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1.
Int J Nanomedicine ; 14: 7431-7446, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31686815

RESUMO

Background: Low density lipoprotein (LDL) has been regarded as a promising antitumor drug vehicle. However some problems, such as rare source, difficulty of large-scale production, and potential safety concerns, hinder its clinical application. Purpose: The objective of this study is to develop a biomimetic LDL nanocarrier by replacing the native apolipoprotein B-100 (apoB-100) with an artificial amphipathic peptide and demonstrate its antitumor efficacy. Methods: The amphipathic hybrid peptide (termed as FPL) consisting of a lipid binding motif of apoB-100 (LBMapoB)-polyethylene glycol (PEG)-folic acid (FA) was synthesized and characterized by 1H NMR and circular dichroism. FPL decorated lipoprotein-mimic nanoparticles (termed as FPLM NPs) were prepared by a modified solvent emulsification method. Paclitaxel (PTX) was incorporated into NPs and its content was quantified by HPLC analysis. The morphology of NPs was observed by transmission electron microscopy (TEM), and the particle size and zeta potential of NPs were determined by dynamic light scattering (DLS). The colloidal stability of FPLM NPs was evaluated in PBS containing bovine serum albumin (BSA). In vitro release of PTX loaded FPLM NPs was evaluated using the dialysis method. Cellular uptake and cytotoxity assayswere evaluated on human cervical cancer cells (HeLa) and lung cancer cells (A549). Tumor inhibition in vivo was investigated in M109 tumor-bearing mice via tail vein injection of Taxol formulation and PTX loaded NPs. Results: The composition of FPLM NPs, including cholesteryl oleate, glyceryl trioleate, cholesterol, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and FPL peptides, was optimized to be 5:1:1:3:10 (w/w). FPLM NPs had a spherical shape with a mean diameter of 83 nm and a negative charge (-12 mV). FPLM NPs with optimum formulation had good colloidal stability in BSA solution.The release of PTX from FPLM NPs was slow and sustained. The uptake of FPLM NPs was higher in folate receptor (FR) overexpressing tumor cells (HeLa cells) than in FR deficient tumor cells (A549 cells). The intracellular distribution indicated that FPLM NPs had the lysosome escape capacity. The internalization mechanism of FPLM NPs was involved with clathrin- and caveolae-mediated endocytosis and FR played a positive role in the internalization of FPLM NPs. The CCK-8 assay demonstrated that FPLM NPs exhibited notably better anti-tumor effect than Taxol formulation in vitro. Moreover, PTX loaded FPLM NPs produced very marked anti-tumor efficiency in M109 tumor-bearing mice in vivo. Conclusion: FPLM NPs is a promising nanocarrier which can improve the therapeutic effect and reduce the side effects of antitumor drugs.


Assuntos
Materiais Biomiméticos/química , Sistemas de Liberação de Medicamentos , Lipídeos/química , Lipoproteínas LDL/química , Nanopartículas/química , Neoplasias/tratamento farmacológico , Paclitaxel/uso terapêutico , Peptídeos/química , Células A549 , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Apolipoproteína B-100/química , Coloides/química , Liberação Controlada de Fármacos , Endocitose , Ácido Fólico/química , Células HeLa , Humanos , Camundongos Endogâmicos BALB C , Nanopartículas/ultraestrutura , Paclitaxel/farmacologia , Tamanho da Partícula , Polietilenoglicóis/química , Eletricidade Estática
3.
Med Hypotheses ; 126: 20-22, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31010493

RESUMO

Carbamylation (or carbamoylation) is a non-enzymatic post-translational modification process of lysine residues and protein N-termini, which occurs throughout the lifespan of both various plasma proteins and low-density lipoprotein (LDL) particles. Carbamylation results from the binding of isocyanates spontaneously derived from high levels of blood urea, environmental pollutants, nutritional sources and leads to the formation of potentially atherogenic carbamylated-LDL (c-LDL) particles. The carbamylation of LDL apolipoproteins is associated unfavorable downstream effects. Ornithine is a non-proteinogenic amino acid, which plays a central role at the urea cycle function. The primary use of ornithine in supplements is to support athletic performance, liver function and wound recovery. Ornithine is structurally highly similar to lysine, and is only one carbon atom shorter in its side-chain. Therefore, we hypothesize that supplemented ornithine could compete with ε-amino groups of lysine residues found in apolipoproteins of native LDL particles in their binding to isocyanates and decrease c-LDL formation. This issue still remains unresolved in current literature and needs to be elucidated in experimental studies.


Assuntos
Aminoácidos/metabolismo , Aterosclerose/metabolismo , Aterosclerose/terapia , Lipoproteínas LDL/metabolismo , Ornitina/uso terapêutico , Carbamilação de Proteínas , Apolipoproteína B-100/química , Apolipoproteínas/química , Aterosclerose/fisiopatologia , Humanos , Lisina/química , Modelos Biológicos , Ornitina/química
4.
Nanomedicine (Lond) ; 13(20): 2657-2668, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30334470

RESUMO

AIM: We analyzed the protein corona of thermoresponsive, poly(N-isopropylacrylamide)- or poly(N-isopropylmethacrylamide)-based nanogels. MATERIALS & METHODS: Traces of protein corona detected after incubation in human serum were characterized by proteomics and dynamic light scattering in undiluted serum. RESULTS: Apolipoprotein B-100 and albumin were the main components of the protein coronae. For dendritic polyglycerol-poly(N-isopropylacrylamide) nanogels at 37°C, an increase in adsorbed immunoglobulin light chains was detected, followed by partially reversible nanogel aggregation. All nanogels in their hydrophilic state are colloidally stable in serum and bear a dysopsonin-rich protein corona. CONCLUSION: We observed strong changes in NG stability upon slight alterations in the composition of the protein coronae according to nanogel solvation state. Nanogels in their hydrophilic state possess safe protein coronae.


Assuntos
Apolipoproteína B-100/química , Nanopartículas/química , Coroa de Proteína/química , Proteômica , Acrilamidas/química , Apolipoproteína B-100/genética , Difusão Dinâmica da Luz , Humanos , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Nanogéis , Nanopartículas/administração & dosagem , Polietilenoglicóis/química , Polietilenoimina/química , Pele/efeitos dos fármacos
5.
Protein J ; 37(6): 548-571, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30259240

RESUMO

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.


Assuntos
Apolipoproteína B-100 , Endocitose/efeitos dos fármacos , Peptídeos , Receptores de LDL , Motivos de Aminoácidos , Apolipoproteína B-100/química , Apolipoproteína B-100/farmacologia , Células HeLa , Humanos , Peptídeos/química , Peptídeos/farmacologia , Domínios Proteicos , Receptores de LDL/agonistas , Receptores de LDL/metabolismo
6.
J Inorg Biochem ; 188: 29-37, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30119015

RESUMO

[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).


Assuntos
Ferro , Lipoproteínas LDL , Macrófagos/metabolismo , Óxidos de Nitrogênio , Animais , Apolipoproteína B-100/química , Apolipoproteína B-100/farmacologia , Ferro/química , Ferro/farmacologia , Lipoproteínas LDL/química , Lipoproteínas LDL/farmacologia , Macrófagos/citologia , Camundongos , Óxidos de Nitrogênio/química , Óxidos de Nitrogênio/farmacologia , Células RAW 264.7
7.
Biosci Rep ; 38(2)2018 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-29545317

RESUMO

Estimation of the function as well as the amount of high-density lipoprotein (HDL) is required to predict the risk of cardiovascular disease development. Cholesterol efflux capacity (CEC) is the key metric for determining the antiatherosclerotic function of HDL. However, the assay methods currently used to calculate CEC are not ideal for clinical use as they require the culture of cells. In the present study, we developed a novel CEC assay using immobilized liposome-bound gel beads (ILGs), containing fluorescently labeled cholesterol, as a substitute for cultured cells. When apolipoprotein B-100 depleted serum, obtained by polyethylene glycol precipitation, was used as the cholesterol acceptors, the basic properties of this method, such as the available range of HDL-cholesterol, efflux temperature and time, and normalization parameters, indicate that this method is sufficient to estimate CEC. Furthermore, the CEC values obtained with this ILG method were also correlated with those obtained with a conventional method using THP-1 macrophages derived foam cells and 3H-cholesterol as a tracer (r = 0.932). Overall, this novel cholesterol efflux assay method is a realistic and effective alternative to current methods in the field while also being easier to use in clinical laboratories as neither cell culture, radioisotope nor ultracentrifugation is required.


Assuntos
Apolipoproteína B-100/química , Colesterol/análise , Lipossomos/química , Polietilenoglicóis/química , Apolipoproteína B-100/metabolismo , Colesterol/metabolismo , Células Espumosas/metabolismo , Células Espumosas/patologia , Humanos , Polietilenoglicóis/metabolismo , Células THP-1
8.
Biochemistry ; 56(31): 4084-4094, 2017 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-28702990

RESUMO

Our previous studies demonstrated that the first 1000 amino acid residues (the ßα1 domain) of human apolipoprotein (apo) B-100, termed apoB:1000, are required for the initiation of lipoprotein assembly and the formation of a monodisperse stable phospholipid (PL)-rich particle. The objectives of this study were (a) to assess the effects on the properties of apoB truncates undergoing sequential inclusion of the amphipathic ß strands in the 700 N-terminal residues of the ß1 domain of apoB-100 and (b) to identify the subdomain in the ß1 domain that is required for the formation of a microsomal triglyceride transfer protein (MTP)-dependent triacylglycerol (TAG)-rich apoB-containing particle. Characterization of particles secreted by stable transformants of McA-RH7777 cells demonstrated the following. (1) The presence of amphipathic ß strands in the 200 N-terminal residues of the ß1 domain resulted in the secretion of apoB truncates (apoB:1050 to apoB:1200) as both lipidated and lipid-poor particles. (2) Inclusion of residues 300-700 of the ß1 domain led to the secretion of apoB:1300, apoB:1400, apoB:1500, and apoB:1700 predominantly as lipidated particles. (3) Particles containing residues 1050-1500 were all rich in PL. (4) There was a marked increase in the lipid loading capacity and TAG content of apoB:1700-containing particles. (5) Only the level of secretion of apoB:1700 was markedly diminished by MTP inhibitor BMS-197636. These results suggest that apoB:1700 marks the threshold for the formation of a TAG-rich particle and support the concept that MTP participates in apoB assembly and secretion at the stage where particles undergo a transition from PL-rich to TAG-rich.


Assuntos
Apolipoproteína B-100/química , Proteínas de Transporte/metabolismo , Hepatócitos/metabolismo , Lipoproteínas VLDL/metabolismo , Animais , Apolipoproteína B-100/genética , Apolipoproteína B-100/metabolismo , Transporte Biológico/efeitos dos fármacos , Proteínas de Transporte/antagonistas & inibidores , Linhagem Celular Tumoral , Fluorenos/farmacologia , Hepatócitos/efeitos dos fármacos , Humanos , Isoindóis/farmacologia , Lipoproteínas VLDL/antagonistas & inibidores , Lipoproteínas VLDL/química , Peso Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosfolipídeos/análise , Fosfolipídeos/metabolismo , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Proteólise/efeitos dos fármacos , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Triglicerídeos/análise , Triglicerídeos/metabolismo
9.
J Biol Phys ; 43(3): 381-395, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28647778

RESUMO

The secondary structure of apolipoprotein B-100 is studied within the bulk phase and at the air/water interface. In these "in viro" experiments, infrared reflection absorption spectroscopy (IRRAS) study was performed at the air/water interface while circular dichroism (CD) was conducted in the bulk phase. In the bulk phase, the conformational structure containing a significant amount of ß-structure, whereas varying amount of α-helix, unordered structures, and ß-sheet were observed at the air/water interface depending on the low-density lipoprotein (LDL) film interfacial pressure. The present IRRAS results demonstrate the importance of interfacial pressure-induced structural conformations on the apoB-100. A correlation between the secondary structure of the apoB-100 protein and the monomolecular film elasticity at the air/water interface was also established. The orientation of apoB-100 with respect to the LDL film-normal was found to depend on the interfacial pressure exhibited by the monomolecular film. These results may shed light on LDL's pivotal role in the progression of atherosclerotic coronary artery disease as demonstrated previously by clinical trials.


Assuntos
Ar , Lipoproteínas LDL/química , Reologia , Água/química , Apolipoproteína B-100/química , Elasticidade , Pressão , Estrutura Secundária de Proteína , Propriedades de Superfície
10.
Proteomics Clin Appl ; 11(7-8)2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28296203

RESUMO

PURPOSE: Apolipoprotein A-I (ApoA-I) and apolipoprotein B-100 (ApoB-100) are amphipathic proteins that are strong predictors of cardiovascular disease risk. The traceable calibration of apolipoprotein assays is a persistent challenge, especially for ApoB-100, which cannot be solubilized in purified form. EXPERIMENTAL DESIGN: A simultaneous quantitation method for ApoA-I and ApoB-100 was developed using tryptic digestion without predigestion reduction and alkylation, followed by LC separation coupled with isotope dilution MS analysis. The accuracy of the method was assured by selecting structurally exposed signature peptides, optimal choice of detergent, protein:enzyme ratio, and incubation time. Peptide calibrators were value assigned by isobaric tagging isotope dilution MS amino acid analysis. RESULTS: The method reproducibility was validated in technical repeats of three serum samples, giving 2-3% intraday CVs (N = 5) and <7% interday CVs (N = 21). The repeated analysis of interlaboratory harmonization standards showed -1% difference for ApoA-I and -12% for ApoB-100 relative to the assigned value. The applicability of the method was demonstrated by repeated analysis of 24 patient samples with a wide range of total cholesterol and triglyceride levels. CONCLUSIONS AND CLINICAL RELEVANCE: The method is applicable for simultaneous analysis of ApoA-I and ApoB-100 in patient samples, and for characterization of serum pool calibrators for other analytical platforms.


Assuntos
Apolipoproteína A-I/química , Apolipoproteína B-100/química , Análise Química do Sangue/métodos , Espectrometria de Massas , Fragmentos de Peptídeos/sangue , Proteólise , Tripsina/metabolismo , Sequência de Aminoácidos , Apolipoproteína A-I/metabolismo , Apolipoproteína B-100/metabolismo , Calibragem , Cromatografia Líquida , Humanos , Isótopos/química , Modelos Lineares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Reprodutibilidade dos Testes
11.
Anal Biochem ; 518: 25-34, 2017 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-27984014

RESUMO

Two complementary instrumental techniques were used, and the data generated was processed with advanced numerical tools to investigate the interactions between anti-human apoB-100 monoclonal antibody (anti-apoB-100 Mab) and apoB-100 containing lipoproteins. Partial Filling Affinity Capillary Electrophoresis (PF-ACE) combined with Adsorption Energy Distribution (AED) calculations provided information on the heterogeneity of the interactions without any a priori model assumptions. The AED calculations evidenced a homogenous binding site distribution for the interactions. Quartz Crystal Microbalance (QCM) studies were used to evaluate thermodynamics and kinetics of the Low-Density Lipoprotein (LDL) and anti-apoB-100 Mab interactions. High affinity and selectivity were observed, and the emerging data sets were analysed with so called Interaction Maps. In thermodynamic studies, the interaction between LDL and anti-apoB-100 Mab was found to be predominantly enthalpy driven. Both techniques were also used to study antibody interactions with Intermediate-Density (IDL) and Very Low-Density (VLDL) Lipoproteins. By screening affinity constants for IDL-VLDL sample in a single injection we were able to distinguish affinity constants for both subpopulations using the numerical Interaction Map tool.


Assuntos
Anticorpos Monoclonais Murinos/química , Apolipoproteína B-100/química , Modelos Químicos , Termodinâmica , Animais , Humanos , Cinética , Camundongos
12.
Sci Rep ; 6: 36324, 2016 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-27824107

RESUMO

Acidification in the endosome causes lipoprotein release by promoting a conformational change in the LDLR allowing its recycling and degradation of LDL. Notwithstanding conformational changes occurring in the LDLR have expanded considerably, structural changes occurring in LDL particles have not been fully explored yet. The objectives of the present work were to study structural changes occurring in apoB100 by infrared spectroscopy (IR) and also LDL size and morphology by dynamic light scattering (DLS) and electron microscopy (EM) at both pH 7.4 and 5.0. We determined by IR that pH acidification from 7.4 to 5.0, resembling that occurring within endosomal environment, induces a huge reversible structural rearrangement of apoB100 that is characterized by a reduction of beta-sheet content in favor of alpha-helix structures. Data obtained from DLS and EM showed no appreciable differences in size and morphology of LDL. These structural changes observed in apoB100, which are likely implied in particle release from lipoprotein receptor, also compromise the apoprotein stability what would facilitate LDL degradation. In conclusion, the obtained results reveal a more dynamic picture of the LDL/LDLR dissociation process than previously perceived and provide new structural insights into LDL/LDLR interactions than can occur at endosomal low-pH milieu.


Assuntos
Apolipoproteína B-100/química , Apolipoproteína B-100/metabolismo , Lipoproteínas LDL/metabolismo , Difusão Dinâmica da Luz , Endossomos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Lipoproteínas LDL/química , Microscopia Eletrônica , Modelos Moleculares , Ligação Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína
13.
Sci Rep ; 5: 18184, 2015 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-26643808

RESUMO

Familial hypercholesterolaemia (FH) is an inherited autosomal dominant disorder resulting from defects in the low-density lipoprotein receptor (LDLR), in the apolipoprotein B (APOB) or in the proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. In the majority of the cases FH is caused by mutations occurring within LDLR, while only few mutations in APOB and PCSK9 have been proved to cause disease. p.(Arg3527Gln) was the first mutation in APOB being identified and characterized. Recently two novel pathogenic APOB variants have been described: p.(Arg1164Thr) and p.(Gln4494del) showing impaired LDLR binding capacity, and diminished LDL uptake. The objective of this work was to analyse the structure of p.(Arg1164Thr) and p.(Gln4494del) variants to gain insight into their pathogenicity. Secondary structure of the human ApoB100 has been investigated by infrared spectroscopy (IR) and LDL particle size both by dynamic light scattering (DLS) and electron microscopy. The results show differences in secondary structure and/or in particle size of p.(Arg1164Thr) and p.(Gln4494del) variants compared with wild type. We conclude that these changes underlie the defective binding and uptake of p.(Arg1164Thr) and p.(Gln4494del) variants. Our study reveals that structural studies on pathogenic variants of APOB may provide very useful information to understand their role in FH disease.


Assuntos
Substituição de Aminoácidos , Apolipoproteínas B/química , Apolipoproteínas B/genética , Códon , Hiperlipoproteinemia Tipo II/genética , Mutação , Apolipoproteína B-100/química , Apolipoproteína B-100/genética , Apolipoproteína B-100/ultraestrutura , Apolipoproteínas B/metabolismo , Apolipoproteínas B/ultraestrutura , Linhagem Celular , Humanos , Hiperlipoproteinemia Tipo II/metabolismo , Lipoproteínas LDL/química , Lipoproteínas LDL/metabolismo , Lipoproteínas LDL/ultraestrutura , Linfócitos/metabolismo , Tamanho da Partícula , Ligação Proteica , Estrutura Secundária de Proteína
14.
J Mass Spectrom ; 50(12): 1386-92, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26634972

RESUMO

Phospholipids are major components of cell membranes and lipoprotein complexes. They are prone to oxidation by endogenous and exogenous reactive oxygen species yielding a large variety of modified lipids including small aliphatic and phospholipid bound aldehydes and ketones. These carbonyls are strong electrophiles that can modify proteins and, thereby, alter their structures and functions triggering various pathophysiological conditions. The analysis of lipid-protein adducts by liquid chromatography-MS is challenged by their mixed chemical nature (polar peptide and hydrophobic lipid), low abundance in biological samples, and formation of multiple isomers. Thus, we investigated traveling wave ion mobility mass spectrometry (TWIMS) to analyze lipid-peptide adducts generated by incubating model peptides corresponding to the amphipathic ß1 sheet sequence of apolipoprotein B-100 with 1-palmitoyl-2-(oxo-nonanoyl)-sn-glycerophosphatidylcholine (PONPC). The complex mixture of peptides, lipids, and peptide-lipid adducts was separated by TWIMS, which was especially important for the identification of two mono-PONPC-peptide isomers containing Schiff bases at different lysine residues. Moreover, TWIMS separated structural conformers of one peptide-lipid adduct possessing most likely different orientations of the hydrophobic sn-1 fatty acyl residue and head group of PONPC, relative to the peptide backbone.


Assuntos
Lipídeos/química , Espectrometria de Massas/métodos , Modelos Químicos , Peptídeos/química , Apolipoproteína B-100/química , Isomerismo , Fosfatidilcolinas/química
15.
Biochem Biophys Res Commun ; 464(1): 306-11, 2015 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-26116775

RESUMO

BACKGROUND: Th1 responses in atherosclerosis are mainly associated with the aggravation of atherosclerotic plaques, whereas Th2 responses lead to a less pronounced disease in mouse models. The fixation of antigens on cells by means of ethylene carbodiimide (ECDI), and subsequent injection of these antigen-coupled splenocytes (Ag-SP) to induce tolerance against the attached antigens, has been successfully used to treat murine type 1 diabetes or encephalomyelitis in. We analyzed this approach in a mouse model for atherosclerosis. METHODS AND RESULTS: OTII-transgenic mice that were treated with a single dose of 5 × 10(7) OVA-coupled splenocytes (OVA-SP), had decreased splenocyte proliferation, and lower IFNγ production in vitro upon antigen recall. However, in vivo CD4 cell activation was increased. To try lipoprotein-derived, "atherosclerosis-associated" antigens, we first tested human oxidized LDL. In wild type mice, an increase of IFNγ production upon in vitro recall was detected in the oxLDL-SP group. In Apolipoprotein E - deficient (ApoE-/-) mice that received oxLDL-SP every 5 weeks for 20 weeks, we did not find any difference of atherosclerotic plaque burden, but again increased IFNγ production. To overcome xenogenous limitations, we then examined the effects of mouse Apolipoprotein B100 peptides P3 and P6. ApoB100-SP treatment again promoted a more IFNγ pronounced response upon in vitro recall. Flow cytometry analysis of cytokine secreting spleen cells revealed CD4 positive T cells to be mainly the source for IFNγ. In ApoE-/- mice that were administered ApoB100-SP during 20 weeks, the atherosclerotic plaque burden in aortic roots as well as total aorta was unchanged compared to PBS treated controls. Splenocyte proliferation upon antigen recall was not significantly altered in ApoB100-SP treated ApoE-/- mice. CONCLUSION: Although we did not observe a relevant anti-atherosclerotic benefit, the treatment with antigen-coupled splenocytes in its present form already impacts the immune responses and deserves further exploration.


Assuntos
Apolipoproteína B-100/imunologia , Apolipoproteínas E/deficiência , Aterosclerose/terapia , Lipoproteínas LDL/imunologia , Placa Aterosclerótica/terapia , Animais , Apolipoproteína B-100/química , Apolipoproteínas E/genética , Apolipoproteínas E/imunologia , Aterosclerose/genética , Aterosclerose/imunologia , Aterosclerose/patologia , Carbodi-Imidas/química , Terapia Baseada em Transplante de Células e Tecidos , Modelos Animais de Doenças , Feminino , Expressão Gênica , Humanos , Injeções Intravenosas , Interferon gama/biossíntese , Interferon gama/metabolismo , Lipoproteínas LDL/química , Transfusão de Linfócitos , Linfócitos/química , Linfócitos/imunologia , Macrófagos/química , Macrófagos/imunologia , Macrófagos/transplante , Masculino , Camundongos , Camundongos Knockout , Monócitos/química , Monócitos/imunologia , Monócitos/transplante , Placa Aterosclerótica/genética , Placa Aterosclerótica/imunologia , Placa Aterosclerótica/patologia , Baço/citologia , Baço/imunologia , Células Th1/imunologia , Células Th1/patologia , Falha de Tratamento
16.
PLoS One ; 10(6): e0131731, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26121471

RESUMO

Recent studies suggest the potential involvement of CD8+ T cells in the pathogenesis of murine hypertension. We recently reported that immunization with apoB-100 related peptide, p210, modified CD8+ T cell function in angiotensin II (AngII)-infused apoE (-/-) mice. In this study, we hypothesized that p210 vaccine modulates blood pressure in AngII-infused apoE (-/-) mice. Male apoE (-/-) mice were immunized with p210 vaccine and compared to unimmunized controls. At 10 weeks of age, mice were subcutaneously implanted with an osmotic pump which released AngII for 4 weeks. At 13 weeks of age, p210 immunized mice showed significantly lower blood pressure response to AngII compared to controls. CD8+ T cells from p210 immunized mice displayed a different phenotype compared to CD8+ T cells from unimmunized controls. Serum creatinine and urine albumin to creatinine ratio were significantly decreased in p210 immunized mice suggesting that p210 vaccine had renal protective effect. At euthanasia, inflammatory genes IL-6, TNF-α, and MCP-1 in renal tissue were down-regulated by p210 vaccine. Renal fibrosis and pro-fibrotic gene expression were also significantly reduced in p210 immunized mice. To assess the role of CD8+ T cells in these beneficial effects of p210 vaccine, CD8+ T cells were depleted by CD8 depleting antibody in p210 immunized mice. p210 immunized mice with CD8+ T cell depletion developed higher blood pressure compared to mice receiving isotype control. Depletion of CD8+ T cells also increased renal fibrotic gene expression compared to controls. We conclude that immunization with p210 vaccine attenuated AngII-induced hypertension and renal fibrosis. CD8+ T cells modulated by p210 vaccine could play an important role in the anti-hypertensive, anti-fibrotic and renal-protective effect of p210 vaccine.


Assuntos
Angiotensina II/efeitos adversos , Apolipoproteína B-100/imunologia , Hipertensão/etiologia , Hipertensão/prevenção & controle , Nefropatias/etiologia , Nefropatias/prevenção & controle , Peptídeos/imunologia , Animais , Apolipoproteína B-100/química , Pressão Sanguínea , Citocinas/metabolismo , Modelos Animais de Doenças , Fibrose , Expressão Gênica , Hipertensão/fisiopatologia , Imunização , Nefropatias/patologia , Depleção Linfocítica , Masculino , Camundongos , Camundongos Knockout , NADH NADPH Oxirredutases/metabolismo , NADPH Oxidase 1 , Peptídeos/administração & dosagem , Peptídeos/química , Espécies Reativas de Oxigênio , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Vacinas de Subunidades
17.
J Clin Endocrinol Metab ; 100(6): 2497-501, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25816050

RESUMO

INTRODUCTION: The objective of the study was to examine post hoc associations between plasma sphingolipids and lipoprotein kinetics in men with the metabolic syndrome after rosuvastatin treatment. MATERIALS AND METHODS: Plasma sphingolipid profiling, determined by tandem mass spectrometry, was performed in a randomized, double-blind, triple-crossover trial (n = 12) of 5-week treatment periods with placebo or rosuvastatin (10 or 40 mg/d) with 2-week washouts between treatments. RESULTS AND DISCUSSION: Baseline plasma ceramides were associated with very low-density lipoprotein (VLDL) apolipoprotein (apo)-B-100 concentration (r = 0.58, P < .05) and inversely with VLDL apoB-100 fractional catabolic rate (FCR; r = -0.67, P = .02). Posttreatment changes with rosuvastatin (40 mg/d) in plasma ceramides were inversely associated with VLDL apoB-100 FCR (r = -0.62, P = .03) independent of changes in plasma triglycerides, cholesterol, and low-density lipoprotein-cholesterol. By contrast, baseline and postrosuvastatin treatment plasma sphingomyelin levels were not associated with apoB-100 kinetics. Plasma ceramides and sphingomyelin were not associated with the kinetics or concentrations of high-density lipoprotein apoA-I, and low-density lipoprotein apoB. In the metabolic syndrome, the ability of rosuvastatin to increase VLDL apoB-100 FCR may reflect ceramide-specific mechanistic actions and/or sphingolipid exchange.


Assuntos
Anticolesterolemiantes/uso terapêutico , Apolipoproteína B-100/sangue , Ceramidas/sangue , Fluorbenzenos/uso terapêutico , Lipoproteínas VLDL/sangue , Síndrome Metabólica/tratamento farmacológico , Pirimidinas/uso terapêutico , Esfingomielinas/sangue , Sulfonamidas/uso terapêutico , Adulto , Apolipoproteína B-100/química , Humanos , Cinética , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Síndrome Metabólica/sangue , Síndrome Metabólica/complicações , Metabolismo/efeitos dos fármacos , Pessoa de Meia-Idade , Obesidade Abdominal/sangue , Obesidade Abdominal/complicações , Obesidade Abdominal/tratamento farmacológico , Rosuvastatina Cálcica
18.
Arch Biochem Biophys ; 573: 40-51, 2015 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-25795019

RESUMO

Atherosclerosis is characterised by the accumulation of lipids within macrophages in the artery wall. Low-density lipoprotein (LDL) is the source of this lipid, owing to the uptake of oxidised LDL by scavenger receptors. Myeloperoxidase (MPO) released by leukocytes during inflammation produces oxidants that are implicated in atherosclerosis. Modification of LDL by the MPO oxidant hypochlorous acid (HOCl), results in extensive lipid accumulation by macrophages. However, the reactivity of the other major MPO oxidant, hypothiocyanous acid (HOSCN) with LDL is poorly characterised, which is significant given that thiocyanate is the favoured substrate for MPO. In this study, we comprehensively compare the reactivity of HOCl and HOSCN with LDL, and show key differences in the profile of oxidative damage observed. HOSCN selectively modifies Cys residues on apolipoprotein B100, and oxidises cholesteryl esters resulting in formation of lipid hydroperoxides, 9-hydroxy-10,12-octadecadienoic acid (9-HODE) and F2-isoprostanes. The modification of LDL by HOSCN results macrophage lipid accumulation, though generally to a lesser extent than HOCl-modified LDL. This suggests that a change in the ratio of HOSCN:HOCl formation by MPO from variations in plasma thiocyanate levels, will influence the nature of LDL oxidation in vivo, and has implications for the progression of atherosclerosis.


Assuntos
Aterosclerose/patologia , Células Espumosas/patologia , Ácido Hipocloroso/metabolismo , Lipoproteínas LDL/metabolismo , Oxidantes/metabolismo , Peroxidase/metabolismo , Tiocianatos/metabolismo , Animais , Apolipoproteína B-100/química , Aterosclerose/metabolismo , Linhagem Celular , Colesterol/biossíntese , Ésteres do Colesterol/biossíntese , Células Espumosas/metabolismo , Humanos , Ácido Hipocloroso/química , Lipoproteínas LDL/química , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Oxidantes/química , Oxirredução , Tiocianatos/química
19.
Mol Biol Cell ; 26(4): 594-604, 2015 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-25518935

RESUMO

Apolipoprotein (apo) B is an obligatory component of very low density lipoprotein (VLDL), and its cotranslational and posttranslational modifications are important in VLDL synthesis, secretion, and hepatic lipid homeostasis. ApoB100 contains 25 cysteine residues and eight disulfide bonds. Although these disulfide bonds were suggested to be important in maintaining apoB100 function, neither the specific oxidoreductase involved nor the direct role of these disulfide bonds in apoB100-lipidation is known. Here we used RNA knockdown to evaluate both MTP-dependent and -independent roles of PDI1 in apoB100 synthesis and lipidation in McA-RH7777 cells. Pdi1 knockdown did not elicit any discernible detrimental effect under normal, unstressed conditions. However, it decreased apoB100 synthesis with attenuated MTP activity, delayed apoB100 oxidative folding, and reduced apoB100 lipidation, leading to defective VLDL secretion. The oxidative folding-impaired apoB100 was secreted mainly associated with LDL instead of VLDL particles from PDI1-deficient cells, a phenotype that was fully rescued by overexpression of wild-type but not a catalytically inactive PDI1 that fully restored MTP activity. Further, we demonstrate that PDI1 directly interacts with apoB100 via its redox-active CXXC motifs and assists in the oxidative folding of apoB100. Taken together, these findings reveal an unsuspected, yet key role for PDI1 in oxidative folding of apoB100 and VLDL assembly.


Assuntos
Apolipoproteína B-100/química , Isomerases de Dissulfetos de Proteínas/fisiologia , Animais , Apolipoproteína B-100/biossíntese , Linhagem Celular , Retículo Endoplasmático/metabolismo , Técnicas de Silenciamento de Genes , Homeostase , Metabolismo dos Lipídeos , Camundongos , Estresse Oxidativo , Isomerases de Dissulfetos de Proteínas/química , Isomerases de Dissulfetos de Proteínas/genética , Ratos
20.
J Pediatr Gastroenterol Nutr ; 60(1): 42-7, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25250685

RESUMO

OBJECTIVES: Crohn disease (CD) and ulcerative colitis (UC), known collectively as inflammatory bowel diseases (IBDs), are chronic immunoinflammatory pathologies of unknown aetiology. Despite the frequent use of biomarkers in medical practice, there is a relative lack of information regarding validated paediatric biomarkers for IBD. Furthermore, biomarkers proved to be efficacious in adults are frequently extrapolated to the paediatric clinical setting without considering that the pathogenesis of many diseases is distinctly different in children. In the present study, proteomics technology was used to monitor differences in protein expression among adult and young patients with CD, identify a panel of candidate protein biomarkers that may be used to improve prognostic-diagnostic accuracy, and advance paediatric medical care. METHODS: Male and female serum samples from 12 adults and 12 children with active CD were subjected to 2-dimensional gel electrophoresis. Following the relative quantitation of protein spots exhibiting a differential expression between the 2 groups by densitometry, the spots were further characterized by matrix-assisted laser desorption tandem time-of-flight mass spectrometer. The results were confirmed by Western blot analysis. RESULTS: Clusterin was found to be significantly overexpressed in adults with CD, whereas ceruloplasmin and apolipoprotein B-100 were found to be significantly overexpressed in children, indicating that the expression of these proteins may be implicated in the onset or progression of CD in these 2 subgroups of patients. CONCLUSIONS: Interestingly, we found a differential expression of several proteins in adults versus paediatric patients with CD. Undoubtedly, future experiments using a larger cohort of patients with CD are needed to evaluate the relevance of our preliminary findings.


Assuntos
Apolipoproteína B-100/sangue , Ceruloplasmina/análise , Clusterina/sangue , Doença de Crohn/sangue , Adulto , Idade de Início , Apolipoproteína B-100/química , Apolipoproteína B-100/metabolismo , Biomarcadores/sangue , Biomarcadores/química , Biomarcadores/metabolismo , Proteínas Sanguíneas/análise , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Western Blotting , Ceruloplasmina/química , Ceruloplasmina/metabolismo , Criança , Clusterina/química , Clusterina/metabolismo , Doença de Crohn/epidemiologia , Doença de Crohn/fisiopatologia , Feminino , Grécia/epidemiologia , Humanos , Masculino , Mapeamento de Peptídeos , Proteômica/métodos , Índice de Gravidade de Doença , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Eletroforese em Gel Diferencial Bidimensional
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