils and mononuclear cells from healthy donors and banked RBCs was investigated by flow-cytomety and confocal microscopy. The assembly of extrinsic tenase, intrinsic tenase, and prothrombinase complexes on these cells was studied using enzymatic assays employing plasma or purified proteins. Lactadherin, a glycoprotein, was utilized as a probe to track PS exposure and as an agent to block exposed PS. Coagulant activity increased approx 5-fold after cells were banked for 14 days and progressively increases. The percent of phosphatidylserine (PS)-positive cells increased from 7.4% on day 14 to 30% on day 42. RBCs with exposed PS were nearly absent in samples at 7 days but readily evident at 14 days on confocal microscopy. Banked RBCs exhibited patched, a rim, and diffuse-pattern that stained with lactadherin but neutrophils and MNC did not, indicating more PS exposure on banked RBCs from 7 days than the other two cell types. These data reveal a positive correlation between PCA and PS exposure on stored RBCs. Inhibition of PCA by lactadherin further confirms that PS supports the PCA in stored RBCs. Thus, we propose the possible role of PCA due to increased PS exposure on aged RBCs as a potential pathogenetic factor leading to organ injury, aggravated infections and finally higher mortality.

scholarly journals Polarized Human Immunodeficiency Virus Budding in Lymphocytes Involves a Tyrosine-Based Signal and Favors Cell-to-Cell Viral Transmission

1999 ◽  
Vol 73 (6) ◽  
pp. 5010-5017 ◽  
Author(s):  
Julie Deschambeault ◽  
Jean-Philippe Lalonde ◽  
Guillermo Cervantes-Acosta ◽  
Robert Lodge ◽  
Éric A. Cohen ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Mononuclear Cells ◽  
Vaccine Development ◽  
Membrane Surface ◽  
Basolateral Membrane ◽  
Cell Types ◽  
Viral Transmission ◽  
Site Directed Mutagenesis ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Maturation and release of human immunodeficiency virus type 1 (HIV-1) is targeted at the pseudopod of infected mononuclear cells. However, the intracellular mechanism or targeting signals leading to this polarized viral maturation are yet to be identified. We have recently demonstrated the presence of a functional YXXL motif for specific targeting of HIV-1 virions to the basolateral membrane surface in polarized epithelial Madin-Darby canine kidney cells (MDCK). Site-directed mutagenesis was used to demonstrate that the membrane-proximal tyrosine in the intracytoplasmic tail of the HIV-1 transmembrane glycoprotein (gp41) is an essential component of this signal. In the present study, immunolocalization of viral budding allowed us to establish that this tyrosine-based signal is involved in determining the exact site of viral release at the surface of infected mononuclear cells. Substitution of the critical tyrosine residue was also shown to increase the amount of envelope glycoprotein at the cell surface, supporting previous suggestions that the tyrosine-based motif can promote endocytosis. Although alteration of the dual polarization-endocytosis motif did not affect the infectivity of cell-free virus, it could play a key role in cell-to-cell viral transmission. Accordingly, chronically infected lymphocytes showed a reduced ability to transmit the mutant virus to a cocultivated cell line. Overall, our data indicate that the YXXL targeting motif of HIV is active in various cell types and could play an important role in viral propagation; this may constitute an alternative target for HIV therapeutics and vaccine development.

Contributions of Phosphatidylserine Exposure on Leukemic Cells to Unregulated Procoagulant Activity in Acute Promyelocytic Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2153-2153
Author(s):  
Jin Zhou ◽  
Fenglin Cao ◽  
Chengfang Lu ◽  
Jinmei Li ◽  
Jinxiao Hou ◽  
...  
Keyword(s):  
Acute Promyelocytic Leukemia ◽  
Mononuclear Cells ◽  
Cell Types ◽  
Promyelocytic Leukemia ◽  
Procoagulant Activity ◽  
Leukemic Cells ◽  
Outer Leaflet ◽  
All Trans Retinoic Acid ◽  
Healthy Donors ◽  
Differentiating Agents

Abstract Acute promyelocytic leukemia (APL) causes coagulation, which can worsen following initiation of chemotherapy and is improved or corrected by the differentiating agents, all trans retinoic acid (ATRA) and arsenic trioxide (As2O3). The relationship of procoagulant activity to phosphatidylserine (PS) exposure on leukemic cells has not been clarified, although prior studies indicate that APL cells expose tissue factor (TF). Procoagulant activity of leukemic cells was measured in a modified prothrombin time in which leukemic cells replaced thromboplastin. The presence of phosphatidylserine (PS) in neutrophils and mononuclear cells (MNC) from healthy donors and APL cells from patients was investigated by flow-cytometry and confocal microscopy. The assembly of extrinsic tenase, intrinsic tenase and prothrombinase complexes on these cells was studied using enzymatic assays employing plasma or purified proteins. Lactadherin was utilized as a probe to track PS exposure and as an agent to block exposed PS. Isolated APL cells exhibited patches that stained with lactadherin, but neutrophils and MNC did not, indicating more PS exposure on APL cells than the other two cell types. Exclusion of propidium iodide by the leukemic cells indicated that PS exposure occurs in the absence of frank apoptosis. Coagulant activity increased approx 20-fold after cells were exposed to 0.1 μM daunorubicin for 24 hr with 70% of apoptosis and decreased by 85% after 24 hr treatment with 1 μM ATRA or As2O3 with partial differentiation. Inhibition of procoagulant activity by ATRA and As2O3 corresponded to decreased PS and decrease activity for all three enzyme complexes. Lactadherin, which blocks PS binding sites, inhibited Xase and prothrombin conversion by their respective APL-assembled activating complexes. APL cells support all procoagulant reactions leading to robust thrombin formation. This ability results from concomitant PS and TF exposure at the outer leaflet of cell membrane. In contrast, less PS on neutrophils and MNC do not support the coagulation.

scholarly journals Induction of monocyte procoagulant activity by murine hepatitis virus type 3 parallels disease susceptibility in mice.

1981 ◽  
Vol 154 (4) ◽  
pp. 1150-1163 ◽  
Author(s):  
G A Levy ◽  
J L Leibowitz ◽  
T S Edgington
Keyword(s):  
Virus Type ◽  
Disease Susceptibility ◽  
Mononuclear Cells ◽  
Hepatitis Virus ◽  
Procoagulant Activity ◽  
Induction Phase ◽  
Murine Hepatitis Virus ◽  
Direct Stimulation ◽  
Peripheral Blood Mononuclear ◽  
Type 3

The in vitro induction of procoagulant activity (PCA) in murine peripheral blood mononuclear cells (PBM) by murine hepatitis virus type 3 (MHV-3) correlates with the disease susceptibility in three strains of mice. PBM from BALB/c mice, a strain in which MHV-3 infection results in fatal acute fulminant hepatitis, responds to the virus with a robust PCA response, whereas PBM from C3H/St mice, a strain which develops mild acute hepatitis followed by chronic hepatitis, only exhibit a modest PCA response. In contrast, PBM from A/J mice, a strain fully resistant to MHV-3, generate no increase in PCA above control levels. The induction phase of MHV-3 PCA is rapid, with an increase within 1-1.5 h, with maximum activity at 18h, and it precedes MHV-3 replication in either 17 CL1 cells, a fully permissive cell line, or in monocytes from these strains of mice. The PCA response of BALB/c PBM exceeds the response to any other known stimulus. No induction occurs upon direct stimulation of monocytes by MHV-3, but in the presence of lymphocyte collaboration, the PCA response is observed first at a lymphocyte:monocyte ratio of 2:1 and reaches a maximum as the lymphocyte:monocyte ratio approaches 4:1. This response appears to provide a functional marker for susceptibility to MHV-3 infection in inbred strains of mice and could be important in the pathogenesis of MHV-3-induced disease.

Role of PKC isozymes in water transport in toad urinary bladder

1991 ◽  
Vol 49 ◽  
pp. 302-303
Author(s):  
A.J. Mia ◽  
L.X. Oakford ◽  
T. Yorio
Keyword(s):  
Urinary Bladder ◽  
Apical Membrane ◽  
Membrane Surface ◽  
Protein A ◽  
Cell Types ◽  
Leukemic Cells ◽  
Toad Urinary Bladder ◽  
Pkc Isozymes ◽  
Antibody Labeling

Protein kinase C (PKC) isozymes, when activated, are translocated to particulate membrane fractions for transport to the apical membrane surface in a variety of cell types. Evidence of PKC translocation was demonstrated in human megakaryoblastic leukemic cells, and in cardiac myocytes and fibroblasts, using FTTC immunofluorescent antibody labeling techniques. Recently, we reported immunogold localizations of PKC subtypes I and II in toad urinary bladder epithelia, following 60 min stimulation with Mezerein (MZ), a PKC activator, or antidiuretic hormone (ADH). Localization of isozyme subtypes I and n was carried out in separate grids using specific monoclonal antibodies with subsequent labeling with 20nm protein A-gold probes. Each PKC subtype was found to be distributed singularly and in discrete isolated patches in the cytosol as well as in the apical membrane domains. To determine if the PKC isozymes co-localized within the cell, a double immunogold labeling technique using single grids was utilized.

scholarly journals Made to Measure: Patient-Tailored Treatment of Multiple Sclerosis Using Cell-Based Therapies

2021 ◽  
Vol 22 (14) ◽  
pp. 7536
Author(s):  
Inez Wens ◽  
Ibo Janssens ◽  
Judith Derdelinckx ◽  
Megha Meena ◽  
Barbara Willekens ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
Autoimmune Diseases ◽  
Stromal Cells ◽  
Mononuclear Cells ◽  
Treatment Options ◽  
Cell Types ◽  
Peripheral Blood Mononuclear ◽  
Tailored Treatment ◽  
Key Issues ◽  
The Central Nervous System

Currently, there is still no cure for multiple sclerosis (MS), which is an autoimmune and neurodegenerative disease of the central nervous system. Treatment options predominantly consist of drugs that affect adaptive immunity and lead to a reduction of the inflammatory disease activity. A broad range of possible cell-based therapeutic options are being explored in the treatment of autoimmune diseases, including MS. This review aims to provide an overview of recent and future advances in the development of cell-based treatment options for the induction of tolerance in MS. Here, we will focus on haematopoietic stem cells, mesenchymal stromal cells, regulatory T cells and dendritic cells. We will also focus on less familiar cell types that are used in cell therapy, including B cells, natural killer cells and peripheral blood mononuclear cells. We will address key issues regarding the depicted therapies and highlight the major challenges that lie ahead to successfully reverse autoimmune diseases, such as MS, while minimising the side effects. Although cell-based therapies are well known and used in the treatment of several cancers, cell-based treatment options hold promise for the future treatment of autoimmune diseases in general, and MS in particular.

scholarly journals Exon Shuffling Played a Decisive Role in the Evolution of the Genetic Toolkit for the Multicellular Body Plan of Metazoa

Genes ◽  
2021 ◽  
Vol 12 (3) ◽  
pp. 382
Author(s):  
Laszlo Patthy
Keyword(s):  
Transcription Factor ◽  
Decisive Role ◽  
Cell Types ◽  
Body Plan ◽  
Exon Shuffling ◽  
Body Parts ◽  
Multidomain Proteins ◽  
Cellular Phenotypes ◽  
Cell Matrix Interactions ◽  
Genetic Toolkit

Division of labor and establishment of the spatial pattern of different cell types of multicellular organisms require cell type-specific transcription factor modules that control cellular phenotypes and proteins that mediate the interactions of cells with other cells. Recent studies indicate that, although constituent protein domains of numerous components of the genetic toolkit of the multicellular body plan of Metazoa were present in the unicellular ancestor of animals, the repertoire of multidomain proteins that are indispensable for the arrangement of distinct body parts in a reproducible manner evolved only in Metazoa. We have shown that the majority of the multidomain proteins involved in cell–cell and cell–matrix interactions of Metazoa have been assembled by exon shuffling, but there is no evidence for a similar role of exon shuffling in the evolution of proteins of metazoan transcription factor modules. A possible explanation for this difference in the intracellular and intercellular toolkits is that evolution of the transcription factor modules preceded the burst of exon shuffling that led to the creation of the proteins controlling spatial patterning in Metazoa. This explanation is in harmony with the temporal-to-spatial transition hypothesis of multicellularity that proposes that cell differentiation may have predated spatial segregation of cell types in animal ancestors.

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