RESUMO
Nanoparticles capable of mimicking natural tissues represent a major technological advancement in regenerative medicine. In this pilot study, the development of a new nanohybrid composed of titanate nanoribbons to mimic the extracellular matrix is reported. During the first phase, nanoribbons were synthesized by hydrothermal treatment. Subsequently, titanate nanoribbons were functionalized by heterobifunctional polyethylene-glycol (PEG) to graft type I collagen on their surface. Biological properties of this new nanobiohybrid such as cytotoxicity to cardiac cells and platelet aggregation ability were evaluated. The so-formed nanobiohybrid permits cellular adhesion and proliferation favoring fine cardiac tissue healing and regeneration.
RESUMO
OBJECTIVE: Earlier in vitro studies suggested a putative role for the plasma phospholipid transfer protein (PLTP) in the modulation of blood coagulation. The effect of PLTP expression on blood coagulation under both basal and oxidative stress conditions was compared here in wild-type and PLTP-deficient (PLTP-/-) mice. METHODS AND RESULTS: Under basal conditions, PLTP deficiency was associated with an extended tail bleeding time despite a significant depletion of vascular α-tocopherol content and an impairment of endothelial function. When acute oxidative stress was generated in vivo in the brain vasculature, the steady state levels of oxidized lipid derivatives, the extent of blood vessel occlusion, and the volume of ischemic lesions were more severe in wild-type than in PLTP-/- mice. CONCLUSIONS: In addition to its recognized hyperlipidemic, proinflammatory, and proatherogenic properties, PLTP increases blood coagulation and worsens the extent of ischemic lesions in response to acute oxidative stress. Thus, PLTP arises here as a cardiovascular risk factor for the late thrombotic events occurring in the acute phase of atherosclerosis.
Assuntos
Coagulação Sanguínea , Infarto Cerebral/prevenção & controle , Endotélio Vascular/metabolismo , Trombose Intracraniana/prevenção & controle , Estresse Oxidativo , Proteínas de Transferência de Fosfolipídeos/deficiência , Animais , Tempo de Sangramento , Infarto Cerebral/sangue , Infarto Cerebral/genética , Infarto Cerebral/patologia , Infarto Cerebral/fisiopatologia , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiopatologia , Trombose Intracraniana/sangue , Trombose Intracraniana/genética , Trombose Intracraniana/patologia , Trombose Intracraniana/fisiopatologia , Ácidos Linoleicos/metabolismo , Metabolismo dos Lipídeos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredução , Proteínas de Transferência de Fosfolipídeos/genética , Vasodilatadores/farmacologia , alfa-Tocoferol/sangueRESUMO
Three-dimensional (3D) microphysiological systems (MPSs) mimicking human organ function in vitro are an emerging alternative to conventional monolayer cell culture and animal models for drug development. Human induced pluripotent stem cells (hiPSCs) have the potential to capture the diversity of human genetics and provide an unlimited supply of cells. Combining hiPSCs with microfluidics technology in MPSs offers new perspectives for drug development. Here, the integration of a newly developed liver MPS with a cardiac MPS-both created with the same hiPSC line-to study drug-drug interaction (DDI) is reported. As a prominent example of clinically relevant DDI, the interaction of the arrhythmogenic gastroprokinetic cisapride with the fungicide ketoconazole was investigated. As seen in patients, metabolic conversion of cisapride to non-arrhythmogenic norcisapride in the liver MPS by the cytochrome P450 enzyme CYP3A4 was inhibited by ketoconazole, leading to arrhythmia in the cardiac MPS. These results establish integration of hiPSC-based liver and cardiac MPSs to facilitate screening for DDI, and thus drug efficacy and toxicity, isogenic in the same genetic background.
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Plasma cholesteryl ester transfer protein (CETP) activity is high in rabbits, intermediate in humans, and nondetectable in rodents. Human apolipoprotein CI (apoCI) was found to be a potent inhibitor of CETP. The aim of this study was to compare the ability of rabbit and human apoCI to modulate the interaction of CETP with HDLs and to evaluate to which extent apoCI contributes to plasma cholesteryl ester transfer rate in normolipidemic humans and rabbits. Rabbit apoCI gene was cloned and sequenced, rabbit and human apoCI were purified to homogeneity, and their ability to modify the surface charge properties and the CETP inhibitory potential of HDL were compared. It is demonstrated that unlike human apoCI, rabbit apoCI does not modulate cholesteryl ester transfer rate in total plasma. Whereas both human and rabbit apoCI readily associate with HDL, only human apoCI was found to modify the electrostatic charge of HDL. In humans, both CETP and apoCI at normal, physiological levels contribute significantly to the plasma cholesteryl ester transfer rate. In contrast, CETP is the sole major determinant of cholesteryl ester transfer in normolipidemic rabbit plasma as a result of the inability of rabbit apoCI to change HDL electronegativity.
Assuntos
Apolipoproteínas C/metabolismo , Proteínas de Transferência de Ésteres de Colesterol/sangue , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Sequência de Aminoácidos , Animais , Apolipoproteínas C/química , Apolipoproteínas C/genética , Clonagem Molecular , Feminino , Humanos , Lipase Lipoproteica/metabolismo , Lipoproteínas HDL/metabolismo , Masculino , Pessoa de Meia-Idade , Coelhos , Análise de Sequência de DNARESUMO
Members of the haloacid dehalogenase (HAD) family of metabolite phosphatases play an important role in regulating multiple pathways in Plasmodium falciparum central carbon metabolism. We show that the P. falciparum HAD protein, phosphoglycolate phosphatase (PGP), regulates glycolysis and pentose pathway flux in asexual blood stages via detoxifying the damaged metabolite 4-phosphoerythronate (4-PE). Disruption of the P. falciparumpgp gene caused accumulation of two previously uncharacterized metabolites, 2-phospholactate and 4-PE. 4-PE is a putative side product of the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase, and its accumulation inhibits the pentose phosphate pathway enzyme, 6-phosphogluconate dehydrogenase (6-PGD). Inhibition of 6-PGD by 4-PE leads to an unexpected feedback response that includes increased flux into the pentose phosphate pathway as a result of partial inhibition of upper glycolysis, with concomitant increased sensitivity to antimalarials that target pathways downstream of glycolysis. These results highlight the role of metabolite detoxification in regulating central carbon metabolism and drug sensitivity of the malaria parasite.IMPORTANCE The malaria parasite has a voracious appetite, requiring large amounts of glucose and nutrients for its rapid growth and proliferation inside human red blood cells. The host cell is resource rich, but this is a double-edged sword; nutrient excess can lead to undesirable metabolic reactions and harmful by-products. Here, we demonstrate that the parasite possesses a metabolite repair enzyme (PGP) that suppresses harmful metabolic by-products (via substrate dephosphorylation) and allows the parasite to maintain central carbon metabolism. Loss of PGP leads to the accumulation of two damaged metabolites and causes a domino effect of metabolic dysregulation. Accumulation of one damaged metabolite inhibits an essential enzyme in the pentose phosphate pathway, leading to substrate accumulation and secondary inhibition of glycolysis. This work highlights how the parasite coordinates metabolic flux by eliminating harmful metabolic by-products to ensure rapid proliferation in its resource-rich niche.
Assuntos
Antimaláricos/farmacologia , Carbono/metabolismo , Resistência a Medicamentos/efeitos dos fármacos , Fosfomicina/análogos & derivados , Monoéster Fosfórico Hidrolases/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/metabolismo , Fosfomicina/farmacologia , Glicólise/efeitos dos fármacos , Humanos , Lactatos/farmacologia , Malária Falciparum/tratamento farmacológico , Malária Falciparum/metabolismo , Açúcares Ácidos/farmacologiaRESUMO
The malaria parasite, Plasmodium falciparum, displays the P. falciparum erythrocyte membrane protein 1 (PfEMP1) on the surface of infected red blood cells (RBCs). We here examine the physical organization of PfEMP1 trafficking intermediates in infected RBCs and determine interacting partners using an epitope-tagged minimal construct (PfEMP1B). We show that parasitophorous vacuole (PV)-located PfEMP1B interacts with components of the PTEX (Plasmodium Translocon of EXported proteins) as well as a novel protein complex, EPIC (Exported Protein-Interacting Complex). Within the RBC cytoplasm PfEMP1B interacts with components of the Maurer's clefts and the RBC chaperonin complex. We define the EPIC interactome and, using an inducible knockdown approach, show that depletion of one of its components, the parasitophorous vacuolar protein-1 (PV1), results in altered knob morphology, reduced cell rigidity and decreased binding to CD36. Accordingly, we show that deletion of the Plasmodium berghei homologue of PV1 is associated with attenuation of parasite virulence in vivo.
Assuntos
Interações Hospedeiro-Patógeno , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Proteínas de Transporte/metabolismo , Adesão Celular , Feminino , Técnicas de Silenciamento de Genes , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Plasmodium berghei/genética , Plasmodium falciparum/patogenicidade , Transporte ProteicoRESUMO
ApoCI (apolipoprotein CI) is a potent inhibitor of plasma CETP [CE (cholesteryl ester) transfer protein]. The relevance of apoCI overexpression as a method for CETP blockade in vivo was addressed in the present study in CETPTg/apoCITg mice (mice expressing both human CETP and apoCI). Despite a significant reduction in specific CETP activity in CETPTg/apoCITg mice compared with CETPTg mice [transgenic mouse to human CETP; 46.8+/-11.1 versus 101.8+/-25.7 pmol x h(-1).(mug of plasma CETP)(-1) respectively; P<0.05], apoCI overexpression increased both the CETP mass concentration (3-fold increase; P<0.05) and the hepatic CETP mRNA level (4-fold increase, P<0.005), leading to an increase in total plasma CE transfer activity (by 39%, P<0.05). The ratio of apoB-containing lipoprotein to HDL (high-density lipoprotein) CE was 10-fold higher in CETPTg/apoCITg mice than in apoCITg mice (P<0.0005). It is proposed that the increased CETP expression in CETPTg/apoCITg mice is a direct consequence of liver X receptor activation in response to the accumulation of cholesterol-rich apoB-containing lipoproteins. In support of the latter view, hepatic mRNA levels of other liver X receptor-responsive genes [ABCG5 (ATP-binding cassette transporter GS) and SREBP-1c (sterol-regulatory-binding protein-1c)] were higher in CETPTg/apoCITg mice compared with CETPTg mice. In conclusion, overexpression of apoCI, while producing a significant inhibitory effect on specific CETP activity, does not represent a suitable method for decreasing total CE transfer activity in CETPTg/apoCITg mice, owing to an hyperlipidaemia-mediated effect on CETP gene expression.
Assuntos
Apolipoproteínas C/genética , Apolipoproteínas C/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Lipoproteínas/sangue , Transgenes/genética , Animais , Apoproteínas/sangue , Proteínas de Transporte/sangue , Proteínas de Transporte/química , Colesterol/sangue , Proteínas de Transferência de Ésteres de Colesterol , Expressão Gênica , Glicoproteínas/sangue , Glicoproteínas/química , Humanos , Lipoproteínas/química , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Peso Molecular , RNA Mensageiro/análise , RNA Mensageiro/genéticaRESUMO
Liver fibrosis, a form of scarring, develops in chronic liver diseases when hepatocyte regeneration cannot compensate for hepatocyte death. Initially, collagen produced by myofibroblasts (MFs) functions to maintain the integrity of the liver, but excessive collagen accumulation suppresses residual hepatocyte function, leading to liver failure. As a strategy to generate new hepatocytes and limit collagen deposition in the chronically injured liver, we developed in vivo reprogramming of MFs into hepatocytes using adeno-associated virus (AAV) vectors expressing hepatic transcription factors. We first identified the AAV6 capsid as effective in transducing MFs in a mouse model of liver fibrosis. We then showed in lineage-tracing mice that AAV6 vector-mediated in vivo hepatic reprogramming of MFs generates hepatocytes that replicate function and proliferation of primary hepatocytes, and reduces liver fibrosis. Because AAV vectors are already used for liver-directed human gene therapy, our strategy has potential for clinical translation into a therapy for liver fibrosis.
Assuntos
Reprogramação Celular , Dependovirus/genética , Vetores Genéticos/metabolismo , Cirrose Hepática/patologia , Cirrose Hepática/terapia , Fígado/citologia , Miofibroblastos/citologia , Animais , Capsídeo/metabolismo , Proliferação de Células , Técnicas de Transferência de Genes , Camundongos Endogâmicos C57BLRESUMO
OBJECTIVE: In order to determine the influence of the lipid status on the ability of cholesteryl ester transfer protein (CETP) to modify the plasma lipoprotein profile, the effect of hypercholesterolemia versus hypertriglyceridemia were compared in wild-type and CETP-transgenic (CETPTg) rats expressing CETP at a constant level. METHODS AND RESULTS: Wild-type and CETPTg rats were fed either a chow diet, a high fat/high cholesterol (HF/HC) diet, or a sucrose diet. As compared to wild-type rats, CETPTg rats fed the standard chow exhibited lower high-density lipoproteins (HDL)-cholesterol concentration (-65%, p<0.01), but similar non-HDL-cholesterol concentrations. Both wild-type and CETPTg rats fed the HF/HC diet displayed pronounced increases in total and non-HDL-cholesterol levels, with no influence of CETP expression in this case. In contrast, the sucrose diet produced significant changes only in CETPTg rats which then exhibited a 82% increase in non-HDL-cholesterol in addition to a 80% reduction in HDL cholesterol when compared to sucrose-fed, wild-type rats (p<0.01 in both cases). The triglyceride to cholesterol ratio in very low-density lipoprotein (VLDL) was 10-fold lower in 'HF/HC' rats than in 'chow' and 'sucrose' rats (p<0.005 and p<0.01, respectively), and VLDL from 'HF/HC' animals were proven to constitute poor cholesteryl ester acceptors. CONCLUSIONS: CETP expression modified dramatically the lipoprotein phenotype in 'sucrose' rats but not in 'HF/HC' rats. These observations suggest that a CETP inhibitor treatment is susceptible to produce profound changes in hypertriglyceridemia or combined hyperlipidemia.
Assuntos
Proteínas de Transporte/biossíntese , Proteínas de Transporte/farmacologia , Dieta , Glicoproteínas/biossíntese , Glicoproteínas/farmacologia , Hiperlipidemias/genética , Hiperlipidemias/fisiopatologia , Lipoproteínas/sangue , Ração Animal , Animais , Animais Geneticamente Modificados , Proteínas de Transferência de Ésteres de Colesterol , Ésteres do Colesterol , Hiperlipidemias/veterinária , Fenótipo , Ratos , Sacarose/metabolismo , Edulcorantes/metabolismo , TriglicerídeosRESUMO
The aim of this study was to investigate the influence of the surface charge and coating of Superparamagnetic Iron Oxide Nanoparticles (SPIONs) on their in vitro and in vivo behaviors. Neutral and negatively-charged PEG-based SPIONs were synthesized and compared to Resovist, a carboxydextran-based SPION currently used in clinics. Their cytotoxicity, cell internalization, and potential as contrast agents for magnetic resonance imaging were assessed. Neutral pegylated SPIONs were internalized less readily by the reticuloendothelial system and showed a lower uptake by the liver, compared to negatively-charged SPIONs (with carboxydextran and PEG). These results suggested that the charge of functionalized SPIONs was more relevant for their biological interactions than the nature of their coating.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Materiais Revestidos Biocompatíveis/química , Dextranos/química , Dextranos/toxicidade , Nanopartículas de Magnetita/química , Nanopartículas de Magnetita/toxicidade , Nanocápsulas/química , Polietilenoglicóis/química , Animais , Linhagem Celular , Materiais Revestidos Biocompatíveis/toxicidade , Dextranos/administração & dosagem , Dextranos/ultraestrutura , Células Hep G2 , Humanos , Macrófagos/efeitos dos fármacos , Nanopartículas de Magnetita/administração & dosagem , Nanopartículas de Magnetita/ultraestrutura , Teste de Materiais , Camundongos , Nanocápsulas/toxicidade , Nanocápsulas/ultraestrutura , Especificidade de Órgãos , Tamanho da Partícula , Eletricidade Estática , Relação Estrutura-Atividade , Distribuição TecidualRESUMO
The ristocetin cofactor activity assay (VWF:RCo) is the reference method for assessing von Willebrand factor (VWF) activity but remains difficult to perform, and the coefficient of variation of the method is high (about 20-30%). This study evaluated and compared the performance for measuring the VWF activity of two newly commercialised assays [VWF:Ac Innovance (VWF:Ac) and VWF:RCo Acustar (VWF:RCo Acu)] with the reference VWF:RCo aggregation in 123 pathological plasma samples. The correlation and concordance between both new tests (VWF:RCo-Acu and VWF:Ac) and the reference VWF:RCo were good. The results of the VWF activity to VWF antigen ratio were also comparable whatever the method for the classification of VWF deficiency in all patients. Our results showed that both new tests could replace the "gold standard" VWF:RCo in aggregometry with several benefits: they are fully automated, easier and faster to perform, better adapted to emergency situations if necessary.
Assuntos
Testes de Coagulação Sanguínea/métodos , Doenças de von Willebrand/sangue , Fator de von Willebrand/análise , Fator de von Willebrand/imunologia , Automação , Coagulação Sanguínea , Calibragem , Estudos de Casos e Controles , Colágeno/química , Ensaio de Imunoadsorção Enzimática , Humanos , Agregação Plaquetária , Estudos Prospectivos , Valores de Referência , Reprodutibilidade dos Testes , Ristocetina/sangue , Sensibilidade e Especificidade , Doenças de von Willebrand/diagnóstico , Fator de von Willebrand/metabolismoRESUMO
Actively contractile cardiomyocyte (CM) monolayer represents an interesting tool to study both cardiac diseases and injuries. However, this model is poorly transfectable with conventional agents. Consequently, there is a need to develop new carriers that could overcome this problem. Titanate nanotubes (TiONts) could be a potential candidate due to possibly higher cell uptake as a direct consequence of their shape. On the basis of this rationale, TiONts were assessed for their cytotoxicity and internalization pathways. Cytotoxicity was assessed for TiONts either functionalized with PEI or unfunctionalized and its spherical counterpart P25 TiO2. No cytotoxic effect was observed under TiONts, TiONts-PEI1800 and P25 TiO2 exposed conditions. The tubular morphology was found to be an important parameter promoting internalization while reversing the charge was assessed as non-additional. Internalization was found to occur by endocytosis and diffusion through the membrane. A preliminary transfection study indicated the potential of TiONts as a nanocarrier.
Assuntos
Nanopartículas Metálicas/toxicidade , Miócitos Cardíacos/efeitos dos fármacos , Titânio/toxicidade , Animais , Animais Recém-Nascidos , Técnicas de Cultura de Células , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Nanopartículas Metálicas/química , Polietilenoimina/química , Ratos , Ratos Wistar , Titânio/químicaRESUMO
Drug discovery and development to date has relied on animal models, which are useful but are often expensive, slow, and fail to mimic human physiology. The discovery of human induced pluripotent stem (iPS) cells has led to the emergence of a new paradigm of drug screening using human and disease-specific organ-like cultures in a dish. Although classical static culture systems are useful for initial screening and toxicity testing, they lack the organization of differentiated iPS cells into microphysiological, organ-like structures deemed necessary for high-content analysis of candidate drugs. One promising approach to produce these organ-like structures is the use of advanced microfluidic systems, which can simulate tissue structure and function at a micron level, and can provide high-throughput testing of different compounds for therapeutic and diagnostic applications. Here, we provide a brief outline on the different approaches, which have been used to engineer in vitro tissue constructs of iPS cell-based myocardium and liver functions on chip. Combining these techniques with iPS cell biology has the potential of reducing the dependence on animal studies for drug toxicity and efficacy screening.
Assuntos
Hepatócitos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Mioblastos Cardíacos/citologia , Diferenciação Celular , Colágeno/química , Inibidores de Ciclo-Oxigenase 2/toxicidade , Combinação de Medicamentos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Hipoglicemiantes/toxicidade , Laminina/química , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Mioblastos Cardíacos/efeitos dos fármacos , Mioblastos Cardíacos/metabolismo , Proteoglicanas/químicaRESUMO
Before transplantation, the heart graft is preserved by the use of cold storage in order to limit ischemia-reperfusion stress. However, sustained exposure to low temperature may induce myocardial ultrastructural damage, particularly microtubules (MT) disruption. Previous data suggested that tubulin-binding agents are able to attenuate cold-induced cytoskeleton alterations. Thus, the aim of the present work was to study the influence of docetaxel (DX, a tubulin-binding taxane) on the effects of deep hypothermia (4 degrees C) and of simulated cold ischemia-reperfusion on the MT network and oxidative stress of cardiomyocyte (CM) in monolayer cultures prepared from newborn rat ventricles. The MT network was explored by immunocytochemistry and Western-blotting, the cell stress by tetrazolium dye assay (MTT) and lactate dehydrogenase (LDH) release, and the superoxide production by the dihydroethidium probe (DHE). The MT assembly remained stable after 4 and 8 h of hypothermia. Tubulin acetylation was promoted in CM subjected to 4-h hypothermia. Low temperature reduced the mitochondrial function and increased the basal LDH release. The cold ischemia during 4 and 8 h preserved MT network. Docetaxel promoted MT polymerization and tubulin acetylation in basal and in cold conditions. This drug decreased the release of LDH induced by cold ischemia. Moreover, hypothermia (4 h) significantly raised the anion superoxide production. Docetaxel decreased this oxidative stress in the control CM and in CM submitted to 4 h of hypothermia. These data demonstrated that stabilizing MT with DX exerted a protective effect on CM subjected to hypothermia and to cold ischemia-reperfusion. Tubulin-ligands should be thus considered to improve the tolerance of the heart graft toward stressing conservative conditions.
Assuntos
Isquemia Fria/efeitos adversos , Citoproteção , Microtúbulos/fisiologia , Miócitos Cardíacos/patologia , Traumatismo por Reperfusão/patologia , Acetilação , Acetiltransferases/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Citoproteção/efeitos dos fármacos , Dimerização , Docetaxel , Radicais Livres/metabolismo , Hipotermia/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Ligação Proteica , Ratos , Ratos Wistar , Traumatismo por Reperfusão/metabolismo , Taxoides/farmacologia , Tubulina (Proteína)/metabolismoRESUMO
OBJECTIVE: To determine the effect of short-term weight loss in obese women on concentrations of plasma cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP), two new risk factors for cardiovascular disease. RESEARCH METHODS AND PROCEDURES: Plasma CETP and PLTP mass concentrations were measured in 38 obese, non-diabetic women before and after a moderate, 4% weight loss that was obtained by a 1250 kcal/d diet for 4 weeks. Anthropometric and biological parameters were measured before and after weight loss. RESULTS: Plasma CETP concentration decreased substantially after weight loss (2.76 +/- 0.79 before and 2.31 +/- 0.69 mg/L after; p = 0.000), and the same was true for plasma PLTP concentration (9.01 +/- 2.44 mg/L before vs. 8.34 +/- 2.57 after; p = 0.043). The HDL profile shifted toward the small-sized range, with significant decreases in the relative abundance of HDL(2b) and HDL(2a) at the expense of HDL(3b) after weight loss. A significant, positive correlation between CETP and PLTP mass concentrations is reported for the first time in obese patients (r = 0.43, p = 0.004), and weight reduction was accompanied by early, concomitant, and parallel decreases in plasma CETP and PLTP levels (r = 0.47, p = 0.003). The significant relationship between CETP and PLTP levels was lost after the dietary intervention (r = 0.27; p = 0.11). DISCUSSION: CETP and PLTP correlate positively and significantly in obese patients. The hypocaloric dietary manipulation constitutes a relevant intervention to reduce rapidly and simultaneously plasma levels of CETP and PLTP. The impact of reduced PLTP activity on HDL size appeared to be more prominent than the impact of concomitant reduction in CETP activity.
Assuntos
Proteínas de Transporte/sangue , Obesidade/dietoterapia , Redução de Peso/fisiologia , Adulto , Índice de Massa Corporal , Restrição Calórica , Proteínas de Transferência de Ésteres de Colesterol/sangue , HDL-Colesterol/análise , HDL-Colesterol/sangue , Dieta Redutora , Feminino , Humanos , Pessoa de Meia-Idade , Obesidade/sangue , Proteínas de Transferência de Fosfolipídeos/sangueRESUMO
Genetically engineered mice demonstrated that apolipoprotein (apo) CI is a potent, physiological inhibitor of plasma cholesteryl ester transfer protein (CETP) activity. The goal of this study was to determine the molecular mechanism of the apoCI-mediated blockade of CETP activity. Kinetic analyses revealed that the inhibitory property of apoCI is independent of the amount of active CETP, but it is tightly dependent on the amount of high density lipoproteins (HDL) in the incubation mixtures. The electrostatic charge of HDL, i.e. the main carrier of apoCI in human plasma, is gradually modified with increasing amounts of apoCI, and the neutralization of apoCI lysine residues by acetylation produces a marked reduction in its inhibitory potential. The inhibitory property of full-length apoCI is shared by its C-terminal alpha-helix with significant electrostratic properties, whereas its N-terminal alpha-helix with no CETP inhibitory property has no effect on HDL electronegativity. Finally, binding experiments demonstrated that apoCI and to a lower extent its C-terminal alpha-helix are able to disrupt CETP-lipoprotein complexes in a concentration-dependent manner. It was concluded that the inhibition of CETP activity by apoCI is in direct link with its specific electrostatic properties, and the apoCI-mediated reduction in the binding properties of lipoproteins results in weaker CETP-HDL interactions and fewer cholesteryl ester transfers.
Assuntos
Apolipoproteínas C/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Glicoproteínas/antagonistas & inibidores , Glicoproteínas/metabolismo , Acetilação , Apolipoproteína C-I , Proteínas de Transporte/sangue , Proteínas de Transferência de Ésteres de Colesterol , Glicoproteínas/sangue , HumanosRESUMO
Analysis of a polymorphic microsatellite locus was applied to 85 Candida albicans strains from healthy individuals. Comparison with strains from nonhealthy individuals previously analyzed in our laboratory showed an overall similarity, suggesting that all commensal strains have the ability to develop as pathogens.
Assuntos
Candida albicans/genética , Candida albicans/isolamento & purificação , Alelos , Candida albicans/classificação , Candida albicans/patogenicidade , DNA Fúngico/genética , Genes Fúngicos , Genótipo , Humanos , Repetições de Microssatélites , Orofaringe/microbiologia , Polimorfismo GenéticoRESUMO
Transgenic mice expressing human cholesteryl ester transfer protein (HuCETPTg mice) were crossed with apolipoprotein CI-knocked out (apoCI-KO) mice. Although total cholesterol levels tended to be reduced as the result of CETP expression in HuCETPTg heterozygotes compared with C57BL6 control mice (-13%, not significant), a more pronounced decrease (-28%, p < 0.05) was observed when human CETP was expressed in an apoCI-deficient background (HuCETPTg/apoCI-KO mice). Gel permeation chromatography analysis revealed a significant, 6.1-fold rise (p < 0.05) in the cholesteryl ester content of very low density lipoproteins in HuCETPTg/apoCI-KO mice compared with control mice, whereas the 2.7-fold increase in HuCETPTg mice did not reach the significance level in these experiments. Approximately 50% decreases in the cholesteryl ester content and cholesteryl ester to triglyceride ratio of high density lipoproteins (HDL) were observed in HuCETPTg/apoCI-KO mice compared with controls (p < 0.05 in both cases), with intermediate -20% changes in HuCETPTg mice. The cholesteryl ester depletion of HDL was accompanied with a significant reduction in their mean apparent diameter (8.68 +/- 0.04 nm in HuCETPTg/apoCI-KO mice versus 8.83 +/- 0.02 nm in control mice; p < 0.05), again with intermediate values in HuCETPTg mice (8.77 +/- 0.04 nm). In vitro purified apoCI was able to inhibit cholesteryl ester exchange when added to either total plasma or reconstituted HDL-free mixtures, and coincidently, the specific activity of CETP was significantly increased in the apoCI-deficient state (173 +/- 75 pmol/microg/h in HuCETPTg/apoCI-KO mice versus 72 +/- 19 pmol/microg/h in HuCETPTg, p < 0.05). Finally, HDL from apoCI-KO mice were shown to interact more readily with purified CETP than control HDL that differ only by their apoCI content. Overall, the present observations provide direct support for a potent specific inhibition of CETP by plasma apoCI in vivo.
Assuntos
Apolipoproteínas C/genética , Proteínas de Transporte/metabolismo , Glicoproteínas , Lipoproteínas/sangue , Animais , Apolipoproteína C-I , Apolipoproteínas C/sangue , Apolipoproteínas C/deficiência , Proteínas de Transporte/genética , Proteínas de Transferência de Ésteres de Colesterol , Humanos , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos TransgênicosRESUMO
In order to investigate the direct effect of cholesteryl ester transfer protein (CETP) on the structure and composition of HDL in vivo, simian CETP was expressed in Fisher rat that spontaneously displays high plasma levels of HDL1. In the new CETPTg rat line, the production of active CETP by the liver induced a significant 48% decrease in plasma HDL cholesterol, resulting in a 34% decrease in total cholesterol level (P < 0.01 in both cases). Among the various plasma HDL subpopulations, the largest HDL were those mostly affected by CETP, with a 74% decrease in HDL1 versus a significantly weaker 38% decrease in smaller HDL2 (P < 0.0001). Apolipoprotein E (apoE)-containing HDL1 were selectively affected by CETP expression, whereas apoA content of HDL remained unmodified. The reduction in the apoE content of serum HDL observed in CETPTg rats compared to controls (53%, P < 0.02) suggests that apoE in HDL may constitute in vivo a major determinant of their ability to interact with CETP. These results bring new insight into the lack of HDL1 in plasma from CETP-deficient heterozygotes despite their substantial 50% decrease in CETP activity. In addition, they indicate that HDL1 constitute reliable and practicable sensors of very low plasma CETP activity in vivo.
Assuntos
Apolipoproteínas E/metabolismo , Proteínas de Transporte/genética , HDL-Colesterol/metabolismo , Colesterol/metabolismo , Glicoproteínas , Animais , Animais Geneticamente Modificados , Proteínas de Transporte/biossíntese , Proteínas de Transporte/metabolismo , Colesterol/sangue , Proteínas de Transferência de Ésteres de Colesterol , Feminino , Heterozigoto , Masculino , RNA Mensageiro , Ratos , Ratos Endogâmicos F344RESUMO
Human plasma, unlike mouse plasma, contains the cholesteryl ester transfer protein (CETP) that may influence the reverse cholesterol transport. Liver X receptor (LXR), an oxysterol-activated nuclear receptor induces CETP transcription via a direct repeat 4 element in the CETP gene promoter. The aim of the study was to assess in vivo the impact of LXR activation on CETP expression and its consequences on plasma lipid metabolism and hepatic and bile lipid content. Wild-type and humanized mice expressing CETP were treated for five days with T0901317 LXR agonist. This treatment produced marked rises in both hepatic CETP mRNA and plasma CETP activity levels. Interestingly, the LXR agonist-mediated, 2-fold rise in both total and HDL cholesterol levels in treated wild-type mice was not observed in CETPTg mice, and the accumulation of cholesterol in the liver of CETPTg mice was reversed by LXR agonist treatment. Moreover, LXR activation induced a 2-fold increase in hepatic LDL-receptor expression in wild-type and CETPTg mice, and it produced a significantly greater rise in biliary cholesterol concentration in CETPTg mice as compared with wild-type mice. In conclusion, induction of CETP constitutes a major determinant of the effect of LXR agonists on cholesterol transport and excretion.