RESUMO
BACKGROUND: This single center, double-blinded, cross-over, placebo controlled clinical trial investigated the effect of oral α-cyclodextrin (α-CD), a soluble dietary fiber, on blood lipid and lipoprotein levels in healthy human subjects. α-CD, a cyclical polymer containing 6 glucose subunits, is currently sold as an over the counter food supplement and is also a common additive in many foods. α-CD forms a hydrophobic central cavity that binds lipids and has been shown in animal studies and in previous clinical trials to alter plasma lipid levels. METHODS: We screened for healthy subjects, males and females, between ages 18 to 75. Out of total 103 subjects interviewed, 75 subjects completed the study. Qualified individuals in each gender group were randomized into two groups in terms of which treatment arm they received first (placebo vs. α-CD, receiving 6 grams P.O. a day, for 12-14 weeks with a 7 day wash out between arms). The primary outcome variable, plasma total cholesterol, as well as other tests related to lipids and lipoprotein and glucose metabolism, were measured at baseline and at the end of each arm of the study. RESULTS: α-CD was well tolerated; no serious adverse events related to α-CD were observed. Approximately 8 % of the subjects on α-CD complained of minor gastrointestinal symptoms versus 3 % on placebo (p = 0.2). Small-LDL particle number decreased 10 % (p < 0.045) for subjects on α-CD versus placebo. Fasting plasma glucose (1.6 %, p < 0.05) and Insulin resistance index (11 %, p < 0.04) were also decreased when on α-CD versus placebo. CONCLUSION: α-CD treatment appears to be safe and well tolerated in healthy individuals and showed a modest reduction in small LDL particles, and an improvement in glucose related parameters. TRIAL REGISTRATION: NCT01131299.
Assuntos
HDL-Colesterol/sangue , LDL-Colesterol/sangue , Fibras na Dieta/administração & dosagem , Triglicerídeos/sangue , alfa-Ciclodextrinas/administração & dosagem , Administração Oral , Adolescente , Adulto , Idoso , Glicemia/metabolismo , Método Duplo-Cego , Jejum , Feminino , Voluntários Saudáveis , Humanos , Insulina/sangue , Resistência à Insulina/fisiologia , Masculino , Pessoa de Meia-IdadeRESUMO
Apolipoprotein A-I (apoA-I) mimetic peptides are currently being developed as possible new agents for the treatment of cardiovascular disease based on their ability to promote cholesterol efflux and their other beneficial antiatherogenic properties. Many of these peptides, however, have been reported to cause transient hypertriglyceridemia due to inhibition of lipolysis by lipoprotein lipase (LPL). We describe a novel bihelical amphipathic peptide (C-II-a) that contains an amphipathic helix (18A) for binding to lipoproteins and stimulating cholesterol efflux as well as a motif based on the last helix of apolipoprotein C-II (apoC-II) that activates lipolysis by LPL. The C-II-a peptide promoted cholesterol efflux from ATP-binding cassette transporter ABCA1-transfected BHK cells similar to apoA-I mimetic peptides. Furthermore, it was shown in vitro to be comparable to the full-length apoC-II protein in activating lipolysis by LPL. When added to serum from a patient with apoC-II deficiency, it restored normal levels of LPL-induced lipolysis and also enhanced lipolysis in serum from patients with type IV and V hypertriglyceridemia. Intravenous injection of C-II-a (30 mg/kg) in apolipoprotein E-knockout mice resulted in a significant reduction of plasma cholesterol and triglycerides of 38 ± 6% and 85 ± 7%, respectively, at 4 hours. When coinjected with the 5A peptide (60 mg/kg), the C-II-a (30 mg/kg) peptide was found to completely block the hypertriglyceridemic effect of the 5A peptide in C57Bl/6 mice. In summary, C-II-a is a novel peptide based on apoC-II, which promotes cholesterol efflux and lipolysis and may therefore be useful for the treatment of apoC-II deficiency and other forms of hypertriglyceridemia.
Assuntos
Apolipoproteínas E/genética , Lipólise/efeitos dos fármacos , Ativadores de Lipase de Lipoproteínas/farmacologia , Lipase Lipoproteica/metabolismo , Peptídeos/farmacologia , Triglicerídeos/sangue , Animais , Colesterol/metabolismo , Dicroísmo Circular , Desenho de Fármacos , Humanos , Hiperlipoproteinemia Tipo I/sangue , Hipertrigliceridemia/sangue , Técnicas In Vitro , Ativadores de Lipase de Lipoproteínas/química , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Moleculares , Peptídeos/químicaRESUMO
The bihelical apolipoprotein mimetic peptide 5A effluxes cholesterol from cells and reduces inflammation and atherosclerosis in animal models. We investigated how hydrophobic residues in the hinge region between the two helices are important in the structure and function of this peptide. By simulated annealing analysis and molecular dynamics modeling, two hydrophobic amino acids, F-18 and W-21, in the hinge region were predicted to be relatively surface-exposed and to interact with the aqueous solvent. Using a series of 5A peptide analogs in which F-18 or W-21 was changed to either F, W, A, or E, only peptides with hydrophobic amino acids in these two positions were able to readily bind and solubilize phospholipid vesicles. Compared with active peptides containing F or W, peptides containing E in either of these two positions were more than 10-fold less effective in effluxing cholesterol by the ABCA1 transporter. Intravenous injection of 5A in C57BL/6 mice increased plasma-free cholesterol (5A: 89.9 ± 13.6 mg/dl; control: 38.7 ± 4.3 mg/dl (mean ± S.D.); P < 0.05) and triglycerides (5A: 887.0 ± 172.0 mg/dl; control: 108.9 ± 9.9 mg/dl; P < 0.05), whereas the EE peptide containing E in both positions had no effect. Finally, 5A increased cholesterol efflux approximately 2.5-fold in vivo from radiolabeled macrophages, whereas the EE peptide was inactive. These results provide a rationale for future design of therapeutic apolipoprotein mimetic peptides and provide new insights into the interaction of hydrophobic residues on apolipoproteins with phospholipids in the lipid microdomain created by the ABCA1 transporter during the cholesterol efflux process.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Aminoácidos/química , Apolipoproteínas A/farmacologia , Colesterol/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Sequência de Aminoácidos , Animais , Linhagem Celular , Dicroísmo Circular , Cricetinae , Feminino , Lipídeos/sangue , Lipoproteínas/sangue , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Mimetismo Molecular , Dados de Sequência Molecular , Fosfolipídeos/metabolismoRESUMO
New treatment approaches are needed for patients with asthma. Apolipoprotein A-I (apoA-I), the major structural protein of high-density lipoproteins, mediates reverse cholesterol transport and has atheroprotective and anti-inflammatory effects. In this study, we hypothesized that an apoA-I mimetic peptide might be effective at inhibiting asthmatic airway inflammation. A 5A peptide, which is a synthetic, bihelical apoA-I mimetic, was administered to wild-type A/J mice via osmotic mini-pump prior to the induction of house dust mite (HDM)-induced asthma. HDM-challenged mice that received the 5A apoA-I mimetic peptide had significant reductions in the number of bronchoalveolar lavage fluid eosinophils, lymphocytes, and neutrophils, as well as in histopathological evidence of airway inflammation. The reduction in airway inflammation was mediated by a reduction in the expression of Th2- and Th17-type cytokines, as well as in chemokines that promote T cell and eosinophil chemotaxis, including CCL7, CCL17, CCL11, and CCL24. Furthermore, the 5A apoA-I mimetic peptide inhibited the alternative activation of pulmonary macrophages in the lungs of HDM-challenged mice. It also abrogated the development of airway hyperresponsiveness and reduced several key features of airway remodeling, including goblet cell hyperplasia and the expression of collagen genes (Col1a1 and Col3a1). Our results demonstrate that the 5A apoA-I mimetic peptide attenuates the development of airway inflammation and airway hyperresponsiveness in an experimental murine model of HDM-induced asthma. These data support the conclusion that strategies using apoA-I mimetic peptides, such as 5A, might be developed further as a possible new treatment approach for asthma.
Assuntos
Apolipoproteína A-I/administração & dosagem , Asma/imunologia , Asma/prevenção & controle , Mimetismo Molecular/imunologia , Fragmentos de Peptídeos/administração & dosagem , Pyroglyphidae/imunologia , Administração por Inalação , Animais , Antiasmáticos/administração & dosagem , Antiasmáticos/uso terapêutico , Apolipoproteína A-I/uso terapêutico , Asma/patologia , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/patologia , Hiper-Reatividade Brônquica/prevenção & controle , Citocinas/antagonistas & inibidores , Citocinas/biossíntese , Modelos Animais de Doenças , Feminino , Humanos , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/metabolismo , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos A , Fragmentos de Peptídeos/uso terapêuticoRESUMO
RATIONALE: Distinct sets of corticosteroid-unresponsive genes modulate disease severity in asthma. OBJECTIVES: To identify corticosteroid-unresponsive genes that provide new insights into disease pathogenesis and asthma therapeutics. METHODS: Experimental murine asthma was induced by nasal administration of house dust mite for 5 days per week. Dexamethasone and apolipoprotein E (apo E) mimetic peptides were administered via osmotic minipumps. MEASUREMENTS AND MAIN RESULTS: Genome-wide expression profiling of the lung transcriptome in a house dust mite-induced model of murine asthma identified increases in apo E mRNA levels that persisted despite corticosteroid treatment. House dust mite-challenged apo Eâ»(/)â» mice displayed enhanced airway hyperreactivity and goblet cell hyperplasia, which could be rescued by administration of an apo E(130-149) mimetic peptide. Administration of the apo E(130-149) mimetic peptide to house dust mite-challenged apo Eâ»(/)â» mice also inhibited eosinophilic airway inflammation, IgE production, and the expression of Th2 and Th17 cytokines. House dust mite-challenged low-density lipoprotein receptor (LDLR) knockout mice displayed a similar phenotype as apo Eâ»(/)â» mice with enhanced airway hyperreactivity, goblet cell hyperplasia, and mucin gene expression, but could not be rescued by the apo E(130-149) mimetic peptide, consistent with a LDLR-dependent mechanism. CONCLUSIONS: These findings for the first time identify an apo E-LDLR pathway as an endogenous negative regulator of airway hyperreactivity and goblet cell hyperplasia in asthma. Furthermore, our results demonstrate that strategies that activate the apo E-LDLR pathway, such as apo E mimetic peptides, might be developed into a novel treatment approach for patients with asthma.
Assuntos
Apolipoproteínas E/fisiologia , Asma/etiologia , Pyroglyphidae/imunologia , Receptores de LDL/fisiologia , Corticosteroides/farmacologia , Animais , Asma/patologia , Asma/fisiopatologia , Hiper-Reatividade Brônquica/induzido quimicamente , Hiper-Reatividade Brônquica/fisiopatologia , Feminino , Perfilação da Expressão Gênica , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/patologia , Células Caliciformes/fisiologia , Hiperplasia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/fisiopatologia , Camundongos , Camundongos Knockout , Microscopia Confocal , Fragmentos de Peptídeos/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Intravenous administration of apolipoprotein (apo) A-I complexed with phospholipid has been shown to rapidly reduce plaque size in both animal models and humans. Short synthetic amphipathic peptides can mimic the antiatherogenic properties of apoA-I and have been proposed as alternative therapeutic agents. In this study, we investigated the atheroprotective effect of the 5A peptide, a bihelical amphipathic peptide that specifically effluxes cholesterol from cells by ATP-binding cassette transporter 1 (ABCA1). 5A stimulated a 3.5-fold increase in ABCA1-mediated efflux from cells and an additional 2.5-fold increase after complexing it with phospholipid (1:7 mol/mol). 5A-palmitoyl oleoyl phosphatidyl choline (POPC), but not free 5A, was also found to promote cholesterol efflux by ABCG1. When incubated with human serum, 5A-POPC bound primarily to high-density lipoprotein (HDL) but also to low-density lipoprotein (LDL) and promoted the transfer of cholesterol from LDL to HDL. Twenty-four hours after intravenous injection of 5A-POPC (30 mg/kg) into apoE-knockout (KO) mice, both the cholesterol (181%) and phospholipid (219%) content of HDL significantly increased. By an in vivo cholesterol isotope dilution study and monitoring of the flux of cholesterol from radiolabeled macrophages to stool, 5A-POPC treatment was observed to increase reverse cholesterol transport. In three separate studies, 5A when complexed with various phospholipids reduced aortic plaque surface area by 29 to 53% (n = 8 per group; p < 0.02) in apoE-KO mice. No signs of toxicity from the treatment were observed during these studies. In summary, 5A promotes cholesterol efflux both in vitro and in vivo and reduces atherosclerosis in apoE-KO mice, indicating that it may be a useful alternative to apoA-I for HDL therapy.
Assuntos
Apolipoproteína A-I/fisiologia , Aterosclerose/tratamento farmacológico , Colesterol/metabolismo , Peptídeos , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Aorta/efeitos dos fármacos , Aorta/patologia , Apolipoproteína A-I/química , Apolipoproteínas E/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Transporte Biológico , Feminino , Humanos , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intercelular , Lipoproteínas/metabolismo , Lipoproteínas HDL/sangue , Lipoproteínas LDL/sangue , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mimetismo Molecular , Peptídeos/química , Fosfatidilcolinas/química , Ratos , Ratos Sprague-DawleyRESUMO
Spliceosome-mediated RNA trans-splicing has emerged as an exciting mode of RNA therapy. Here we describe a novel trans-splicing strategy, which targets highly abundant pre-mRNAs, to produce therapeutic proteins in vivo. First, we used a pre-trans-splicing molecule (PTM) that mediated trans-splicing of human apolipoprotein A-I (hapoA-I) into the highly abundant mouse albumin exon 1. Hydrodynamic tail vein injection of the hapoA-I PTM plasmid in mice followed by analysis of the chimeric transcripts and protein, confirmed accurate and efficient trans-splicing into albumin pre-mRNA and production of hapoA-I protein. The versatility of this approach was demonstrated by producing functional human papillomavirus type-16 E7 (HPV16-E7) single-chain antibody in C57BL/6 mice and functional factor VIII (FVIII) and phenotypic correction in hemophilia A mice. Altogether, these studies demonstrate that trans-splicing to highly abundant albumin transcripts can be used as a general platform to produce therapeutic proteins in vivo.
Assuntos
Albuminas/genética , Trans-Splicing/genética , Animais , Apolipoproteína A-I/genética , Apolipoproteína A-I/fisiologia , Éxons/genética , Feminino , Terapia Genética/métodos , Vetores Genéticos/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Precursores de RNA/genética , Splicing de RNA/genética , Splicing de RNA/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Spliceossomos/genética , Spliceossomos/metabolismo , Trans-Splicing/fisiologiaRESUMO
Familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) is a rare genetic disease characterized by low HDL-C levels, low plasma cholesterol esterification, and the formation of Lipoprotein-X (Lp-X), an abnormal cholesterol-rich lipoprotein particle. LCAT deficiency causes corneal opacities, normochromic normocytic anemia, and progressive renal disease due to Lp-X deposition in the glomeruli. Recombinant LCAT is being investigated as a potential therapy for this disorder. Several hepatic disorders, namely primary biliary cirrhosis, primary sclerosing cholangitis, cholestatic liver disease, and chronic alcoholism also develop Lp-X, which may contribute to the complications of these disorders. We aimed to test the hypothesis that an increase in plasma LCAT could prevent the formation of Lp-X in other diseases besides FLD. We generated a murine model of intrahepatic cholestasis in LCAT-deficient (KO), wild type (WT), and LCAT-transgenic (Tg) mice by gavaging mice with alpha-naphthylisothiocyanate (ANIT), a drug well known to induce intrahepatic cholestasis. Three days after the treatment, all mice developed hyperbilirubinemia and elevated liver function markers (ALT, AST, Alkaline Phosphatase). The presence of high levels of LCAT in the LCAT-Tg mice, however, prevented the formation of Lp-X and other plasma lipid abnormalities in WT and LCAT-KO mice. In addition, we demonstrated that multiple injections of recombinant human LCAT can prevent significant accumulation of Lp-X after ANIT treatment in WT mice. In summary, LCAT can protect against the formation of Lp-X in a murine model of cholestasis and thus recombinant LCAT could be a potential therapy to prevent the formation of Lp-X in other diseases besides FLD.
Assuntos
1-Naftilisotiocianato/efeitos adversos , Colestase Intra-Hepática/tratamento farmacológico , Lipoproteína-X/sangue , Fosfatidilcolina-Esterol O-Aciltransferase/uso terapêutico , Animais , Colestase Intra-Hepática/induzido quimicamente , Colestase Intra-Hepática/metabolismo , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Humanos , Lipoproteína-X/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Fosfatidilcolina-Esterol O-Aciltransferase/genética , Fosfatidilcolina-Esterol O-Aciltransferase/farmacologiaRESUMO
Farnesoid X receptor (FXR), a bile-acid-activated member of the nuclear receptor superfamily, is essential in regulating bile-acid, cholesterol, and triglyceride homeostasis. Disruption of the FXR gene in mice results in a proatherosclerotic lipid profile with increased serum cholesterols and triglycerides. However, the role of FXR in foam-cell formation and atherosclerosis development remains unclear. The current study showed that the peritoneal macrophages isolated from FXR-null mice took up less oxidized LDL-cholesterol (oxLDL-C), which was accompanied by a marked reduction in CD36 expression in these cells. This result appears to be FXR-independent, as FXR was not detected in the peritoneal macrophages. To assess to what extent FXR modulates atherosclerosis development, FXR/ApoE double-null mice were generated. Female mice were used for atherosclerosis analysis. Compared to ApoE-null mice, the FXR/ApoE double-null mice were found to have less atherosclerotic lesion area in the aorta, despite a further increase in the serum cholesterols and triglycerides. Our results indicate that disruption of the FXR gene could attenuate atherosclerosis development, most likely resulting from reduced oxLDL-C uptake by macrophages. Our study cautions the use of serum lipid levels as a surrogate marker to determine the efficiency of FXR modulators in treating hyperlipidemia.
Assuntos
Aterosclerose/etiologia , Proteínas de Ligação a DNA/metabolismo , Células Espumosas/metabolismo , Células Espumosas/patologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Transporte Biológico Ativo , LDL-Colesterol/metabolismo , Citocinas/biossíntese , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Feminino , Expressão Gênica , Homeostase , Técnicas In Vitro , Lipídeos/sangue , Lipoproteínas LDL/metabolismo , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Citoplasmáticos e Nucleares/deficiência , Receptores Citoplasmáticos e Nucleares/genética , Fatores de Transcrição/deficiência , Fatores de Transcrição/genéticaRESUMO
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secretory protein that controls cholesterol homeostasis by enhancing endosomal and lysosomal degradation of the low-density lipoprotein receptor (LDL-R). Mutations that cause increased activity of PCSK9 are associated with hypercholesterolemia, atherosclerosis and early cardiovascular disease (CVD), whereas individuals with loss-of-function mutations in PCSK9 are apparently healthy but are hypocholesterolemic and have a dramatically decreased risk of CVD. In this study, we generated virus-like particle (VLP)-based vaccines targeting PCSK9. Mice and macaques vaccinated with bacteriophage VLPs displaying PCSK9-derived peptides developed high titer IgG antibodies that bound to circulating PCSK9. Vaccination was associated with significant reductions in total cholesterol, free cholesterol, phospholipids, and triglycerides. A vaccine targeting PCSK9 may, therefore, be an attractive alternative to monoclonal antibody-based therapies.
Assuntos
Colesterol/sangue , Pró-Proteína Convertases/antagonistas & inibidores , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Animais , Autoanticorpos/sangue , Bacteriófagos/genética , Portadores de Fármacos , Feminino , Imunoglobulina G/sangue , Macaca , Masculino , Camundongos Endogâmicos BALB C , Fosfolipídeos/sangue , Pró-Proteína Convertases/imunologia , Resultado do Tratamento , Triglicerídeos/sangue , Vacinas de Partículas Semelhantes a Vírus/genéticaRESUMO
Lecithin-cholesterol acyltransferase (LCAT), a key enzyme in high-density lipoprotein (HDL) metabolism, has been proposed to have atheroprotective properties by promoting reverse cholesterol transport. Overexpression of LCAT in various animal models, however, has led to conflicting results on its overall effect on lipoproteins and atherosclerosis. In this study, the effect of overexpression of LCAT in nonhuman primates on lipoprotein metabolism is examined. Human LCAT was expressed with adenovirus in squirrel monkeys (n = 8), resulting on day 4 in a 22-fold increase of LCAT activity (257 +/- 23 vs 5618 +/- 799 nmol mL(-1) h(-1), P < .0001). At its peak, LCAT was found to nearly double the level of HDL cholesterol from baseline (113 +/- 7 vs 260 +/- 24 mg/dL, P < .01). High-density lipoprotein formed after treatment with the adenovirus was larger in size, as assessed by fast protein liquid chromatography (FPLC) analysis. By kinetic studies, it was determined that there was a decrease in apolipoprotein (Apo) A-I resident time (0.373 +/- 0.027 vs 0.685 +/- 0.045 d(-1), P < .0001) and almost a doubling in the ApoA-I synthetic rate (22 +/- 2 vs 41 +/- 3 mg kg(-1) d(-1), P < .0001), but no overall change in ApoA-I levels. In addition, increased expression of LCAT was associated with a 37% reduction of ApoB levels (12 +/- 1 vs 19 +/- 1 mg/dL, P < .05) due to increased low-density lipoprotein catabolism (fractional catabolic rate = 1.7 +/- 0.1 d(-1) in controls vs 4.2 +/- 0.3 d(-1) in LCAT-treated group, P < .05). In summary, overexpression of LCAT in nonhuman primates leads to an antiatherogenic lipoprotein profile by increasing HDL cholesterol and lowering ApoB, thus making LCAT a potential drug target for reducing atherosclerosis.
Assuntos
Adenoviridae/genética , Aterosclerose/genética , Lipoproteínas HDL/sangue , Lipoproteínas LDL/sangue , Fosfatidilcolina-Esterol O-Aciltransferase/genética , Animais , Cromatografia Líquida , Humanos , Cinética , Masculino , Fenótipo , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismo , SaimiriRESUMO
To elucidate the separate contributions of the lipolytic versus ligand-binding functions of hepatic lipase (HL) to lipoprotein metabolism and atherosclerosis, and to investigate the role of the low density lipoprotein receptor (LDLr) in these processes, we compared mice expressing catalytically active HL (HL-WT) with mice expressing inactive HL (HL-S145G) in a background lacking endogenous HL and the LDLr (LDLr-KOxHL-KO). HL-WT and HL-S145G reduced (P < 0.05 for all) cholesterol (55% vs. 20%), non-HDL-cholesterol (63% vs. 22%), and apolipoprotein B (apoB; 34% vs. 16%) by enhancing the catabolism of autologous (125)I-apoB-intermediate density lipoprotein (IDL)/LDL (fractional catabolic rate in day(-1): 6.07 +/- 0.25, LDLr-KOxHL-WT; 4.76 +/- 0.30, LDLr-KOxHL-S145G; 3.70 +/- 0.13, LDLr-KOxHL-KO); HL-WT had a greater impact on the concentration, composition, particle size, and catabolism of apoB-containing lipoproteins (apoB-Lps) and HDL. Importantly, consistent with the changes in apoB-Lps, atherosclerosis in LDLr-KOxHL-KO mice fed a regular chow diet (RCD) was reduced by both HL-WT and HL-S145G (by 71% and 51% in cross-sectional analysis, and by 85% and 67% in en face analysis; P < 0.05 for all). These data identify physiologically relevant but distinct roles for the lipolytic versus ligand-binding functions of HL in apoB-Lp metabolism and atherosclerosis and demonstrate that their differential effects on these processes are mediated by changes in catabolism via non-LDLr pathways. These changes, evident even in the presence of apoE, establish an antiatherogenic role of the ligand-binding function of HL in LDLr-deficient mice.
Assuntos
Aterosclerose/prevenção & controle , Lipase/metabolismo , Lipólise , Fígado/enzimologia , Receptores de LDL/deficiência , Animais , Feminino , Ligantes , Lipoproteínas/sangue , Lipoproteínas/química , Lipoproteínas HDL/deficiência , Lipoproteínas HDL/genética , Masculino , Camundongos , Camundongos Knockout , RNA/genética , RNA/isolamento & purificação , Receptores de LDL/genética , Receptores de Lipoproteínas/deficiência , Receptores de Lipoproteínas/genética , Caracteres SexuaisRESUMO
Mutations in Proprotein Convertase Subtilisin Kexin 9 (PCSK9) have been associated with autosomal dominant hypercholesterolemia. In vivo kinetic studies indicate that LDL catabolism was impaired and apolipoprotein B (apoB)-containing lipoprotein synthesis was enhanced in two patients presenting with the S127R mutation on PCSK9. To understand the physiological role of PCSK9, we overexpressed human PCSK9 in mouse and cellular models as well as attenuated the endogenous expression of PCSK9 in HuH7 hepatoma cells using RNA interference. Here, we show that PCSK9 dramatically impairs the expression of the low density lipoprotein receptor (LDLr) and, in turn, LDL cellular binding as well as LDL clearance from the plasma compartment in C57BL6/J mice but not in LDLr-deficient mice, establishing a definitive role for PCSK9 in the modulation of the LDLr metabolic pathway. In contrast to data obtained in S127R-PCSK9 patients presenting with increased apoB production, our study indicates that wild-type PCSK9 does not significantly alter the production and/or secretion of VLDL apoB in either cultured cells or mice. Finally, we show that unlike PCSK9 overexpression in mice, the S127R mutation in patients led to increased VLDL apoB levels, suggesting a potential gain of function for S127R-PCSK9 in humans.
Assuntos
Apolipoproteínas B/química , Lipoproteínas LDL/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/fisiologia , Adenoviridae/genética , Animais , Western Blotting , Carcinoma Hepatocelular/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Inativação Gênica , Heterozigoto , Humanos , Immunoblotting , Cinética , Lipase/metabolismo , Metabolismo dos Lipídeos , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Pró-Proteína Convertase 9 , Pró-Proteína Convertases , Ligação Proteica , Interferência de RNA , Proteínas Recombinantes/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina Endopeptidases/metabolismo , Fatores de TempoRESUMO
To address the importance of the farnesoid X-receptor (FXR; NR1H4) for normal cholesterol homeostasis, we evaluated the major pathways of cholesterol metabolism in the FXR-deficient (-/-) mouse model. Compared with wild-type, FXR(-/-) mice have increased plasma high density lipoprotein (HDL) cholesterol and a markedly reduced rate of plasma HDL cholesterol ester clearance. Concomitantly, FXR(-/-) mice exhibit reduced expression of hepatic genes involved in reverse cholesterol transport, most notably, that for scavenger receptor BI. FXR(-/-) mice also have increased: (i) plasma non-HDL cholesterol and triglyceride levels, (ii) apolipoprotein B-containing lipoprotein synthesis, and (iii) intestinal cholesterol absorption. Surprisingly, biliary cholesterol elimination was increased in FXR(-/-) mice, despite decreased expression of hepatic genes thought to be involved in this process. These data demonstrate that FXR is a critical regulator of normal cholesterol metabolism and that genetic changes affecting FXR function have the potential to be pro-atherogenic.
Assuntos
Colesterol/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/fisiologia , Animais , Apolipoproteínas B/sangue , Transporte Biológico , Northern Blotting , Western Blotting , Colesterol/sangue , Proteínas de Ligação a DNA , Íleo/metabolismo , Cinética , Metabolismo dos Lipídeos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Fatores de Tempo , Fatores de Transcrição , Triglicerídeos/sangueRESUMO
To identify regulatory elements in the proximal human ATP-binding cassette transporter A1 (hABCA1) gene promoter we transfected RAW cells with plasmids containing mutations in the E-box, AP1, and liver X receptor (LXR) elements as well as the two Sp1 motifs. Point mutations in either Sp1 site or in the AP1 site had only a minor effect whereas mutation of the LXR element decreased promoter activity. In contrast, mutation or deletion of the E-box motif caused a 3-fold increase in transcriptional activity under basal conditions. Gel shift and DNaseI footprint analysis showed binding of a protein or protein complex to this region. Preincubation of nuclear extracts with antibodies established that USF1, USF2, and fos related antigen (Fra) 2 bind to DNA sequences in the human ABCA1 promoter that contains the intact E-box but not the mutant or deleted E-box. Co-transfection of USF1 and USF2 enhanced, but Fra2 repressed, ABCA1 promoter activity. Thus, a complex consisting of USF1, USF2, and Fra2 binds the E-box motif 147 bp upstream of the transcriptional start site and facilitates repression of the human ABCA1 promoter. These combined studies identify a novel site in the human ABCA1 promoter involved in the regulation of ABCA1 gene expression.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Elementos E-Box/genética , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/biossíntese , Animais , Sítios de Ligação , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Antígeno 2 Relacionado a Fos , Humanos , Camundongos , Mutação , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Transcrição Gênica , Fatores Estimuladores UpstreamRESUMO
To investigate the separate contributions of the lipolytic versus ligand-binding function of hepatic lipase (HL) to plasma lipoprotein metabolism and atherosclerosis, we compared mice expressing catalytically active wild-type HL (HL-WT) and inactive HL (HL-S145G) with no endogenous expression of mouse apoE or HL (E-KO x HL-KO, where KO is knockout). HL-WT and HL-S145G reduced plasma cholesterol (by 40 and 57%, respectively), non-high density lipoprotein cholesterol (by 48 and 61%, respectively), and apoB (by 36 and 44%, respectively) (p < 0.01), but only HL-WT decreased high density lipoprotein cholesterol (by 67%) and apoA-I (by 54%). Compared with E-KO x HL-KO mice, both active and inactive HL lowered the pro-atherogenic lipoproteins by enhancing the catabolism of autologous (125)I-apoB very low density/intermediate density lipoprotein (VLDL/IDL) (fractional catabolic rates of 2.87 +/- 0.04/day for E-KO x HL-KO, 3.77 +/- 0.03/day for E-KO x HL-WT, and 3.63 +/- 0.09/day for E-KO x HL-S145G mice) and (125)I-apoB-48 low density lipoprotein (LDL) (fractional catabolic rates of 5.67 +/- 0.34/day for E-KO x HL-KO, 18.88 +/- 1.72/day for E-KO x HL-WT, and 9.01 +/- 0.14/day for E-KO x HL-S145G mice). In contrast, the catabolism of apoE-free, (131)I-apoB-100 LDL was not increased by either HL-WT or HL-S145G. Infusion of the receptor-associated protein (RAP), which blocks LDL receptor-related protein function, decreased plasma clearance and hepatic uptake of (131)I-apoB-48 LDL induced by HL-S145G. Despite their similar effects on lowering pro-atherogenic apoB-containing lipoproteins, HL-WT enhanced atherosclerosis by up to 50%, whereas HL-S145G markedly reduced aortic atherosclerosis by up to 96% (p < 0.02) in both male and female E-KO x HL-KO mice. These data identify a major receptor pathway (LDL receptor-related protein) by which the ligand-binding function of HL alters remnant lipoprotein uptake in vivo and delineate the separate contributions of the lipolytic versus ligand-binding function of HL to plasma lipoprotein size and metabolism, identifying an anti-atherogenic role of the ligand-binding function of HL in vivo.
Assuntos
Arteriosclerose/prevenção & controle , Lipase/fisiologia , Animais , Apolipoproteína B-48 , Apolipoproteínas B/análise , Apolipoproteínas B/sangue , Arteriosclerose/etiologia , Catálise , Feminino , Humanos , Ligantes , Lipólise , Lipoproteínas LDL/metabolismo , Lipoproteínas VLDL/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
The individual roles of hepatic versus intestinal ABCG5 and ABCG8 in sterol transport have not yet been investigated. To determine the specific contribution of liver ABCG5/G8 to sterol transport and atherosclerosis, we generated transgenic mice that overexpress human ABCG5 and ABCG8 in the liver but not intestine (liver G5/G8-Tg) in three different genetic backgrounds: C57Bl/6, apoE-KO, and low density lipoprotein receptor (LDLr)-KO. Hepatic overexpression of ABCG5/G8 enhanced hepatobiliary secretion of cholesterol and plant sterols by 1.5-2-fold, increased the amount of intestinal cholesterol available for absorption and fecal excretion by up to 27%, and decreased the accumulation of plant sterols in plasma by approximately 25%. However, it did not alter fractional intestinal cholesterol absorption, fecal neutral sterol excretion, hepatic cholesterol concentrations, or hepatic cholesterol synthesis. Consequently, overexpression of ABCG5/G8 in only the liver had no effect on the plasma lipid profile, including cholesterol, HDL-C, and non-HDL-C, or on the development of proximal aortic atherosclerosis in C57Bl/6, apoE-KO, or LDLr-KO mice. Thus, liver ABCG5/G8 facilitate the secretion of liver sterols into bile and serve as an alternative mechanism, independent of intestinal ABCG5/G8, to protect against the accumulation of dietary plant sterols in plasma. However, in the absence of changes in fractional intestinal cholesterol absorption, increased secretion of sterols into bile induced by hepatic overexpression of ABCG5/G8 was not sufficient to alter hepatic cholesterol balance, enhance cholesterol removal from the body or to alter atherogenic risk in liver G5/G8-Tg mice. These findings demonstrate that overexpression of ABCG5/G8 in the liver profoundly alters hepatic but not intestinal sterol transport, identifying distinct roles for liver and intestinal ABCG5/G8 in modulating sterol metabolism.
Assuntos
Transportadores de Cassetes de Ligação de ATP/biossíntese , Lipoproteínas/biossíntese , Fígado/metabolismo , Esteróis/metabolismo , Membro 5 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Membro 8 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Aorta/metabolismo , Aorta/fisiopatologia , Arteriosclerose/metabolismo , Arteriosclerose/fisiopatologia , Sistema Biliar/metabolismo , Transporte Biológico , Colesterol na Dieta/administração & dosagem , Dieta , Regulação da Expressão Gênica , Humanos , Lipoproteínas/genética , Camundongos , Camundongos TransgênicosRESUMO
Identification of mutations in the ABCA1 transporter (ABCA1) as the genetic defect in Tangier disease has generated interest in modulating atherogenic risk by enhancing ABCA1 gene expression. To investigate the role of ABCA1 in atherogenesis, we analyzed diet-induced atherosclerosis in transgenic mice overexpressing human ABCA1 (hABCA1-Tg) and spontaneous lesion formation in hABCA1-Tg x apoE-knockout (KO) mice. Overexpression of hABCA1 in C57BL/6 mice resulted in a unique anti-atherogenic profile characterized by decreased plasma cholesterol (63%), cholesteryl ester (63%), free cholesterol (67%), non-high density lipoprotein (HDL)-cholesterol (53%), and apolipoprotein (apo) B (64%) but markedly increased HDL-cholesterol (2.8-fold), apoA-I (2.2-fold), and apoE (2.8-fold) levels. These beneficial changes in the lipid profile led to significantly lower (65%) aortic atherosclerosis in hABCA1-Tg mice. In marked contrast, ABCA1 overexpression had a minimal effect on the plasma lipid profile of apoE-KO mice and resulted in a 2- to 2.6-fold increase in aortic lesion area. These combined results indicate that overexpression of ABCA1 in C57BL/6 mice on a high cholesterol diet results in an atheroprotective lipoprotein profile and decreased atherosclerosis, and thus provide previously undocumented in vivo evidence of an anti-atherogenic role for the ABCA1 transporter. In contrast, overexpression of ABCA1 in an apoE-KO background led to increased atherosclerosis, further substantiating the important role of apoE in macrophage cholesterol metabolism and atherogenesis. In summary, these results establish that, in the presence of apoE, overexpression of ABCA1 modulates HDL as well as apoB-containing lipoprotein metabolism and reduces atherosclerosis in vivo, and indicate that pharmacological agents that will increase ABCA1 expression may reduce atherogenic risk in humans.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/fisiologia , Aorta/patologia , Apolipoproteínas E/genética , Arteriosclerose/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Apolipoproteína A-I/biossíntese , Apolipoproteínas E/biossíntese , Colesterol/sangue , Colesterol/metabolismo , HDL-Colesterol/sangue , Cruzamentos Genéticos , Dieta , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica , Fatores SexuaisRESUMO
The current model for reverse cholesterol transport proposes that HDL transports excess cholesterol derived primarily from peripheral cells to the liver for removal. However, recent studies in ABCA1 transgenic mice suggest that the liver itself may be a major source of HDL cholesterol (HDL-C). To directly investigate the hepatic contribution to plasma HDL-C levels, we generated an adenovirus (rABCA1-GFP-AdV) that targets expression of mouse ABCA1-GFP in vivo to the liver. Compared with mice injected with control AdV, infusion of rABCA1-GFP-AdV into C57Bl/6 mice resulted in increased expression of mouse ABCA1 mRNA and protein in the liver. ApoA-I-dependent cholesterol efflux was increased 2.6-fold in primary hepatocytes isolated 1 day after rABCA1-GFP-AdV infusion. Hepatic ABCA1 expression in C57Bl/6 mice (n = 15) raised baseline levels of TC, PL, FC, HDL-C, apoE, and apoA-I by 150-300% (P < 0.05 all). ABCA1 expression led to significant compensatory changes in expression of genes that increase hepatic cholesterol, including HMG-CoA reductase (3.5-fold), LDLr (2.1-fold), and LRP (5-fold) in the liver. These combined results demonstrate that ABCA1 plays a key role in hepatic cholesterol efflux, inducing pathways that modulate cholesterol homeostasis in the liver, and establish the liver as a major source of plasma HDL-C.