RESUMEN
BACKGROUND: Inhibition of cholesteryl ester transfer protein (CETP) has been shown to have a substantial effect on plasma lipoprotein levels. We investigated whether torcetrapib, a potent CETP inhibitor, might reduce major cardiovascular events. The trial was terminated prematurely because of an increased risk of death and cardiac events in patients receiving torcetrapib. METHODS: We conducted a randomized, double-blind study involving 15,067 patients at high cardiovascular risk. The patients received either torcetrapib plus atorvastatin or atorvastatin alone. The primary outcome was the time to the first major cardiovascular event, which was defined as death from coronary heart disease, nonfatal myocardial infarction, stroke, or hospitalization for unstable angina. RESULTS: At 12 months in patients who received torcetrapib, there was an increase of 72.1% in high-density lipoprotein cholesterol and a decrease of 24.9% in low-density lipoprotein cholesterol, as compared with baseline (P<0.001 for both comparisons), in addition to an increase of 5.4 mm Hg in systolic blood pressure, a decrease in serum potassium, and increases in serum sodium, bicarbonate, and aldosterone (P<0.001 for all comparisons). There was also an increased risk of cardiovascular events (hazard ratio, 1.25; 95% confidence interval [CI], 1.09 to 1.44; P=0.001) and death from any cause (hazard ratio, 1.58; 95% CI, 1.14 to 2.19; P=0.006). Post hoc analyses showed an increased risk of death in patients treated with torcetrapib whose reduction in potassium or increase in bicarbonate was greater than the median change. CONCLUSIONS: Torcetrapib therapy resulted in an increased risk of mortality and morbidity of unknown mechanism. Although there was evidence of an off-target effect of torcetrapib, we cannot rule out adverse effects related to CETP inhibition. (ClinicalTrials.gov number, NCT00134264 [ClinicalTrials.gov].).
Asunto(s)
Anticolesterolemiantes/efectos adversos , Enfermedades Cardiovasculares/tratamiento farmacológico , Enfermedades Cardiovasculares/mortalidad , Proteínas de Transferencia de Ésteres de Colesterol/antagonistas & inhibidores , HDL-Colesterol/sangre , Enfermedad Coronaria/prevención & control , Quinolinas/efectos adversos , Anciano , Anticolesterolemiantes/uso terapéutico , Atorvastatina , Enfermedades Cardiovasculares/inducido químicamente , LDL-Colesterol/sangre , Enfermedad Coronaria/mortalidad , Método Doble Ciego , Quimioterapia Combinada , Femenino , Ácidos Heptanoicos/efectos adversos , Ácidos Heptanoicos/uso terapéutico , Humanos , Masculino , Persona de Mediana Edad , Mortalidad , Pirroles/efectos adversos , Pirroles/uso terapéutico , Quinolinas/uso terapéuticoRESUMEN
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.
Asunto(s)
Albúminas/genética , Trans-Empalme/genética , Animales , Apolipoproteína A-I/genética , Apolipoproteína A-I/fisiología , Exones/genética , Femenino , Terapia Genética/métodos , Vectores Genéticos/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Precursores del ARN/genética , Empalme del ARN/genética , Empalme del ARN/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Empalmosomas/genética , Empalmosomas/metabolismo , Trans-Empalme/fisiologíaRESUMEN
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.
Asunto(s)
Aterosclerosis/etiología , Proteínas de Unión al ADN/metabolismo , Células Espumosas/metabolismo , Células Espumosas/patología , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Transporte Biológico Activo , LDL-Colesterol/metabolismo , Citocinas/biosíntesis , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Femenino , Expresión Génica , Homeostasis , Técnicas In Vitro , Lípidos/sangre , Lipoproteínas LDL/metabolismo , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Citoplasmáticos y Nucleares/deficiencia , Receptores Citoplasmáticos y Nucleares/genética , Factores de Transcripción/deficiencia , Factores de Transcripción/genéticaRESUMEN
To elucidate the function of PPARgamma in leptin-deficient mouse (ob/ob) liver, a PPARgamma liver-null mouse on an ob/ob background, ob/ob-PPARgamma(fl/fl)AlbCre(+), was produced using a floxed PPARgamma allele, PPARgamma(fl/fl), and Cre recombinase under control of the albumin promoter (AlbCre). The liver of ob/ob-PPARgamma(fl/fl)AlbCre(+) mice had a deletion of exon 2 and a corresponding loss of full-length PPARgamma mRNA and protein. The PPARgamma-deficient liver in ob/ob mice was smaller and had a dramatically decreased triglyceride (TG) content compared with equivalent mice lacking the AlbCre transgene (ob/ob-PPARgamma(fl/fl)AlbCre(-)). Messenger RNA levels of the hepatic lipogenic genes, fatty acid synthase, acetyl-CoA carboxylase, and stearoyl-CoA desaturase-1, were reduced in ob/ob-PPARgamma(fl/fl)AlbCre(+) mice, and the levels of serum TG and FFA in ob/ob-PPARgamma(fl/fl)AlbCre(+) mice were significantly higher than in the control ob/ob-PPARgamma(fl/fl)AlbCre(-) mice. Rosiglitazone treatment exacerbated the fatty liver in ob/ob-PPARgamma(fl/fl)AlbCre(-) mice compared with livers from nonobese Cre(-) mice; there was no effect of rosiglitazone in ob/ob-PPARgamma(fl/fl)AlbCre(+) mice. The deficiency of hepatic PPARgamma further aggravated the severity of diabetes in ob/ob mice due to decreased insulin sensitivity in muscle and fat. These data indicate that hepatic PPARgamma plays a critical role in the regulation of TG content and in the homeostasis of blood glucose and insulin resistance in steatotic diabetic mice.
Asunto(s)
Hígado Graso/etiología , Hiperglucemia/etiología , Resistencia a la Insulina , Leptina/deficiencia , Hígado/fisiología , Receptores Citoplasmáticos y Nucleares/fisiología , Tiazolidinedionas , Factores de Transcripción/fisiología , Animales , Glucemia/análisis , Ácidos Grasos no Esterificados/sangre , Hígado Graso/terapia , Lipoproteína Lipasa/metabolismo , Lipoproteínas VLDL/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Receptores de LDL/metabolismo , Receptores de Leptina , Rosiglitazona , Tiazoles/uso terapéutico , Triglicéridos/metabolismoRESUMEN
To investigate structural requirement of helical apolipoprotein to phosphorylate and stabilize ATP-binding cassette transporter A1 (ABCA1), synthetic peptides (Remaley, A. T., Thomas, F., Stonik, J. A., Demosky, S. J., Bark, S. E., Neufeld, E. B., Bocharov, A. V., Vishnyakova, T. G., Patterson, A. P., Eggerman, T. L., Santamarina-Fojo, S., and Brewer, H. B. (2003) J. Lipid Res. 44, 828-836) were examined for these activities. L37pA, an L amino acid peptide that contains two class-A amphiphilic helices, and D37pA, the same peptide with all D amino acids, both removed cholesterol and phospholipid from differentiated THP-1 cells more than apolipoproteins (apos) A-I, A-II, and E. Both peptides also mediated lipid release from human fibroblasts WI-38 similar to apoA-I. L2D37pA, an L-peptide whose valine and tyrosine were replaced with D amino acids also promoted lipid release from WI-38 but less so with THP-1, whereas L3D37pA, in which alanine, lysine, and asparatic acid were replaced with D amino acids was ineffective in lipid release for both cell lines. ABCA1 protein in THP-1 and WT-38 was stabilized against proteolytic degradation by apoA-I, apoA-II, and apoE and by all the peptides tested except for L3D37pA, and ABCA1 phosphorylation closely correlated with its stabilization. The analysis of the relationship among these parameters indicated that removal of phospholipid triggers signals for phosphorylation and stabilization of ABCA1. We thus concluded that an amphiphilic helical motif is the minimum structural requirement for a protein to stabilize ABCA1 against proteolytic degradation.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Péptidos/química , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/química , Alanina/química , Apolipoproteína A-I/química , Apolipoproteína A-II/química , Apolipoproteínas E/química , Ácido Aspártico/química , Línea Celular , Colesterol/química , Fibroblastos/metabolismo , Humanos , Metabolismo de los Lípidos , Lisina/química , Fosforilación , Factores de Tiempo , Tirosina/química , Valina/químicaRESUMEN
Overexpression of human APOA5 in mice results in dramatically decreased plasma triglyceride levels. In this study we explored the mechanism underlying this hypotriglyceridemic effect. Initially we found that triglyceride turnover was faster in hAPOA5 transgenic mice compared to controls, and this strongly correlated with increased LPL activity in postheparin plasma. Furthermore, we show that in vitro recombinant apoAV interacts physically with lipoprotein lipase and significantly increased its activity. We show that both apoB and apoCIII are decreased in hAPOA5 transgenic mice indicating a decrease in VLDL number. To further investigate the mechanism of hAPOA5 in a hyperlipidemic background, we inter-crossed hAPOA5 and hAPOC3 transgenic mice. We found a marked decrease in VLDL triglyceride and cholesterol, as well as apolipoprotein B and CIII levels. These data indicated that apoAV induces a decrease in VLDL size by activating lipolysis and an increase of VLDL clearance. In a postprandial state, the normal triglyceride response found in wild-type mice was significantly reduced in hAPOA5 transgenics. In addition, we demonstrated that in response to this fat load in hAPOA5xhAPOC3 mice, apoAV, but not apoCIII, was redistributed from primarily HDL to VLDL. This shift of apoAV in VLDL appears to limit the increase of triglyceride by activating the lipoprotein lipase.