RESUMEN
Hunter syndrome is a rare x-linked recessive genetic disorder that affects lysosomal metabolism due to deficiency of iduronate-2-sulfatase (IDS), with subsequent accumulation of glycosaminoglycans heparan and dermatan sulfates (GAG). Enzyme replacement therapy is the only FDA-approved remedy and is an expensive life-time treatment that alleviates some symptoms of the disease without neurocognitive benefit. We previously reported successful treatment in a mouse model of mucopolysaccharidosis type II (MPS II) using adeno-associated viral vector serotype 9 encoding human IDS (AAV9.hIDS) via intracerebroventricular injection. As a less invasive and more straightforward procedure, here we report intravenously administered AAV9.hIDS in a mouse model of MPS II. In animals administered 1.5 × 1012 vg of AAV9.hIDS at 2 months of age, we observed supraphysiological levels of IDS enzyme activity in the circulation (up to 9100-fold higher than wild-type), in the tested peripheral organs (up to 560-fold higher than wild-type), but only 4% to 50% of wild type levels in the CNS. GAG levels were normalized on both sides of the blood-brain-barrier (BBB) in most of tissues tested. Despite low levels of the IDS observed in the CNS, this treatment prevented neurocognitive decline as shown by testing in the Barnes maze and by fear conditioning. This study demonstrates that a single dose of IV-administered AAV9.hIDS may be an effective and non-invasive procedure to treat MPS II that benefits both sides of the BBB, with implications for potential use of IV-administered AAV9 for other neuronopathic lysosomal diseases.
RESUMEN
Mucopolysaccharidosis type I (MPS I) is an inherited metabolic disorder caused by deficiency of the lysosomal enzyme alpha-L-iduronidase (IDUA). The two current treatments [hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT)], are insufficiently effective in addressing neurologic disease, in part due to the inability of lysosomal enzyme to cross the blood brain barrier. With a goal to more effectively treat neurologic disease, we have investigated the effectiveness of AAV-mediated IDUA gene delivery to the brain using several different routes of administration. Animals were treated by either direct intracerebroventricular (ICV) injection, by intrathecal (IT) infusion into the cerebrospinal fluid, or by intranasal (IN) instillation of AAV9-IDUA vector. AAV9-IDUA was administered to IDUA-deficient mice that were either immunosuppressed with cyclophosphamide (CP), or immunotolerized at birth by weekly injections of human iduronidase. In animals treated by ICV or IT administration, levels of IDUA enzyme ranged from 3- to 1000-fold that of wild type levels in all parts of the microdissected brain. In animals administered vector intranasally, enzyme levels were 100-fold that of wild type in the olfactory bulb, but enzyme expression was close to wild type levels in other parts of the brain. Glycosaminoglycan levels were reduced to normal in ICV and IT treated mice, and in IN treated mice they were normalized in the olfactory bulb, or reduced in other parts of the brain. Immunohistochemical analysis showed extensive IDUA expression in all parts of the brain of ICV treated mice, while IT treated animals showed transduction that was primarily restricted to the hind brain with some sporadic labeling seen in the mid- and fore brain. At 6 months of age, animals were tested for spatial navigation, memory, and neurocognitive function in the Barnes maze; all treated animals were indistinguishable from normal heterozygous control animals, while untreated IDUA deficient animals exhibited significant learning and spatial navigation deficits. We conclude that IT and IN routes are acceptable and alternate routes of administration, respectively, of AAV vector delivery to the brain with effective IDUA expression, while all three routes of administration prevent the emergence of neurocognitive deficiency in a mouse MPS I model.
RESUMEN
Mucopolysaccharidosis type I (MPS I) is an inherited metabolic disorder caused by deficiency of alpha-L-iduronidase (IDUA), resulting in accumulation of heparan and dermatan sulfate glycosaminoglycans (GAGs). Individuals with the most severe form of the disease (Hurler syndrome) suffer from neurodegeneration, intellectual disability, and death by age 10. Current treatments for this disease include allogeneic hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT). However, these treatments do not address CNS manifestations of the disease. In this study we compared the ability of intravenously administered AAV serotypes 9 and rh10 (AAV9 and AAVrh10) for delivery and expression of the IDUA gene in the CNS. Adult C57BL/6 MPS I mice were infused intravenously with either AAV9 or AAVrh10 vector encoding the human IDUA gene. Treated animals demonstrated supraphysiological levels and widespread restoration of IDUA enzyme activity in the plasma and all organs including the CNS. High levels of IDUA enzyme activity were observed in the plasma, brain and spinal cord ranging from 10 to 100-fold higher than heterozygote controls, while levels in peripheral organs were also high, ranging from 1000 to 10,000-fold higher than control animals. In general, levels of IDUA expression were slightly higher in peripheral organs for AAVrh10 administered animals although these differences were not significant except for the lung. Levels of IDUA expression between AAV 9 and rh10 were roughly equivalent in the brain. Urinary and tissue GAGs were significantly reduced starting at 3 weeks after vector infusion, with restoration of normal GAG levels by the end of the study in animals treated with either AAV9 or rh10. These results demonstrate that non-invasive intravenous AAV9 or AAVrh10-mediated IDUA gene therapy is a potentially effective treatment for both systemic and CNS manifestations of MPS I, with implications for the treatment of other metabolic and neurological diseases as well.
RESUMEN
Mucopolysaccharidosis type II (MPS II; Hunter syndrome) is a rare X-linked recessive lysosomal disorder caused by defective iduronate-2-sulfatase (IDS), resulting in accumulation of heparan sulfate and dermatan sulfate glycosaminoglycans (GAGs). Enzyme replacement is the only Food and Drug Administration-approved therapy available for MPS II, but it is expensive and does not improve neurologic outcomes in MPS II patients. This study evaluated the effectiveness of adeno-associated virus (AAV) vector encoding human IDS delivered intracerebroventricularly in a murine model of MPS II. Supraphysiological levels of IDS were observed in the circulation (160-fold higher than wild type) for at least 28 weeks post injection and in most tested peripheral organs (up to 270-fold) at 10 months post injection. In contrast, only low levels of IDS were observed (7-40% of wild type) in all areas of the brain. Sustained IDS expression had a profound effect on normalization of GAG in all tested tissues and on prevention of hepatomegaly. Additionally, sustained IDS expression in the central nervous system (CNS) had a prominent effect in preventing neurocognitive deficit in MPS II mice treated at 2 months of age. This study demonstrates that CNS-directed, AAV9 mediated gene transfer is a potentially effective treatment for Hunter syndrome, as well as other monogenic disorders with neurologic involvement.
Asunto(s)
Dependovirus/genética , Técnicas de Transferencia de Gen , Terapia Genética , Vectores Genéticos/genética , Iduronato Sulfatasa/genética , Mucopolisacaridosis II/genética , Mucopolisacaridosis II/psicología , Animales , Sistema Nervioso Central/metabolismo , Cognición , Modelos Animales de Enfermedad , Activación Enzimática , Femenino , Expresión Génica , Orden Génico , Vectores Genéticos/administración & dosificación , Glicosaminoglicanos/metabolismo , Humanos , Iduronato Sulfatasa/sangre , Iduronato Sulfatasa/metabolismo , Masculino , Ratones , Mucopolisacaridosis II/sangre , Mucopolisacaridosis II/terapia , Pruebas Neuropsicológicas , Proyectos Piloto , Factores de Tiempo , Distribución Tisular , Transducción GenéticaRESUMEN
Mucopolysaccharidosis type I (MPS I) is a progressive, multi-systemic, inherited metabolic disease caused by deficiency of α-L-iduronidase (IDUA). Current treatments for this disease are ineffective in treating central nervous system (CNS) disease due to the inability of lysosomal enzymes to traverse the blood-brain barrier. A noninvasive and effective approach was taken in the treatment of CNS disease by intranasal administration of an IDUA-encoding adeno-associated virus serotype 9 (AAV9) vector. Adult IDUA-deficient mice aged 3 months were instilled intranasally with AAV9-IDUA vector. Animals sacrificed 5 months post instillation exhibited IDUA enzyme activity levels that were up to 50-fold that of wild-type mice in the olfactory bulb, with wild-type levels of enzyme restored in all other parts of the brain. Intranasal treatment with AAV9-IDUA also resulted in the reduction of tissue glycosaminoglycan storage materials in the brain. There was strong IDUA immunofluorescence staining of tissue sections observed in the nasal epithelium and olfactory bulb, but there was no evidence of the presence of transduced cells in other portions of the brain. This indicates that reduction of storage materials most likely occurred as a result of enzyme diffusion from the olfactory bulb and the nasal epithelium into deeper areas of the brain. At 8 months of age, neurocognitive testing using the Barnes maze to assess spatial navigation demonstrated that treated IDUA-deficient mice were no different from normal control animals, while untreated IDUA-deficient mice exhibited significant learning and navigation deficits. This novel, noninvasive strategy for intranasal AAV9-IDUA instillation could potentially be used to treat CNS manifestations of human MPS I.
Asunto(s)
Sistema Nervioso Central/metabolismo , Dependovirus/metabolismo , Técnicas de Transferencia de Gen , Iduronidasa/genética , Iduronidasa/uso terapéutico , Mucopolisacaridosis I/patología , Mucopolisacaridosis I/terapia , Enfermedades del Sistema Nervioso/prevención & control , Administración Intranasal , Animales , Sistema Nervioso Central/patología , Sistema Nervioso Central/fisiopatología , Cognición , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Iduronidasa/metabolismo , Lisosomas/metabolismo , Ratones , Mucopolisacaridosis I/fisiopatología , Mucosa Nasal/metabolismo , Mucosa Nasal/patología , Enfermedades del Sistema Nervioso/patología , Enfermedades del Sistema Nervioso/fisiopatología , Neuronas/metabolismo , Bulbo Olfatorio/metabolismo , Bulbo Olfatorio/patología , Transducción GenéticaRESUMEN
During the acute-phase reaction, SAA (serum amyloid A) replaces apoA-I (apolipoprotein A-I) as the major HDL (high-density lipoprotein)-associated apolipoprotein. A remarkable portion of SAA exists in a lipid-free/lipid-poor form and promotes ABCA1 (ATP-binding cassette transporter A1)-dependent cellular cholesterol efflux. In contrast with lipid-free apoA-I and apoE, lipid-free SAA was recently reported to mobilize SR-BI (scavenger receptor class B, type I)-dependent cellular cholesterol efflux [Van der Westhuyzen, Cai, de Beer and de Beer (2005) J. Biol. Chem. 280, 35890-35895]. This unique property could strongly affect cellular cholesterol mobilization during inflammation. However, in the present study, we show that overexpression of SR-BI in HEK-293 cells (human embryonic kidney cells) (devoid of ABCA1) failed to mobilize cholesterol to lipid-free or lipid-poor SAA. Only reconstituted vesicles containing phospholipids and SAA promoted SR-BI-mediated cholesterol efflux. Cholesterol efflux from HEK-293 and HEK-293[SR-BI] cells to lipid-free and lipid-poor SAA was minimal, while efficient efflux was observed from fibroblasts and CHO cells (Chinese-hamster ovary cells) both expressing functional ABCA1. Overexpression of SR-BI in CHO cells strongly attenuated cholesterol efflux to lipid-free SAA even in the presence of an SR-BI-blocking IgG. This implies that SR-BI attenuates ABCA1-mediated cholesterol efflux in a way that is not dependent on SR-BI-mediated re-uptake of cholesterol. The present in vitro experiments demonstrate that the lipidation status of SAA is a critical factor governing cholesterol acceptor properties of this amphipathic apolipoprotein. In addition, we demonstrate that SAA mediates cellular cholesterol efflux via the ABCA1 and/or SR-BI pathway in a similar way to apoA-I.
Asunto(s)
Antígenos CD36/metabolismo , Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Proteína Amiloide A Sérica/metabolismo , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Apolipoproteína A-I/metabolismo , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Humanos , Metabolismo de los Lípidos , Fosfolípidos/metabolismo , Transducción de SeñalRESUMEN
The etiology of human female infertility is often uncertain. The sterility of high-density lipoprotein (HDL) receptor-negative (SR-BI(-/-)) female mice suggests a link between female infertility and abnormal lipoprotein metabolism. SR-BI(-/-) mice exhibit elevated plasma total cholesterol [with normal-sized and abnormally large HDL and high unesterified to total plasma cholesterol (UC:TC) ratio]. We explored the influence of hepatic SR-BI on female fertility by inducing hepatic SR-BI expression in SR-BI(-/-) animals by adenovirus transduction or stable transgenesis. For transgenes, we used both wild-type SR-BI and a double-point mutant, Q402R/Q418R (SR-BI-RR), which is unable to bind to and mediate lipid transfer from wild-type HDL normally, but retains virtually normal lipid transport activities with low-density lipoprotein. Essentially wild-type levels of hepatic SR-BI expression in SR-BI(-/-) mice restored to nearly normal the HDL size distribution and plasma UC:TC ratio, whereas approximately 7- to 40-fold overexpression dramatically lowered plasma TC and increased biliary cholesterol secretion. In contrast, SR-BI-RR overexpression had little effect on SR-BI(+/+) mice, but in SR-BI(-/-) mice, it substantially reduced levels of abnormally large HDL and normalized the UC:TC ratio. In all cases, hepatic transgenic expression restored female fertility. Overexpression in SR-BI(-/-) mice of lecithin:cholesterol acyl transferase, which esterifies plasma HDL cholesterol, did not normalize the UC:TC ratio, probably because the abnormal HDL was a poor substrate, and did not restore fertility. Thus, hepatic SR-BI-mediated lipoprotein metabolism influences murine female fertility, raising the possibility that dyslipidemia might contribute to human female infertility and that targeting lipoprotein metabolism might complement current assisted reproductive technologies.
Asunto(s)
Fertilidad , Lipoproteínas HDL/metabolismo , Hígado/metabolismo , Receptores Depuradores de Clase B/fisiología , Adenoviridae/genética , Animales , Colesterol/metabolismo , Femenino , Ratones , Ratones Endogámicos C57BL , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Receptores Depuradores de Clase B/genéticaRESUMEN
The hypercholesterolemia characteristic of apolipoprotein (apoE)-deficient mice fed on a regular chow diet is caused by the abnormal accumulation of apoB-48-carrying remnants of chylomicrons and very low density lipoproteins in the plasma. Treatment of apoE-deficient mice with ciprofibrate or other peroxisome proliferator-activated receptor alpha agonists severely aggravates their hypercholesterolemia by interfering with one or more mechanisms of remnant removal from the circulation that do not require mediation by apoE (Fu, T., Kashireddy, P., and Borensztajn, J. (2003) Biochem. J. 373, 941-947). In the present investigation we report that ciprofibrate treatment causes the down-regulation of hepatic scavenger receptor class B, type I (SR-BI) protein expression in the livers of apoE-deficient mice. On cessation of the treatment SR-BI expression returns to its pretreatment levels, coinciding with a reversal of the hypercholesterolemia to base-line concentrations. Restoration of SR-BI expression in ciprofibrate-treated apoE-deficient mice by recombinant adenoviral gene transfer abolishes the ciprofibrate-induced over accumulation of apoB-48-carrying remnants in the plasma. We also report that remnants isolated from the plasma of ciprofibrate-treated apoE-deficient mice bind to murine SR-BI expressed in stably transfected cultured cells. These observations suggest that, in addition to its well established role as high density lipoprotein receptor, SR-BI can also function as a remnant receptor responsible for the clearance of remnants from the circulation of apoE-deficient mice.
Asunto(s)
Adenoviridae/genética , Apolipoproteínas E/fisiología , Antígenos CD36/metabolismo , Colesterol/análogos & derivados , Ácido Clofíbrico , Ácido Clofíbrico/análogos & derivados , Hipercolesterolemia/metabolismo , Proteínas de la Membrana , Receptores Inmunológicos , Receptores de Lipoproteína , Animales , Apolipoproteínas E/genética , Células CHO , Colesterol/metabolismo , Colesterol/farmacología , Ácido Clofíbrico/farmacología , Cricetinae , Regulación hacia Abajo , Femenino , Ácidos Fíbricos , Técnicas de Transferencia de Gen , Vectores Genéticos , Hipolipemiantes/farmacología , Immunoblotting , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Proliferadores de Peroxisomas/farmacología , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Depuradores , Receptores Depuradores de Clase B , Succinimidas/farmacología , Factores de Tiempo , Factores de Transcripción/antagonistas & inhibidores , TransfecciónRESUMEN
BACKGROUND: Recombinant adenoviruses are an established tool for somatic gene transfer to multiple cell types in animals as well as in tissue culture. However, generation of adenoviruses expressing transgenes that are potentially toxic to the host cell line represents a practical problem. The aim of this study was to construct an adenoviral expression system that prevents transgene expression during the generation and propagation of the virus, and allows efficient gene transfer to lung and liver, major target organs of gene therapy. METHODS: Using the tet-off system we constructed tetracycline (tet) regulatable recombinant adenoviruses expressing the marker gene LacZ (Adtet-off.LacZ) as well as a secretory protein, human group IIA secretory phospholipase A(2) (Adtet-off.hsPLA(2)). Expression (Western blot, activity assay) was tested in vitro (HeLa cells), and in vivo by gene transfer to lung and liver. RESULTS: Without addition of tetracycline we demonstrated expression of LacZ (Adtet-off.LacZ) and sPLA(2) (Adtet-off.hsPLA(2)) in HeLa cells. Providing additional tet-transactivator (tTA) protein either by stable transfection or coinfection with a tTA-expressing adenovirus resulted in a further increase of LacZ and sPLA(2) expression. Transgene expression in vitro was eliminated by the addition of tetracycline to the culture medium. Adtet-off.LacZ and Adtet-off.hsPLA(2) allowed successful gene transfer in vivo to lung and liver. While the expression was highly efficient within the lungs, however, additional tTA was necessary to achieve high-level expression within liver. CONCLUSIONS: Tet-regulatable adenoviral expression systems may facilitate the construction of recombinant adenoviruses encoding potentially toxic transgenes and permit regulated transgene expression.
Asunto(s)
Adenoviridae/metabolismo , Técnicas de Transferencia de Gen , Hígado , Pulmón , Fosfolipasas A/genética , Tetraciclina/farmacología , Transgenes , Adenoviridae/genética , Animales , Células Cultivadas , Femenino , Regulación de la Expresión Génica , Marcación de Gen , Vectores Genéticos , Fosfolipasas A2 Grupo II , Células HeLa , Humanos , Operón Lac/fisiología , Hígado/metabolismo , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Fosfolipasas A/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transactivadores/fisiologíaRESUMEN
The nuclear bile acid receptor FXR has been proposed to play a central role in the feedback repression of the gene encoding cholesterol 7 alpha-hydroxylase (CYP7A1), the first and rate-limiting step in the biosynthesis of bile acids. We demonstrate that FXR directly regulates expression of fibroblast growth factor-19 (FGF-19), a secreted growth factor that signals through the FGFR4 cell-surface receptor tyrosine kinase. In turn, FGF-19 strongly suppresses expression of CYP7A1 in primary cultures of human hepatocytes and mouse liver through a c-Jun N-terminal kinase (JNK)-dependent pathway. This signaling cascade defines a novel mechanism for feedback repression of bile acid biosynthesis and underscores the vital role of FXR in the regulation of multiple pathways of cholesterol catabolism in the liver.
Asunto(s)
Ácidos y Sales Biliares/biosíntesis , Colesterol 7-alfa-Hidroxilasa/genética , Proteínas de Unión al ADN/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Hepatocitos/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Animales , Antracenos/farmacología , Línea Celular , Células Cultivadas , Ácido Quenodesoxicólico/farmacología , Colesterol 7-alfa-Hidroxilasa/metabolismo , Proteínas de Unión al ADN/agonistas , Proteínas de Unión al ADN/genética , Represión Enzimática , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/farmacología , Regulación de la Expresión Génica , Humanos , Isoxazoles/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos , Ratones , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-jun/metabolismo , Receptores Citoplasmáticos y Nucleares , Proteínas Recombinantes/farmacología , Elementos de Respuesta , Factores de Transcripción/agonistas , Factores de Transcripción/genética , TransfecciónRESUMEN
Hepatoma cell lines serve as a suitable model to study hepatic clearance of lipoprotein-associated cholesteryl esters (CEs). The present study aimed at investigating holoparticle-association of and selective CE-uptake from human high density lipoprotein subclass 3 (HDL3) by non-malignant adult (Chang-liver) and non-malignant fetal (WRL-68) epithelial cell lines as well as a hepatocellular carcinoma (HUH-7) cell line. Binding properties of 125I-HDL3 at 4 and 37 degrees C were similar for all three cell lines while degradation rates were highest for Chang-liver cells. Calculating the selective uptake of HDL3-associated CEs as the difference between [3H]CE- and 125I-HDL3 cell-association revealed that the selective lipid uptake and holoparticle-association was similar in Chang-liver while in WRL-68 and HUH-7 cells pronounced capacity for lipid tracer uptake in excess of holoparticle uptake was measured. Using RT-PCR, Northern and Western blot analysis, as well as immunocytochemical technique pronounced expression of scavenger receptor class B, type I (SR-BI) but not SR-BII (a splice variant of SR-BI less efficient for selective CE-uptake than SR-BI) could be identified in HUH-7 and WRL-68 cells. A polyclonal antiserum raised against SR-BI significantly decreased cell-association of [3H]CE-HDL3 in HUH-7 and WRL-68. The present findings suggest that the capacity for selective cholesteryl ester-uptake from high density lipoprotein by malignant and normal epithelial cells from the liver depends on expression of the scavenger receptor class B, type I.
Asunto(s)
Antígenos CD36/metabolismo , Ésteres del Colesterol/metabolismo , Hepatocitos/metabolismo , Lipoproteínas HDL/metabolismo , Proteínas de la Membrana , Receptores de Lipoproteína , Sialoglicoproteínas , Animales , Células CHO , Carcinoma Hepatocelular/metabolismo , Cricetinae , Células Epiteliales/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Proteínas de Membrana de los Lisosomas , Receptores Depuradores , Receptores Depuradores de Clase B , Células Tumorales CultivadasRESUMEN
Fibrates are normolipidemic drugs used in atherogenic dyslipidemia because of their ability to raise high density lipoprotein (HDL) and decrease triglyceride levels. They exert multiple effects on lipid metabolism by activating the peroxisome proliferator-activated receptor-alpha (PPAR-alpha), which controls the transcriptional regulation of genes involved in hepatic fatty acid, cholesterol, and lipoprotein metabolism. The hepatic expression of the scavenger receptor class B type I (SR-BI) plays a critical role in lipoprotein metabolism, mainly due to its ability to mediate selective cholesterol uptake. Because fibrates and PPAR-alpha agonists up-regulate SR-BI expression in human and murine macrophages, we tested whether fibrates raised a similar regulatory response on hepatic SR-BI expression in mice. Surprisingly, fibrate treatment suppressed SR-BI protein expression in the liver without changing steady state SR-BI mRNA levels. Decreased hepatic SR-BI protein expression correlated with enlarged HDL particle size. This effect was concomitant with down-regulation of CLAMP, a putative SR-BI-stabilizing protein found in the hepatic plasma membrane, which was also not associated to changes in CLAMP mRNA levels. The post-transcriptional regulatory effect of fibrates over hepatic SR-BI protein levels was dependent on PPAR-alpha expression, because it was absent in PPAR-alpha-deficient mice. Restoring hepatic SR-BI expression in fibrate-treated mice by recombinant adenoviral gene transfer abolished fibrate-mediated HDL particle size enlargement. This study describes a novel effect of fibrates on hepatic SR-BI expression providing an alternative mechanism by which this drug family modulates HDL metabolism in vivo.
Asunto(s)
Antígenos CD36/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Hipolipemiantes/farmacología , Proteínas de la Membrana , Receptores Inmunológicos , Receptores de Lipoproteína , Adenoviridae/genética , Animales , Antígenos CD36/genética , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Depuradores , Receptores Depuradores de Clase B , TransfecciónRESUMEN
Hypochlorous acid/hypochlorite (HOCl/OCl(-)), a potent oxidant generated in vivo by the myeloperoxidase-H(2)O(2)-chloride system of activated phagocytes, alters the physiological properties of high density lipoprotein (HDL) by generating a proatherogenic lipoprotein particle. On endothelial cells lectin-like oxidized low density lipoprotein receptor 1 (LOX-1) and scavenger receptor class B, type I (SR-BI), act in concert by mediating the holoparticle of and selective cholesteryl ester uptake from HOCl-HDL. We therefore investigated the ligand specificity of HOCl-HDL to SR-BI-overexpressing Chinese hamster ovary cells. Binding of HOCl-HDL was saturable, and the degree of HOCl modification was the determining factor for increased binding affinity to SR-BI. Competition experiments further confirmed that HOCl-HDL binds with increased affinity to the same or overlapping domain(s) of SR-BI as does native HDL. Furthermore, SR-BI-mediated selective HDL-cholesteryl ester association as well as time- and concentration-dependent cholesterol efflux from SR-BI overexpressing Chinese hamster ovary cells were, depending on the degree of HOCl modification of HDL, markedly impaired. The most significant findings of this study were that the presence of very low concentrations of HOCl-HDL severely impaired SR-BI-mediated bidirectional cholesterol flux mediated by native HDL. The colocalization of immunoreactive HOCl-modified epitopes with apolipoprotein A-I along with deposits of lipids in serial sections of human atheroma shown here indicates that the myeloperoxidase-H(2)O(2)-halide system contributes to oxidative damage of HDL in vivo.