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1.
APMIS ; 128(1): 10-19, 2020 Jan.
Article in English | MEDLINE | ID: mdl-31642122

ABSTRACT

Atherogenesis is associated with chronic gut infections; however, the mechanisms are not clear. The aim of the study was to determine whether lipopolysaccharide of E. coli (E. coli LPS) may affect endothelial barrier and modify IL-10 expression in dendritic cells (DCs). Human umbilical vein endothelial cells (HUVECs) and monocyte-derived DCs were treated with E. coli LPS, apolipoprotein B100 (ApoB100) and 7-ketocholesterol (7-kCH) - harmful oxidized form of cholesterol. The effect of E. coli LPS, 7-kCH and ApoB100 on the barrier functions of HUVECs in real-time cell electric impedance sensing system (RTCA-DP) was assessed. Furthermore, the effect of 7-kCH and ApoB100 on barrier functions of HUVECs co-cultured with DCs previously treated with LPS was analyzed. Both E. coli LPS and 7-kCH decreased barrier functions of HUVECs and reduced tight junction protein mRNA expression, whereas ApoB100 increased endothelial barrier. In DCs, ApoB100 and E. coli LPS decreased IL-10 mRNA expression. In HUVECs co-cultured with DCs treated with LPS and subsequently pulsed with ApoB100 or 7-kCH, IL-10 mRNA expression was lower. E. coli LPS-exposed DCs diminished the protective effect of ApoB100 on endothelial integrity and led to the decrease in occludin mRNA expression. LPS potentially derived from gut microflora may destabilize endothelial barrier together with oxidized cholesterol and intensify the immunogenicity of ApoB100.


Subject(s)
Dendritic Cells/drug effects , Escherichia coli/immunology , Human Umbilical Vein Endothelial Cells/drug effects , Interleukin-10/genetics , Lipopolysaccharides/immunology , Tight Junctions/drug effects , Apolipoprotein B-100/pharmacology , Cells, Cultured , Coculture Techniques , Dendritic Cells/immunology , Escherichia coli/chemistry , Humans , Ketocholesterols/pharmacology , Lipopolysaccharides/pharmacology , Occludin/genetics , Signal Transduction/drug effects , Tight Junction Proteins/genetics
2.
Inflammation ; 43(2): 722-730, 2020 Apr.
Article in English | MEDLINE | ID: mdl-31858317

ABSTRACT

The inflammasome is responsible for maturation of interleukin-1ß (IL-1ß) and interleukin-18 (IL-18) contributing to the inflammatory process in atherosclerosis. It is shown here that an electronegative low-density lipoprotein [LDL (-)] apoB-100 mimetic peptide can activate the transcriptional and posttranslational signs needed for complete inflammasome activation. This peptide, named p2C7, can activate the Toll-like receptor 4 (TLR4) that induces NF-κB activation and the transcription of inflammasome components. After blocking TLR4 with a neutralizing antibody, inflammasome component (NLRP3, CASP1, and ASC) and IL1b and IL18 gene downregulation occurred in human-derived macrophages stimulated with p2C7 or LDL (-). Moreover, the posttranslational signal was activated by the interaction between p2C7 and the lectin-type oxidized LDL receptor 1 (LOX-1), as demonstrated by the induction of caspase-1 cleavage in macrophages. The blockage of either TLR4 or LOX-1 decreased IL-1ß and IL-18 secretion by human-derived macrophages as both pathways are necessary for complete inflammasome activation. These findings suggest a mechanism by which macrophages transduce the pro-inflammatory signal provided by LDL (-) ApoB-100 and its mimetic peptides to activate the inflammasome protein complex what may be relevant for the inflammatory process in atherosclerosis.


Subject(s)
Apolipoprotein B-100/pharmacology , Biomimetic Materials/pharmacology , Inflammasomes/metabolism , Lipoproteins, LDL/pharmacology , Macrophages/metabolism , Cells, Cultured , Humans , Macrophages/drug effects , Monocytes/drug effects , Monocytes/metabolism
3.
Protein J ; 37(6): 548-571, 2018 12.
Article in English | MEDLINE | ID: mdl-30259240

ABSTRACT

LDL, VLDL and other members of the low-density lipoparticles (LLPs) enter cells through a large family of receptors. The actual receptor ligand(s) in apolipoprotein B100, one of the main proteins of LLP, remain(s) unknown. The objective of this study was to identify true receptor ligand(s) in apo B100, a molecule of 4563 residues. Apo B100 contains 33 analogues of Cardin-Weintraub arginine/lysine-based receptor ligand motifs and shares key lysine motifs and sequence similarity with the LDL receptor-associated protein, MESD, and heat shock proteins. Eleven FITC-labeled synthetic peptides of 21-42 residues, with at least one ligand, were tested for binding and internalization using HeLa cells. All peptides bind but display different binding capacities and patterns. Peptides B0013, B0582, B2366, and B2932 mediate endocytosis and appear in distinct sites in the cytoplasm. B0708 and B3181 bind and remain on the cell surface as aggregates/clusters. Peptides B3119 (Site A) and B3347 (Site B), the putative ligands, showed low binding and no cell entry capacity. Apo B100 regions in this study share similarities with related proteins of known function including chaperone proteins and Apo BEC stimulating protein, and not directly related proteins, e.g., the DNA-binding domain of interferon regulatory factors, MSX2-interacting protein, and snake venom Zinc metalloproteinase-disintegrin-like proteins.


Subject(s)
Apolipoprotein B-100 , Endocytosis/drug effects , Peptides , Receptors, LDL , Amino Acid Motifs , Apolipoprotein B-100/chemistry , Apolipoprotein B-100/pharmacology , HeLa Cells , Humans , Peptides/chemistry , Peptides/pharmacology , Protein Domains , Receptors, LDL/agonists , Receptors, LDL/metabolism
4.
J Inorg Biochem ; 188: 29-37, 2018 11.
Article in English | MEDLINE | ID: mdl-30119015

ABSTRACT

[Fe(NO)2] - modified nanoparticles of low-density protein (DNICLDL) can serve as conveyors of iron in the form of stable complexes with ApoB100 protein. As reported recently, in human hepatoma cells DNICLDL significantly increased the total iron content, while showing low toxicity. In the present work, we focused on the effects of internalization of DNIC-modified lipoproteins in macrophages, with special regards to cytotoxicity. DNICLDL was administered to a model macrophage cell line, RAW 264.7. Administration of DNICLDL considerably increased total iron content. High increase of iron was accompanied by moderate toxicity. As shown by in vitro plasmid nicking assay, chelation of iron in the form of DNIC strongly reduced the iron-related reactive oxygen species (ROS) -induced DNA damage. In addition, DNICLDL, plausibly due to its NO-donating activity, did not induce inducible nitric oxide synthase (iNOS) expression, as opposed to other forms of low-density protein (LDL).


Subject(s)
Iron , Lipoproteins, LDL , Macrophages/metabolism , Nitrogen Oxides , Animals , Apolipoprotein B-100/chemistry , Apolipoprotein B-100/pharmacology , Iron/chemistry , Iron/pharmacology , Lipoproteins, LDL/chemistry , Lipoproteins, LDL/pharmacology , Macrophages/cytology , Mice , Nitrogen Oxides/chemistry , Nitrogen Oxides/pharmacology , RAW 264.7 Cells
5.
Eur J Immunol ; 42(11): 2983-9, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22806614

ABSTRACT

Plasma lipoproteins such as LDL (low-density lipoprotein) are important therapeutic targets as they play a crucial role in macrophage biology and metabolic disorders. The impact of lipoprotein profiles on host defense pathways against Gram-positive bacteria is poorly understood. In this report, we discovered that human serum lipoproteins bind to lipoteichoic acid (LTA) from Staphylococcus aureus and thereby alter the immune response to these bacteria. Size-exclusion chromatography and solid-phase-binding analysis of serum revealed the direct interaction of LTA with apolipoproteins (Apo) B100, ApoA1, and ApoA2. Only ApoB100 and the corresponding LDL exerted biological effects as this binding significantly inhibited LTA-induced cytokine releases from human and murine immune cells. Serum from hypercholesterolemic mice or humans significantly diminished cytokine induction in response to S. aureus or its LTA. Sera taken from the patients with familial hypercholesterolemia before and after ApoB100-directed immuno-apheresis confirmed that ApoB100 inhibited LTA-induced inflammation in humans. In addition, mice in which LDL secretion was pharmacologically inhibited, displayed significantly increased serum cytokine levels upon infection with S. aureus in vivo. The present study identifies ApoB100 as an important suppressor of innate immune activation in response to S. aureus and its LTA.


Subject(s)
Apolipoprotein B-100/pharmacology , Lipopolysaccharides/immunology , Staphylococcal Infections/immunology , Staphylococcus aureus/immunology , Teichoic Acids/immunology , Animals , Female , Humans , Hypercholesterolemia/immunology , Immunity, Innate/immunology , Leukocytes, Mononuclear/immunology , Lipopolysaccharides/antagonists & inhibitors , Mice , Mice, Inbred C57BL , Mice, Knockout , Specific Pathogen-Free Organisms , Staphylococcal Infections/microbiology , Teichoic Acids/antagonists & inhibitors
6.
Diabetes Obes Metab ; 14(7): 662-5, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22268579

ABSTRACT

Dipeptidyl peptidase-4 inhibitors (DPP-4i) improve glycaemic control in type 2 diabetes, but their benefits on reverse cholesterol transport (RCT) remain unknown. We evaluated the effects of DPP-4i sitagliptin 500 mg/kg/day on RCT in obese insulin-resistant CETP-apoB100 transgenic mice. Metformin 300 mg/kg/day orally was used as a reference compound. Both metformin and sitagliptin showed the expected effects on glucose parameters. Although no significant effect was observed on total cholesterol and high-density lipoprotein (HDL) cholesterol levels, sitagliptin, but not metformin, increased faecal cholesterol mass excretion by 132% (p < 0.001 vs. vehicle), suggesting a potent effect on cholesterol metabolism. Mice were then injected i.p. with (3) H-cholesterol labelled macrophages to measure RCT over 48 h. Compared with vehicle, sitagliptin significantly increased macrophage-derived (3) H-cholesterol faecal excretion by 39%. Administration of (14) C-cholesterol labelled olive oil orally showed a significant reduction of (14) C-tracer plasma appearance over time with sitagliptin, indicating that this drug promotes RCT through reduced intestinal cholesterol absorption.


Subject(s)
Apolipoprotein B-100/pharmacology , Diabetes Mellitus, Type 2/metabolism , Hypoglycemic Agents/pharmacology , Macrophages/metabolism , Metformin/pharmacology , Pyrazines/pharmacology , Triazoles/pharmacology , Animals , Biological Transport/drug effects , Biomarkers/metabolism , Blood Glucose/metabolism , Cholesterol/metabolism , Cholesterol Ester Transfer Proteins/metabolism , Diabetes Mellitus, Type 2/blood , Diabetes Mellitus, Type 2/drug therapy , Dipeptidyl-Peptidase IV Inhibitors/pharmacology , Intestinal Absorption/drug effects , Lipoproteins, HDL/metabolism , Male , Mice , Mice, Obese , Mice, Transgenic , Sitagliptin Phosphate
7.
J Control Release ; 149(2): 196-205, 2011 Jan 20.
Article in English | MEDLINE | ID: mdl-20946921

ABSTRACT

Doxorubicin is one of the most employed anticancer drugs, but its efficacy is limited by the onset of adverse effects such as drug resistance, due to the drug efflux via P-glycoprotein (Pgp). Several factors are associated to a high Pgp activity, including the amount of cholesterol in plasma membrane, which is essential to maintain the pump function. In this work we started from the following observations: 1) the drug-resistant colon cancer HT29-dx cells had a higher content of cholesterol in plasma membrane than drug-sensitive HT29 cells and a higher activity of Pgp, which was decreased by the cholesterol-lowering agent ß-methyl-cyclodextrin; 2) HT29-dx cells showed a higher synthesis of endogenous cholesterol and a higher expression of the low-density lipoprotein receptor (LDLR); 3) the anti-cholesterolemic drug simvastatin reduced the cholesterol synthesis, increased the synthesis of LDLR and lowered the Pgp activity in resistant cells. In order to circumvent drug resistance we designed a new liposomal doxorubicin, conjugated with a recombinant LDLR-binding peptide from human apoB100: this LDL-masked doxorubicin ("apo-Lipodox") was efficiently internalized by a LDLR-driven endocytosis and induced cytotoxic effects in HT29-dx cells, reversing their drug resistance. Its efficacy was further increased by simvastatin, which up-regulates the LDLR levels and contemporarily reduces the Pgp activity, thus increasing the liposomes uptake and limiting the drug efflux. We propose that the association of liposomal doxorubicin and statins may be a future promising strategy to reverse drug-resistance in human cancer cells.


Subject(s)
Antibiotics, Antineoplastic/pharmacology , Doxorubicin/pharmacology , Drug Resistance, Neoplasm/drug effects , Receptors, LDL/antagonists & inhibitors , Amino Acid Sequence , Antibiotics, Antineoplastic/administration & dosage , Apolipoprotein B-100/chemistry , Apolipoprotein B-100/pharmacology , Binding Sites , Cell Culture Techniques , Cell Survival/drug effects , Cholesterol/biosynthesis , Doxorubicin/administration & dosage , HT29 Cells , Humans , Liposomes , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/pharmacology
8.
J Exp Med ; 207(5): 1081-93, 2010 May 10.
Article in English | MEDLINE | ID: mdl-20439543

ABSTRACT

Immune responses to oxidized low-density lipoprotein (oxLDL) are proposed to be important in atherosclerosis. To identify the mechanisms of recognition that govern T cell responses to LDL particles, we generated T cell hybridomas from human ApoB100 transgenic (huB100(tg)) mice that were immunized with human oxLDL. Surprisingly, none of the hybridomas responded to oxidized LDL, only to native LDL and the purified LDL apolipoprotein ApoB100. However, sera from immunized mice contained IgG antibodies to oxLDL, suggesting that T cell responses to native ApoB100 help B cells making antibodies to oxLDL. ApoB100 responding CD4(+) T cell hybridomas were MHC class II-restricted and expressed a single T cell receptor (TCR) variable (V) beta chain, TRBV31, with different Valpha chains. Immunization of huB100(tg)xLdlr(-/-) mice with a TRBV31-derived peptide induced anti-TRBV31 antibodies that blocked T cell recognition of ApoB100. This treatment significantly reduced atherosclerosis by 65%, with a concomitant reduction of macrophage infiltration and MHC class II expression in lesions. In conclusion, CD4(+) T cells recognize epitopes on native ApoB100 protein, this response is associated with a limited set of clonotypic TCRs, and blocking TCR-dependent antigen recognition by these T cells protects against atherosclerosis.


Subject(s)
Atherosclerosis/immunology , Lipoproteins, LDL/immunology , T-Lymphocytes/immunology , Animals , Antigen-Presenting Cells/immunology , Antigen-Presenting Cells/physiology , Antigens, CD/immunology , Antigens, Differentiation, Myelomonocytic/immunology , Apolipoprotein B-100/blood , Apolipoprotein B-100/immunology , Apolipoprotein B-100/pharmacology , Atherosclerosis/pathology , Atherosclerosis/therapy , Humans , Hybridomas/immunology , Hybridomas/pathology , Immunity, Cellular , Lipoproteins, LDL/pharmacology , Malondialdehyde/pharmacology , Mice , Receptors, Antigen, T-Cell/immunology , T-Lymphocytes/drug effects , T-Lymphocytes/pathology
9.
Life Sci ; 84(25-26): 935-9, 2009 Jun 19.
Article in English | MEDLINE | ID: mdl-19409916

ABSTRACT

AIMS: Apolipoprotein B-100 (apoB-100) has been implicated in hyperlipidemia, which contributes to the pathogenesis of vascular disorders. Our aim was to investigate whether the expression of human apoB-100 in transgenic mice and/or a high-cholesterol diet cause cerebral microvascular lesions, and whether these conditions augment ischemia-related capillary damage. MAIN METHODS: Human apoB-100 overexpressing transgenic (Tg(apoB-100), n=23) and wild-type mice (C5/B6, Wt, n=26) were supplied with standard or 2% cholesterol-enriched diet for 17-19 weeks. Cerebral ischemia was induced by unilateral common carotid artery occlusion. Cortical samples were embedded for electron microscopy. Microvascular density (number of microvascular profiles/examined area), lumen diameter, the swelling of astrocytic endfeet, the occurrence of endothelial microvilli (affected capillaries expressed as ratio of all capillaries encountered), and the ratio of intact capillaries (devoid of all the above pathology) were calculated. KEY FINDINGS: The expression of apoB-100 coincided with decreased cortical microvascular density (195+/-7 vs. 223+/-8 vessels/mm(2), vs. Wt; P<0.008) and increased capillary lumen diameter (3.16+/-0.5 vs. 2.88+/-0.6 microm, vs. Wt; P<0.001). Cerebral ischemia promoted the swelling of perivascular astrocytes (62.1+/-4.2 vs. 36.5+/-4.0%, vs. contralateral, Wt; P<0.001), and reduced the ratio of intact capillaries (32.1+/-5.6 vs. 65.2+/-3.7%, vs. contralateral, Wt; P<0.001). Hyperlipidemia did not exacerbate the injury. SIGNIFICANCE: The overexpression of human apoB-100 alters the density of the microvascular network and the diameter of capillaries, which may compromise cerebrovascular reactivity during ischemia.


Subject(s)
Apolipoprotein B-100/pharmacology , Brain Ischemia/complications , Brain/blood supply , Brain/drug effects , Capillaries/pathology , Cholesterol, Dietary/metabolism , Animals , Apolipoprotein B-100/genetics , Apolipoprotein B-100/metabolism , Brain/pathology , Brain Ischemia/surgery , Capillaries/ultrastructure , Female , Gene Expression , Humans , Hyperlipidemias/chemically induced , Hyperlipidemias/complications , Lipids/blood , Male , Mice , Mice, Transgenic
10.
J Neuroimmunol ; 201-202: 41-9, 2008 Sep 15.
Article in English | MEDLINE | ID: mdl-18649950

ABSTRACT

CD4(+) T cells specific for the acetylcholine receptor (AChR) are assumed to play an important role in pathogenesis of myasthenia gravis (MG). A large and diverse number of potential T cell epitopes have been reported for different experimental setups aiming at the identification of disease-relevant T cells in MG. Investigating the T cell response to the epsilon subunit of human AChR, we explore complementary in vitro and in vivo approaches (PBMC from MG patients and mice transgenic for HLA-DR3 and human CD4) to address the possibilities and limitations of different strategies for elucidating natural autoimmune T cell epitopes.


Subject(s)
Epitope Mapping , Epitopes/physiology , Myasthenia Gravis/pathology , Receptors, Nicotinic/chemistry , T-Lymphocytes, Helper-Inducer/immunology , Amino Acid Sequence , Animals , Apolipoprotein B-100/pharmacology , CD4 Antigens/genetics , Dose-Response Relationship, Drug , HLA-DR3 Antigen/genetics , Humans , Mice , Mice, Transgenic , Myasthenia Gravis/blood , Peptide Fragments/immunology , Protein Binding/drug effects , Receptors, Nicotinic/immunology , Receptors, Nicotinic/metabolism
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