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1.
Circ Res ; 127(6): 778-792, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32495699

RESUMO

RATIONALE: The HDL (high-density lipoprotein)-mediated stimulation of cellular cholesterol efflux initiates macrophage-specific reverse cholesterol transport (m-RCT), which ends in the fecal excretion of macrophage-derived unesterified cholesterol (UC). Early studies established that LDL (low-density lipoprotein) particles could act as efficient intermediate acceptors of cellular-derived UC, thereby preventing the saturation of HDL particles and facilitating their cholesterol efflux capacity. However, the capacity of LDL to act as a plasma cholesterol reservoir and its potential impact in supporting the m-RCT pathway in vivo both remain unknown. OBJECTIVE: We investigated LDL contributions to the m-RCT pathway in hypercholesterolemic mice. METHODS AND RESULTS: Macrophage cholesterol efflux induced in vitro by LDL added to the culture media either alone or together with HDL or ex vivo by plasma derived from subjects with familial hypercholesterolemia was assessed. In vivo, m-RCT was evaluated in mouse models of hypercholesterolemia that were naturally deficient in CETP (cholesteryl ester transfer protein) and fed a Western-type diet. LDL induced the efflux of radiolabeled UC from cultured macrophages, and, in the simultaneous presence of HDL, a rapid transfer of the radiolabeled UC from HDL to LDL occurred. However, LDL did not exert a synergistic effect on HDL cholesterol efflux capacity in the familial hypercholesterolemia plasma. The m-RCT rates of the LDLr (LDL receptor)-KO (knockout), LDLr-KO/APOB100, and PCSK9 (proprotein convertase subtilisin/kexin type 9)-overexpressing mice were all significantly reduced relative to the wild-type mice. In contrast, m-RCT remained unchanged in HAPOB100 Tg (human APOB100 transgenic) mice with fully functional LDLr, despite increased levels of plasma APO (apolipoprotein)-B-containing lipoproteins. CONCLUSIONS: Hepatic LDLr plays a critical role in the flow of macrophage-derived UC to feces, while the plasma increase of APOB-containing lipoproteins is unable to stimulate m-RCT. The results indicate that, besides the major HDL-dependent m-RCT pathway via SR-BI (scavenger receptor class B type 1) to the liver, a CETP-independent m-RCT path exists, in which LDL mediates the transfer of cholesterol from macrophages to feces. Graphical Abstract: A graphical abstract is available for this article.


Assuntos
HDL-Colesterol/sangue , LDL-Colesterol/sangue , Hiperlipoproteinemia Tipo II/sangue , Fígado/metabolismo , Macrófagos/metabolismo , Receptores de LDL/metabolismo , Animais , Apolipoproteína B-100/sangue , Apolipoproteína B-100/genética , Transporte Biológico , Linhagem Celular , Proteínas de Transferência de Ésteres de Colesterol/genética , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Modelos Animais de Doenças , Fezes/química , Humanos , Hiperlipoproteinemia Tipo II/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de LDL/deficiência , Receptores de LDL/genética , Receptores Depuradores Classe B/metabolismo
2.
Arterioscler Thromb Vasc Biol ; 40(3): 597-610, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31996021

RESUMO

OBJECTIVE: By binding to its high-affinity receptor FcεR1, IgE activates mast cells, macrophages, and other inflammatory and vascular cells. Recent studies support an essential role of IgE in cardiometabolic diseases. Plasma IgE level is an independent predictor of human coronary heart disease. Yet, a direct role of IgE and its mechanisms in cardiometabolic diseases remain incompletely understood. Approach and Results: Using atherosclerosis prone Apoe-/- mice and IgE-deficient Ige-/- mice, we demonstrated that IgE deficiency reduced atherosclerosis lesion burden, lesion lipid deposition, smooth muscle cell and endothelial cell contents, chemokine MCP (monocyte chemoattractant protein)-1 expression and macrophage accumulation. IgE deficiency also reduced bodyweight gain and increased glucose and insulin sensitivities with significantly reduced plasma cholesterol, triglyceride, insulin, and inflammatory cytokines and chemokines, including IL (interleukin)-6, IFN (interferon)-γ, and MCP-1. From atherosclerotic lesions and peritoneal macrophages from Apoe-/-Ige-/- mice that consumed an atherogenic diet, we detected reduced expression of M1 macrophage markers (CD68, MCP-1, TNF [tumor necrosis factor]-α, IL-6, and iNOS [inducible nitric oxide synthase]) but increased expression of M2 macrophage markers (Arg [arginase]-1 and IL-10) and macrophage-sterol-responsive-network molecules (complement C3, lipoprotein lipase, LDLR [low-density lipoprotein receptor]-related protein 1, and TFR [transferrin]) that suppress macrophage foam cell formation. These IgE activities can be reproduced in bone marrow-derived macrophages from wild-type mice, but muted in cells from FcεR1-deficient mice, or blocked by anti-IgE antibody or complement C3 deficiency. CONCLUSIONS: IgE deficiency protects mice from diet-induced atherosclerosis, obesity, glucose tolerance, and insulin resistance by regulating macrophage polarization, macrophage-sterol-responsive-network gene expression, and foam cell formation.


Assuntos
Aorta/metabolismo , Aterosclerose/metabolismo , Células Espumosas/metabolismo , Imunoglobulina E/metabolismo , Inflamação/metabolismo , Ativação de Macrófagos , Macrófagos Peritoneais/metabolismo , Obesidade/metabolismo , Animais , Aorta/imunologia , Aorta/patologia , Aterosclerose/imunologia , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Glicemia/metabolismo , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Células Espumosas/imunologia , Células Espumosas/patologia , Redes Reguladoras de Genes , Imunoglobulina E/deficiência , Imunoglobulina E/genética , Inflamação/imunologia , Inflamação/patologia , Inflamação/prevenção & controle , Mediadores da Inflamação/metabolismo , Resistência à Insulina , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Obesidade/imunologia , Obesidade/patologia , Obesidade/prevenção & controle , Fenótipo , Placa Aterosclerótica , Receptores de IgE/genética , Receptores de IgE/metabolismo , Transdução de Sinais , Esteróis/metabolismo
3.
Arterioscler Thromb Vasc Biol ; 38(11): 2750-2754, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30354236

RESUMO

Objective- The ability of HDL (high-density lipoprotein) to promote macrophage cholesterol efflux is considered the main HDL cardioprotective function. Abdominal aortic aneurysm (AAA) is usually characterized by cholesterol accumulation and macrophage infiltration in the aortic wall. Here, we aim to evaluate the composition of circulating HDL particles and their potential for promoting macrophage cholesterol efflux in AAA subjects. Approach and Results- First, we randomly selected AAA and control subjects from Spain. The AAA patients in the Spanish cohort showed lower plasma apoA-I levels concomitantly associated with low levels of plasma HDL cholesterol and the amount of preß-HDL particles. We determined macrophage cholesterol efflux to apoB-depleted plasma, which contains mature HDL, preß-HDL particles and HDL regulatory proteins. ApoB-depleted plasma from AAA patients displayed an impaired ability to promote macrophage cholesterol efflux. Next, we replicated the experiments with AAA and control subjects derived from Danish cohort. Danish AAA patients also showed lower apoA-I levels and a defective HDL-mediated macrophage cholesterol efflux. Conclusions- AAA patients show impaired HDL-facilitated cholesterol removal from macrophages, which could be mechanistically linked to AAA.


Assuntos
Aneurisma da Aorta Abdominal/sangue , HDL-Colesterol/sangue , Macrófagos/metabolismo , Idoso , Aneurisma da Aorta Abdominal/diagnóstico por imagem , Apolipoproteína A-I/sangue , Apolipoproteína B-100/sangue , Estudos de Casos e Controles , Dinamarca , Feminino , Lipoproteínas de Alta Densidade Pré-beta/sangue , Humanos , Masculino , Espanha
4.
Int J Mol Sci ; 20(3)2019 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-30717414

RESUMO

Human apolipoprotein A-I (hApoA-I) overexpression improves high-density lipoprotein (HDL) function and the metabolic complications of obesity. We used a mouse model of diabesity, the db/db mouse, to examine the effects of hApoA-I on the two main functional properties of HDL, i.e., macrophage-specific reverse cholesterol transport (m-RCT) in vivo and the antioxidant potential, as well as the phenotypic features of obesity. HApoA-I transgenic (hA-I) mice were bred with nonobese control (db/+) mice to generate hApoA-I-overexpressing db/+ offspring, which were subsequently bred to obtain hA-I-db/db mice. Overexpression of hApoA-I significantly increased weight gain and the incidence of fatty liver in db/db mice. Weight gain was mainly explained by the increased caloric intake of hA-I-db/db mice (>1.2-fold). Overexpression of hApoA-I also produced a mixed type of dyslipidemia in db/db mice. Despite these deleterious effects, the overexpression of hApoA-I partially restored m-RCT in db/db mice to levels similar to nonobese control mice. Moreover, HDL from hA-I-db/db mice also enhanced the protection against low-density lipoprotein (LDL) oxidation compared with HDL from db/db mice. In conclusion, overexpression of hApoA-I in db/db mice enhanced two main anti-atherogenic HDL properties while exacerbating weight gain and the fatty liver phenotype. These adverse metabolic side-effects were also observed in obese mice subjected to long-term HDL-based therapies in independent studies and might raise concerns regarding the use of hApoA-I-mediated therapy in obese humans.


Assuntos
Apolipoproteína A-I/genética , Colesterol/metabolismo , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Expressão Gênica , Macrófagos/metabolismo , Animais , Transporte Biológico , Peso Corporal , Modelos Animais de Doenças , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Perfilação da Expressão Gênica , Humanos , Camundongos
5.
Lipids Health Dis ; 17(1): 285, 2018 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-30545366

RESUMO

BACKGROUND: The focus of studies on high-density lipoproteins (HDL) has shifted from HDL-cholesterol (HDL-C) to HDL function. We recently demonstrated that low USF1 expression in mice and humans associates with high plasma HDL-C and low triglyceride levels, as well as protection against obesity, insulin resistance, and atherosclerosis. Here, we studied the impact of USF1 deficiency on HDL functional capacity and macrophage atherogenic functions, including inflammation, cholesterol efflux, and cholesterol accumulation. METHODS: We used a congenic Usf1 deficient mice in C57Bl/6JRccHsd background and blood samples were collected to isolate HDL for structural and functional studies. Lentiviral preparations containing the USF1 silencing shRNA expression vector were used to silence USF1 in human THP-1 and Huh-7 cells. Cholesterol efflux from acetyl-LDL loaded THP-1 macrophages was measured using HDL and plasma as acceptors. Gene expression analysis from USF1 silenced peritoneal macrophages was carried out using Affymetrix protocols. RESULTS: We show that Usf1 deficiency not only increases HDL-C levels in vivo, consistent with elevated ABCA1 protein expression in hepatic cell lines, but also improves the functional capacity of HDL particles. HDL particles derived from Usf1 deficient mice remove cholesterol more efficiently from macrophages, attributed to their higher contents of phospholipids. Furthermore, silencing of USF1 in macrophages enhanced the cholesterol efflux capacity of these cells. These findings are consistent with reduced inflammatory burden of USF1 deficient macrophages, manifested by reduced secretion of pro-inflammatory cytokines MCP-1 and IL-1ß and protection against inflammation-induced macrophage cholesterol accumulation in a cell-autonomous manner. CONCLUSIONS: Our findings identify USF1 as a novel factor regulating HDL functionality, showing that USF1 inactivation boosts cholesterol efflux, reduces macrophage inflammation and attenuates macrophage cholesterol accumulation, linking improved macrophage cholesterol metabolism and inflammatory pathways to the antiatherogenic function of USF1 deficiency.


Assuntos
HDL-Colesterol/genética , Colesterol/genética , Lipoproteínas HDL/genética , Fatores Estimuladores Upstream/genética , Transportador 1 de Cassete de Ligação de ATP/genética , Animais , Quimiocina CCL2/genética , Colesterol/sangue , Expressão Gênica/genética , Humanos , Inflamação/sangue , Inflamação/genética , Inflamação/patologia , Resistência à Insulina/genética , Lipoproteínas HDL/sangue , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/patologia , Masculino , Camundongos , Camundongos Knockout , Obesidade/sangue , Obesidade/genética , Obesidade/patologia
6.
Arterioscler Thromb Vasc Biol ; 36(2): 274-84, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26681753

RESUMO

OBJECTIVE: Apolipoprotein A-I (apoA-I) has been shown to possess several atheroprotective functions, including inhibition of inflammation. Protease-secreting activated mast cells reside in human atherosclerotic lesions. Here we investigated the effects of the neutral proteases released by activated mast cells on the anti-inflammatory properties of apoA-I. APPROACH AND RESULTS: Activation of human mast cells triggered the release of granule-associated proteases chymase, tryptase, cathepsin G, carboxypeptidase A, and granzyme B. Among them, chymase cleaved apoA-I with the greatest efficiency and generated C-terminally truncated apoA-I, which failed to bind with high affinity to human coronary artery endothelial cells. In tumor necrosis factor-α-activated human coronary artery endothelial cells, the chymase-cleaved apoA-I was unable to suppress nuclear factor-κB-dependent upregulation of vascular cell adhesion molecule-1 (VCAM-1) and to block THP-1 cells from adhering to and transmigrating across the human coronary artery endothelial cells. Chymase-cleaved apoA-I also had an impaired ability to downregulate the expression of tumor necrosis factor-α, interleukin-1ß, interleukin-6, and interleukin-8 in lipopolysaccharide-activated GM-CSF (granulocyte-macrophage colony-stimulating factor)- and M-CSF (macrophage colony-stimulating factor)-differentiated human macrophage foam cells and to inhibit reactive oxygen species formation in PMA (phorbol 12-myristate 13-acetate)-activated human neutrophils. Importantly, chymase-cleaved apoA-I showed reduced ability to inhibit lipopolysaccharide-induced inflammation in vivo in mice. Treatment with chymase blocked the ability of the apoA-I mimetic peptide L-4F, but not of the protease-resistant D-4F, to inhibit proinflammatory gene expression in activated human coronary artery endothelial cells and macrophage foam cells and to prevent reactive oxygen species formation in activated neutrophils. CONCLUSIONS: The findings identify C-terminal cleavage of apoA-I by human mast cell chymase as a novel mechanism leading to loss of its anti-inflammatory functions. When targeting inflamed protease-rich atherosclerotic lesions with apoA-I, infusions of protease-resistant apoA-I might be the appropriate approach.


Assuntos
Apolipoproteína A-I/metabolismo , Aterosclerose/enzimologia , Quimases/metabolismo , Células Endoteliais/metabolismo , Inflamação/enzimologia , Mastócitos/enzimologia , Apolipoproteína A-I/farmacologia , Aterosclerose/imunologia , Aterosclerose/prevenção & controle , Adesão Celular , Linhagem Celular Tumoral , Colesterol/metabolismo , Técnicas de Cocultura , Citocinas/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/imunologia , Células Espumosas/imunologia , Células Espumosas/metabolismo , Humanos , Inflamação/imunologia , Inflamação/prevenção & controle , Mediadores da Inflamação/metabolismo , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , NF-kappa B/metabolismo , Ativação de Neutrófilo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Peptídeos/farmacologia , Estrutura Terciária de Proteína , Proteólise , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Migração Transendotelial e Transepitelial , Molécula 1 de Adesão de Célula Vascular/metabolismo
7.
Scand J Clin Lab Invest ; 77(8): 601-609, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28972399

RESUMO

Genetic variants of angiopoietin-like protein 3 (ANGPTL3) are associated with serum triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) concentration in GWASs. ANGPTL3 deficiency causes declined TG, total cholesterol (TC), LDL-C, high-density lipoprotein cholesterol (HDL-C), apolipoprotein B (apoB) and apolipoprotein A-I (apoA-I) serum concentration, a phenotype defined as familial combined hypolipidaemia (FHBL2). Our aim is to establish whether ANGPTL3 serum protein concentration correlates with lipoproteins and lipids in hyper- or hypolipidaemic subjects, and whether ANGPTL3 sequence variants are associated with untypical lipid profiles. Additionally, 10 subjects with very low lipoprotein concentrations were sequenced for ANGPTL3 for possible loss-of-function (LOF) variants. Study subjects were selected from Finnish FINRISK and Health 2000 surveys. ANGPTL protein concentrations were measured by ELISA method. As a result, ANGPTL3 serum concentration correlated positively with age, phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein (CETP) activities, but not with any of the lipid or lifestyle attributes. No ANGPTL3 variants were found among sequenced samples. Subjects who carried ANGPTL3 sequence variants rs12563308 (n = 4) and rs199772471 (n = 1) had abnormally high TC and LDL-C concentrations. Whole exome sequencing data of these five subjects were further analyzed for rare and deleterious missense variants in genes associated with cholesterol metabolism. In conclusion, ANGPTL3 serum protein concentration did not predict lipid concentrations, unlike apolipoprotein C-III (apoC-III) which positively correlated with most of the lipid attributes. ANGPTL3 variant screen yielded five carriers with abnormally high TC concentration; the actual genetic causality, however, could not be verified.


Assuntos
Proteínas Semelhantes a Angiopoietina/sangue , Hipercolesterolemia/genética , Adulto , Idoso , Proteína 3 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina/genética , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Estudos Transversais , Análise Mutacional de DNA , Feminino , Finlândia , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Hipercolesterolemia/sangue , Masculino , Pessoa de Meia-Idade , Mutação , Polimorfismo de Nucleotídeo Único , Fatores de Risco
8.
J Lipid Res ; 57(6): 1097-107, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27040449

RESUMO

The consequences of angiopoietin-like protein 3 (ANGPTL3) deficiency on postprandial lipid and lipoprotein metabolism has not been investigated in humans. We studied 7 homozygous (undetectable circulating ANGPTL3 levels) and 31 heterozygous (50% of circulating ANGPTL3 levels) subjects with familial combined hypolipidemia (FHBL2) due to inactivating ANGPTL3 mutations in comparison with 35 controls. All subjects were evaluated at fasting and during 6 h after a high fat meal. Postprandial lipid and lipoprotein changes were quantified by calculating the areas under the curve (AUCs) using the 6 h concentration data. Plasma changes of ß-hydroxybutyric acid (ß-HBA) were measured as marker of hepatic oxidation of fatty acids. Compared with controls, homozygotes showed lower incremental AUCs (iAUCs) of total TG (-69%, P < 0.001), TG-rich lipoproteins (-90%, P < 0.001), apoB-48 (-78%, P = 0.032), and larger absolute increase of FFA (128%, P < 00.1). Also, heterozygotes displayed attenuated postprandial lipemia, but the difference was significant only for the iAUC of apoB-48 (-28%; P < 0.05). During the postprandial period, homozygotes, but not heterozygotes, showed a lower increase of ß-HBA. Our findings demonstrate that complete ANGPTL3 deficiency associates with highly reduced postprandial lipemia probably due to faster catabolism of intestinally derived lipoproteins, larger expansion of the postprandial FFA pool, and decreased influx of dietary-derived fatty acids into the liver. These results add information on mechanisms underlying hypolipidemia in FHBL2.


Assuntos
Angiopoietinas/genética , Ácidos Graxos não Esterificados/sangue , Hipobetalipoproteinemias/sangue , Lipídeos/sangue , Proteína 3 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Angiopoietinas/sangue , Angiopoietinas/deficiência , Apolipoproteína B-48/sangue , Feminino , Heterozigoto , Homozigoto , Humanos , Hipobetalipoproteinemias/genética , Hipobetalipoproteinemias/patologia , Lipoproteínas/sangue , Masculino , Pessoa de Meia-Idade , Mutação , Período Pós-Prandial , Triglicerídeos/sangue
9.
J Biol Chem ; 290(48): 28977-87, 2015 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-26468283

RESUMO

The alternative pathway of complement is an important part of the innate immunity response against foreign particles invading the human body. To avoid damage to host cells, it needs to be efficiently down-regulated by plasma factor H (FH) as exemplified by various diseases caused by mutations in its domains 19-20 (FH19-20) and 5-7 (FH5-7). These regions are also the main interaction sites for microbial pathogens that bind host FH to evade complement attack. We previously showed that inhibition of FH binding by a recombinant FH5-7 construct impairs survival of FH binding pathogens in human blood. In this study we found that upon exposure to full blood, the addition of FH5-7 reduces survival of, surprisingly, also those microbes that are not able to bind FH. This effect was mediated by inhibition of complement regulation and subsequently enhanced neutrophil phagocytosis by FH5-7. We found that although FH5-7 does not reduce complement regulation in the actual fluid phase of plasma, it reduces regulation on HDL particles in plasma. Using affinity chromatography and mass spectrometry we revealed that FH interacts with serum apolipoprotein E (apoE) via FH5-7 domains. Furthermore, binding of FH5-7 to HDL was dependent on the concentration of apoE on the HDL particles. These findings explain why the addition of FH5-7 to plasma leads to excessive complement activation and phagocytosis of microbes in full anticoagulated blood. In conclusion, our data show how FH interacts with apoE molecules via domains 5-7 and regulates alternative pathway activation on plasma HDL particles.


Assuntos
Apolipoproteínas E/química , Fator H do Complemento/química , Lipoproteínas HDL/química , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Cromatografia de Afinidade , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Humanos , Lipoproteínas HDL/genética , Lipoproteínas HDL/metabolismo , Espectrometria de Massas , Ligação Proteica , Estrutura Terciária de Proteína
10.
Cardiovasc Diabetol ; 15: 26, 2016 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-26852231

RESUMO

BACKGROUND: Diabetes is a risk factor for atherosclerosis associated with oxidative stress, inflammation and cell proliferation. The purpose of this study was to evaluate arterial choline uptake and its relationship to atherosclerotic inflammation in diabetic and non-diabetic hypercholesterolemic mice. METHODS: Low-density lipoprotein-receptor deficient mice expressing only apolipoprotein B100, with or without type 2 diabetes caused by pancreatic overexpression of insulin-like growth factor II (IGF-II/LDLR(-/-)ApoB(100/100) and LDLR(-/-)ApoB(100/100)) were studied. Distribution kinetics of choline analogue (18)F-fluoromethylcholine ((18)F-FMCH) was assessed in vivo by positron emission tomography (PET) imaging. Then, aortic uptakes of (18)F-FMCH and glucose analogue (18)F-fluorodeoxyglucose ((18)F-FDG), were assessed ex vivo by gamma counting and autoradiography of tissue sections. The (18)F-FMCH uptake in atherosclerotic plaques was further compared with macrophage infiltration and the plasma levels of cytokines and metabolic markers. RESULTS: The aortas of all hypercholesterolemic mice showed large, macrophage-rich atherosclerotic plaques. The plaque burden and densities of macrophage subtypes were similar in diabetic and non-diabetic animals. The blood clearance of (18)F-FMCH was rapid. Both the absolute (18)F-FMCH uptake in the aorta and the aorta-to-blood uptake ratio were higher in diabetic than in non-diabetic mice. In autoradiography, the highest (18)F-FMCH uptake co-localized with macrophage-rich atherosclerotic plaques. (18)F-FMCH uptake in plaques correlated with levels of total cholesterol, insulin, C-peptide and leptin. In comparison with (18)F-FDG, (18)F-FMCH provided similar or higher plaque-to-background ratios in diabetic mice. CONCLUSIONS: Type 2 diabetes enhances the uptake of choline that reflects inflammation in atherosclerotic plaques in mice. PET tracer (18)F-FMCH is a potential tool to study vascular inflammation associated with diabetes.


Assuntos
Aorta/diagnóstico por imagem , Doenças da Aorta/diagnóstico por imagem , Aterosclerose/diagnóstico por imagem , Colina/análogos & derivados , Diabetes Mellitus Tipo 2/complicações , Angiopatias Diabéticas/diagnóstico por imagem , Radioisótopos de Flúor , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos/farmacocinética , Animais , Aorta/metabolismo , Doenças da Aorta/sangue , Doenças da Aorta/etiologia , Doenças da Aorta/metabolismo , Aterosclerose/sangue , Aterosclerose/etiologia , Aterosclerose/metabolismo , Biomarcadores/sangue , Colina/administração & dosagem , Colina/farmacocinética , Citocinas/sangue , Angiopatias Diabéticas/sangue , Angiopatias Diabéticas/etiologia , Angiopatias Diabéticas/metabolismo , Modelos Animais de Doenças , Hipercolesterolemia , Macrófagos/metabolismo , Macrófagos/efeitos da radiação , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Placa Aterosclerótica , Valor Preditivo dos Testes , Compostos Radiofarmacêuticos/administração & dosagem , Distribuição Tecidual
11.
FASEB J ; 29(4): 1435-45, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25550459

RESUMO

Various cell types in atherosclerotic lesions express matrix metalloproteinase (MMP)-8. We investigated whether MMP-8 affects the structure and antiatherogenic function of apolipoprotein (apo) A-I, the main protein component of HDL particles. Furthermore, we studied serum lipid profiles and cholesterol efflux capacity in MMP-8-deficient mouse model. Incubation of apoA-I (28 kDa) with activated MMP-8 yielded 22 kDa and 25 kDa apoA-I fragments. Mass spectrometric analyses revealed that apoA-I was cleaved at its carboxyl-terminal part. Treatment of apoA-I and HDL with MMP-8 resulted in significant reduction (up to 84%, P < 0.001) in their ability to facilitate cholesterol efflux from cholesterol-loaded THP-1 macrophages. The cleavage of apoA-I by MMP-8 and the reduction in its cholesterol efflux capacity was inhibited by doxycycline. MMP-8-deficient mice had significantly lower serum triglyceride (TG) levels (P = 0.003) and larger HDL particles compared with wild-type (WT) mice. However, no differences were observed in the apoA-I levels or serum cholesterol efflux capacities between the mouse groups. Proteolytic modification of apoA-I by MMP-8 may impair the first steps of reverse cholesterol transport, leading to increased accumulation of cholesterol in the vessel walls. Eventually, inhibition of MMPs by doxycycline may reduce the risk for atherosclerotic vascular diseases.


Assuntos
Apolipoproteína A-I/metabolismo , Colesterol/metabolismo , Metaloproteinase 8 da Matriz/metabolismo , Sequência de Aminoácidos , Animais , Apolipoproteína A-I/química , Apolipoproteína A-I/genética , Aterosclerose/etiologia , Aterosclerose/metabolismo , Sítios de Ligação/genética , Transporte Biológico Ativo , Linhagem Celular , Colesterol/sangue , Feminino , Humanos , Lipoproteínas HDL/metabolismo , Macrófagos/metabolismo , Masculino , Metaloproteinase 8 da Matriz/deficiência , Metaloproteinase 8 da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteólise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Triglicerídeos/sangue
12.
Biochemistry ; 54(38): 5856-66, 2015 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-26337529

RESUMO

Phospholipid transfer protein (PLTP), a main protein in lipid and lipoprotein metabolism, exists in high-activity (HA-PLTP) and low-activity (LA-PLTP) forms in human plasma. Proper phospholipid transfer activity of PLTP is modulated by interactions with various apolipoproteins (apo) including apoE. The domains of apoE involved in interactions with PLTP are not known. Here we analyzed the capacity of recombinant apoE isoforms and apoE4 mutants with progressive carboxyl-terminal deletions to bind to and activate HA-PLTP and LA-PLTP. Our analyses demonstrated that lipid-free apoE isoforms bind to both HA-PLTP and LA-PLTP, resulting in phospholipid transfer activation, with apoE3 inducing the highest PLTP activation. The isoform-specific differences in apoE/PLTP binding and PLTP activation were abolished following apoE lipidation. Lipid-free apoE4[Δ(260-299)], apoE4[Δ(230-299)], apoE4[Δ(203-299)], and apoE4[Δ(186-299)] activated HA-PLTP by 120-160% compared to full-length apoE4. Lipid-free apoE4[Δ(186-299)] also activated LA-PLTP by 85% compared to full-length apoE4. All lipidated truncated apoE4 forms displayed a similar effect on HA-PLTP and LA-PLTP activity as full-length apoE4. Strikingly, lipid-free or lipidated full-length apoE4 and apoE4[Δ(186-299)] demonstrated similar binding capacity to LA-PLTP and HA-PLTP. Biophysical studies showed that the carboxyl-terminal truncations of apoE4 resulted in small changes of the structural or thermodynamic properties of lipidated apoE4, that were much less pronounced compared to changes observed previously for lipid-free apoE4. Overall, our findings show an isoform-dependent binding to and activation of PLTP by lipid-free apoE. Furthermore, the domain of apoE4 required for PLTP activation resides within its amino-terminal 1-185 region. The apoE/PLTP interactions can be modulated by the conformation and lipidation state of apoE.


Assuntos
Apolipoproteína E4/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Apolipoproteína E4/química , Apolipoproteína E4/genética , Apolipoproteínas E/química , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Linhagem Celular , Humanos , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência
13.
J Biol Chem ; 289(8): 4683-98, 2014 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-24369175

RESUMO

Phospholipid transfer protein (PLTP) is a key protein involved in biogenesis and remodeling of plasma HDL. Several neuroprotective properties have been ascribed to HDL. We reported earlier that liver X receptor (LXR) activation promotes cellular cholesterol efflux and formation of HDL-like particles in an established in vitro model of the blood-brain barrier (BBB) consisting of primary porcine brain capillary endothelial cells (pBCEC). Here, we report PLTP synthesis, regulation, and its key role in HDL metabolism at the BBB. We demonstrate that PLTP is highly expressed and secreted by pBCEC. In a polarized in vitro model mimicking the BBB, pBCEC secreted phospholipid-transfer active PLTP preferentially to the basolateral ("brain parenchymal") compartment. PLTP expression levels and phospholipid transfer activity were enhanced (up to 2.5-fold) by LXR activation using 24(S)-hydroxycholesterol (a cerebral cholesterol metabolite) or TO901317 (a synthetic LXR agonist). TO901317 administration elevated PLTP activity in BCEC from C57/BL6 mice. Preincubation of HDL3 with human plasma-derived active PLTP resulted in the formation of smaller and larger HDL particles and enhanced the capacity of the generated HDL particles to remove cholesterol from pBCEC by up to 3-fold. Pre-ß-HDL, detected by two-dimensional crossed immunoelectrophoresis, was generated from HDL3 in pBCEC-derived supernatants, and their generation was markedly enhanced (1.9-fold) upon LXR activation. Furthermore, RNA interference-mediated PLTP silencing (up to 75%) reduced both apoA-I-dependent (67%) and HDL3-dependent (30%) cholesterol efflux from pBCEC. Based on these findings, we propose that PLTP is actively involved in lipid transfer, cholesterol efflux, HDL genesis, and remodeling at the BBB.


Assuntos
Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/metabolismo , Células Endoteliais/metabolismo , Lipoproteínas HDL/biossíntese , Proteínas de Transferência de Fosfolipídeos/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Animais , Apolipoproteína A-I/metabolismo , Transporte Biológico , Capilares/citologia , Polaridade Celular , Colesterol/metabolismo , Inativação Gênica , Humanos , Receptores X do Fígado , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Receptores Nucleares Órfãos/agonistas , Receptores Nucleares Órfãos/metabolismo , Estrutura Quaternária de Proteína , Sus scrofa , Regulação para Cima
14.
Analyst ; 140(9): 3175-82, 2015 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-25751597

RESUMO

In this work, a method to study and analyze the interaction data in free solution by exploiting partial filling affinity capillary electrophoresis (PF-ACE) followed by adsorption energy distribution calculations (AED) prior model fit to adsorption isotherms will be demonstrated. PF-ACE-AED approach allowed the possibility to distinguish weak and strong interactions of the binding processes between the most common apolipoprotein E protein isoforms (apoE2, apoE3, apoE4) of high density lipoprotein (HDL) and apoE-containing HDL2 with major glycosaminoglycan (GAG) chain of proteoglycans (PGs), chondroitin-6-sulfate (C6S). The AED analysis clearly revealed the heterogeneity of the binding processes. The major difference was that they were heterogeneous with two different adsorption sites for apoE2 and apoE4 isoforms, whereas interestingly for apoE3 and apoE-containing HDL2, the binding was homogeneous (one site) adsorption process. Moreover, our results allowed the evaluation of differences in the binding process strengths giving the following order with C6S: apoE-containing HDL2 > apoE2 > apoE4 > apoE3. In addition, the affinity constant values determined could be compared with those obtained in our previous studies for the interactions between apoE isoforms and another important GAG chain of PGs - dermatan sulfate (DS). The success of the combination of AED calculations prior to non-linear adsorption isotherm model fit with PF-ACE when the concentration range was extended, confirmed the power of the system in the clarification of the heterogeneity of biological processes studied.


Assuntos
Apolipoproteínas E/metabolismo , Eletroforese Capilar/métodos , Mapeamento de Interação de Proteínas/métodos , Adsorção , Apolipoproteínas E/isolamento & purificação , Ligação Proteica , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismo
15.
Biophys J ; 107(1): 114-25, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24988346

RESUMO

Estradiol (E2) and E2 oleate associate with high-density lipoproteins (HDLs). Their orientation in HDLs is unknown. We studied the orientation of E2 and E2 oleate in membranes and reconstituted HDLs, finding that E2 and E2 oleate are membrane-associated and highly mobile. Our combination of NMR measurements, molecular dynamics simulation, and analytic theory identifies three major conformations where the long axis of E2 assumes a parallel, perpendicular, or antiparallel orientation relative to the membrane's z-direction. The perpendicular orientation is preferred, and furthermore, in this orientation, E2 strongly favors a particular roll angle, facing the membrane with carbons 6, 7, 15, and 16, whereas carbons 1, 2, 11, and 12 point toward the aqueous phase. In contrast, the long axis of E2 oleate is almost exclusively oriented at an angle of ∼60° to the z-direction. In such an orientation, the oleoyl chain is firmly inserted into the membrane. Thus, both E2 and E2 oleate have a preference for interface localization in the membrane. These orientations were also found in HDL discs, suggesting that only lipid-E2 interactions determine the localization of the molecule. The structural mapping of E2 and E2 oleate may provide a design platform for specific E2-HDL-targeted pharmacological therapies.


Assuntos
Estradiol/química , Lipoproteínas HDL/química , Lipossomos/química , Simulação de Dinâmica Molecular , Ácido Oleico/química
16.
Biochim Biophys Acta ; 1831(4): 691-7, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23328279

RESUMO

Hepatic lipase (HL) and endothelial lipase (EL) are negative regulators of plasma HDL cholesterol (HDLc) levels and presumably could affect two main HDL atheroprotective functions, macrophage-to-feces reverse cholesterol transport (RCT) and HDL antioxidant properties. In this study, we assessed the effects of both HL and EL deficiency on macrophage-specific RCT process and HDL ability to protect against LDL oxidation. HL- and EL-deficient and wild-type mice were injected intraperitoneally with [(3)H]cholesterol-labeled mouse macrophages, after which the appearance of [(3)H]cholesterol in plasma, liver, and feces was determined. The degree of HDL oxidation and the protection of oxidative modification of LDL co-incubated with HDL were evaluated by measuring conjugated diene kinetics. Plasma levels of HDLc, HDL phospholipids, apoA-I, and platelet-activated factor acetyl-hydrolase were increased in both HL- and EL-deficient mice. These genetically modified mice displayed increased levels of radiolabeled, HDL-bound [(3)H]cholesterol 48h after the label injection. The magnitude of macrophage-derived [(3)H]cholesterol in feces was also increased in both the HL- and EL-deficient mice. HDL from the HL- and EL-deficient mice was less prone to oxidation and had a higher ability to protect LDL from oxidation, compared with the HDL derived from the wild-type mice. These changes were correlated with plasma apoA-I and apoA-I/HDL total protein levels. In conclusion, targeted inactivation of both HL and EL in mice promoted macrophage-to-feces RCT and enhanced HDL antioxidant properties.


Assuntos
Colesterol/metabolismo , Fezes/química , Lipase/deficiência , Lipase/metabolismo , Macrófagos/metabolismo , Animais , Apolipoproteína A-I/sangue , Transporte Biológico/fisiologia , Colesterol/sangue , HDL-Colesterol/sangue , Lipase/genética , Lipoproteínas HDL/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Oxirredução
17.
Circ Res ; 111(11): 1459-69, 2012 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-22931956

RESUMO

RATIONALE: Psychological stress is associated with an increased risk of cardiovascular diseases. However, the connecting mechanisms of the stress-inducing activation of the hypothalamic-pituitary-adrenal axis with atherosclerosis are not well-understood. OBJECTIVE: To study the effect of acute psychological stress on reverse cholesterol transport (RCT), which transfers peripheral cholesterol to the liver for its ultimate fecal excretion. METHODS AND RESULTS: C57Bl/6J mice were exposed to restraint stress for 3 hours to induce acute psychological stress. RCT in vivo was quantified by measuring the transfer of [(3)H]cholesterol from intraperitoneally injected mouse macrophages to the lumen of the small intestine within the stress period. Surprisingly, stress markedly increased the contents of macrophage-derived [(3)H]cholesterol in the intestinal lumen. In the stressed mice, intestinal absorption of [(14)C]cholesterol was significantly impaired, the intestinal mRNA expression level of peroxisome proliferator-activated receptor-α increased, and that of the sterol influx transporter Niemann-Pick C1-like 1 decreased. The stress-dependent effects on RCT rate and peroxisome proliferator-activated receptor-α gene expression were fully mimicked by administration of the stress hormone corticosterone (CORT) to nonstressed mice, and they were blocked by the inhibition of CORT synthesis in stressed mice. Moreover, the intestinal expression of Niemann-Pick C1-like 1 protein decreased when circulating levels of CORT increased. Of note, when either peroxisome proliferator-activated receptor α or liver X receptor α knockout mice were exposed to stress, the RCT rate remained unchanged, although plasma CORT increased. This indicates that activities of both transcription factors were required for the RCT-accelerating effect of stress. CONCLUSIONS: Acute psychological stress accelerated RCT by compromising intestinal cholesterol absorption. The present results uncover a novel functional connection between the hypothalamic-pituitary-adrenal axis and RCT that can be triggered by a stress-induced increase in circulating CORT.


Assuntos
Colesterol/metabolismo , Corticosterona/sangue , Estresse Psicológico/fisiopatologia , Doença Aguda , Animais , Transporte Biológico/efeitos dos fármacos , Western Blotting , Linhagem Celular , Colesterol/farmacocinética , Corticosterona/farmacologia , Feminino , Expressão Gênica , Humanos , Absorção Intestinal/fisiologia , Intestino Delgado/metabolismo , Lipídeos/sangue , Fígado/metabolismo , Receptores X do Fígado , Macrófagos/metabolismo , Masculino , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptores Nucleares Órfãos/genética , Receptores Nucleares Órfãos/metabolismo , PPAR gama/genética , PPAR gama/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
18.
Arterioscler Thromb Vasc Biol ; 33(7): 1706-13, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23661675

RESUMO

OBJECTIVE: Angiopoietin-like 3 (Angptl3) is a regulator of lipoprotein metabolism at least by inhibiting lipoprotein lipase activity. Loss-of-function mutations in ANGPTL3 cause familial combined hypolipidemia through an unknown mechanism. APPROACH AND RESULTS: We compared lipolytic activities, lipoprotein composition, and other lipid-related enzyme/lipid transfer proteins in carriers of the S17X loss-of-function mutation in ANGPTL3 and in age- and sex-matched noncarrier controls. Gel filtration analysis revealed a severely disturbed lipoprotein profile and a reduction in size and triglyceride content of very low density lipoprotein in homozygotes as compared with heterozygotes and noncarriers. S17X homozygotes had significantly higher lipoprotein lipase activity and mass in postheparin plasma, whereas heterozygotes showed no difference in these parameters when compared with noncarriers. No changes in hepatic lipase, endothelial lipase, paraoxonase 1, phospholipid transfer protein, and cholesterol ester transfer protein activities were associated with the S17X mutation. Plasma free fatty acid, insulin, glucose, and homeostatic model assessment of insulin resistance were significantly lower in homozygous subjects compared with heterozygotes and noncarriers subjects. CONCLUSIONS: These results indicate that, although partial Angptl3 deficiency did not affect the activities of lipolytic enzymes, the complete absence of Angptl3 results in an increased lipoprotein lipase activity and mass and low circulating free fatty acid levels. This latter effect is probably because of decreased mobilization of free fatty acid from fat stores in human adipose tissue and may result in reduced hepatic very low density lipoprotein synthesis and secretion via attenuated hepatic free fatty acid supply. Altogether, Angptl3 may affect insulin sensitivity and play a role in modulating both lipid and glucose metabolism.


Assuntos
Angiopoietinas/deficiência , Ácidos Graxos não Esterificados/sangue , Hipobetalipoproteinemias/enzimologia , Resistência à Insulina , Lipase Lipoproteica/sangue , Adulto , Idoso , Análise de Variância , Proteína 3 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Angiopoietinas/genética , Biomarcadores/sangue , Glicemia/análise , Estudos de Casos e Controles , Distribuição de Qui-Quadrado , Regulação para Baixo , Feminino , Heterozigoto , Homozigoto , Humanos , Hipobetalipoproteinemias/sangue , Hipobetalipoproteinemias/genética , Hipobetalipoproteinemias/fisiopatologia , Insulina/sangue , Itália , Modelos Lineares , Lipase/sangue , Lipoproteínas LDL/sangue , Masculino , Pessoa de Meia-Idade , Mutação , Triglicerídeos/sangue , Regulação para Cima
19.
Anal Bioanal Chem ; 406(17): 4137-46, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24788890

RESUMO

Adsorption energy distribution (AED) calculations were successfully applied to partial-filling affinity capillary electrophoresis (PF-ACE) to facilitate more detailed studies of biomolecular interactions. PF-ACE with AED calculations was employed to study the interactions between two isoforms of apolipoprotein E (apoE) and dermatan sulfate (DS), and a quartz crystal microbalance (QCM) was used in combination with AED calculations to examine the interactions of the 15-amino-acid peptide fragment of apoE with DS. The heterogeneity of the interactions was elucidated. Microscale thermophoresis was used to validate the results. The interactions studied are of interest because, in vivo, apolipoprotein E localizes on DS-containing regions in the extracellular matrix of human vascular subendothelium. Two-site binding was demonstrated for the isoform apoE3 and DS, but only one-site binding for apoE2-DS. Comparable affinity constants were obtained for the apoE2-DS, apoE3-D3, and 15-amino-acid peptide of apoE-DS using the three techniques. The results show that combining AED calculations with modern biosensing techniques can open up another dimension in studies on the heterogeneity and affinity constants of biological molecules.


Assuntos
Apolipoproteínas E/química , Dermatan Sulfato/química , Eletroforese Capilar/métodos , Técnicas de Microbalança de Cristal de Quartzo/métodos , Sítios de Ligação , Humanos , Ligação Proteica , Isoformas de Proteínas/química
20.
Nat Cardiovasc Res ; 3(3): 356-371, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-39196121

RESUMO

Apolipoprotein-B (APOB)-containing lipoproteins cause atherosclerosis. Whether the vasculature is the initially responding site or if atherogenic dyslipidemia affects other organs simultaneously is unknown. Here we show that the liver responds to a dyslipidemic insult based on inducible models of familial hypercholesterolemia and APOB tracing. An acute transition to atherogenic APOB lipoprotein levels resulted in uptake by Kupffer cells and rapid accumulation of triglycerides and cholesterol in the liver. Bulk and single-cell RNA sequencing revealed a Kupffer-cell-specific transcriptional program that was not activated by a high-fat diet alone or detected in standard liver function or pathological assays, even in the presence of fulminant atherosclerosis. Depletion of Kupffer cells altered the dynamic of plasma and liver lipid concentrations, indicating that these liver macrophages help restrain and buffer atherogenic lipoproteins while simultaneously secreting atherosclerosis-modulating factors into plasma. Our results place Kupffer cells as key sentinels in organizing systemic responses to lipoproteins at the initiation of atherosclerosis.


Assuntos
Aterosclerose , Modelos Animais de Doenças , Células de Kupffer , Fígado , Células de Kupffer/metabolismo , Animais , Fígado/metabolismo , Fígado/patologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Masculino , Hiperlipoproteinemia Tipo II/metabolismo , Hiperlipoproteinemia Tipo II/complicações , Hiperlipoproteinemia Tipo II/sangue , Hiperlipoproteinemia Tipo II/patologia , Dislipidemias/metabolismo , Camundongos Endogâmicos C57BL , Triglicerídeos/sangue , Triglicerídeos/metabolismo , Apolipoproteínas B/metabolismo , Apolipoproteínas B/sangue , Colesterol/metabolismo , Colesterol/sangue , Dieta Hiperlipídica/efeitos adversos , Apolipoproteína B-100/metabolismo , Feminino
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