RESUMEN
Metachromatic leukodystrophy (MLD) is a rare, inherited, demyelinating lysosomal storage disorder caused by mutations in the arylsulfatase-A gene (ARSA). In patients, levels of functional ARSA enzyme are diminished and lead to deleterious accumulation of sulfatides. Herein, we demonstrate that intravenous administration of HSC15/ARSA restored the endogenous murine biodistribution of the corresponding enzyme, and overexpression of ARSA corrected disease biomarkers and ameliorated motor deficits in Arsa KO mice of either sex. In treated Arsa KO mice, when compared with intravenously administered AAV9/ARSA, significant increases in brain ARSA activity, transcript levels, and vector genomes were observed with HSC15/ARSA Durability of transgene expression was established in neonate and adult mice out to 12 and 52 weeks, respectively. Levels and correlation between changes in biomarkers and ARSA activity required to achieve functional motor benefit was also defined. Finally, we demonstrated blood-nerve, blood-spinal and blood-brain barrier crossing as well as the presence of circulating ARSA enzyme activity in the serum of healthy nonhuman primates of either sex. Together, these findings support the use of intravenous delivery of HSC15/ARSA-mediated gene therapy for the treatment of MLD.SIGNIFICANCE STATEMENT Herein, we describe the method of gene therapy adeno-associated virus (AAV) capsid and route of administration selection leading to an efficacious gene therapy in a mouse model of metachromatic leukodystrophy. We demonstrate the therapeutic outcome of a new naturally derived clade F AAV capsid (AAVHSC15) in a disease model and the importance of triangulating multiple end points to increase the translation into higher species via ARSA enzyme activity and biodistribution profile (with a focus on the CNS) with that of a key clinically relevant biomarker.
Asunto(s)
Arilsulfatasas , Terapia Genética , Leucodistrofia Metacromática , Animales , Ratones , Macaca fascicularis , Arilsulfatasas/genética , Ratones Noqueados , Leucodistrofia Metacromática/genética , Leucodistrofia Metacromática/fisiopatología , Leucodistrofia Metacromática/terapia , Modelos Animales de Enfermedad , Dependovirus/genética , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Encéfalo/enzimología , Trastornos Motores/genética , Trastornos Motores/terapia , Administración Intravenosa , Biomarcadores/análisis , Barrera Hematoencefálica , Masculino , Femenino , HumanosRESUMEN
Lipoprotein lipase (LPL) is responsible for the intravascular processing of triglyceride-rich lipoproteins. The LPL within capillaries is bound to GPIHBP1, an endothelial cell protein with a three-fingered LU domain and an N-terminal intrinsically disordered acidic domain. Loss-of-function mutations in LPL or GPIHBP1 cause severe hypertriglyceridemia (chylomicronemia), but structures for LPL and GPIHBP1 have remained elusive. Inspired by our recent discovery that GPIHBP1's acidic domain preserves LPL structure and activity, we crystallized an LPL-GPIHBP1 complex and solved its structure. GPIHBP1's LU domain binds to LPL's C-terminal domain, largely by hydrophobic interactions. Analysis of electrostatic surfaces revealed that LPL contains a large basic patch spanning its N- and C-terminal domains. GPIHBP1's acidic domain was not defined in the electron density map but was positioned to interact with LPL's large basic patch, providing a likely explanation for how GPIHBP1 stabilizes LPL. The LPL-GPIHBP1 structure provides insights into mutations causing chylomicronemia.
Asunto(s)
Lipoproteína Lipasa/metabolismo , Plasma/metabolismo , Receptores de Lipoproteína/metabolismo , Triglicéridos/sangre , Triglicéridos/metabolismo , Animales , Células CHO , Capilares/metabolismo , Línea Celular , Cricetulus , Cristalografía por Rayos X/métodos , Células Endoteliales/metabolismo , Humanos , Hidrólisis , Hipertrigliceridemia/metabolismoRESUMEN
Studies in human populations have shown a significant correlation between procollagen C-endopeptidase enhancer protein 2 (PCPE2) single nucleotide polymorphisms and plasma HDL cholesterol concentrations. PCPE2, a 52-kDa glycoprotein located in the extracellular matrix, enhances the cleavage of C-terminal procollagen by bone morphogenetic protein 1 (BMP1). Our studies here focused on investigating the basis for the elevated concentration of enlarged plasma HDL in PCPE2-deficient mice to determine whether they protected against diet-induced atherosclerosis. PCPE2-deficient mice were crossed with LDL receptor-deficient mice to obtain LDLr(-/-), PCPE2(-/-) mice, which had elevated HDL levels compared with LDLr(-/-) mice with similar LDL concentrations. We found that LDLr(-/-), PCPE2(-/-) mice had significantly more neutral lipid and CD68+ infiltration in the aortic root than LDLr(-/-) mice. Surprisingly, in light of their elevated HDL levels, the extent of aortic lipid deposition in LDLr(-/-), PCPE2(-/-) mice was similar to that reported for LDLr(-/-), apoA-I(-/-) mice, which lack any apoA-I/HDL. Furthermore, LDLr(-/-), PCPE2(-/-) mice had reduced HDL apoA-I fractional clearance and macrophage to fecal reverse cholesterol transport rates compared with LDLr(-/-) mice, despite a 2-fold increase in liver SR-BI expression. PCPE2 was shown to enhance SR-BI function by increasing the rate of HDL-associated cholesteryl ester uptake, possibly by optimizing SR-BI localization and/or conformation. We conclude that PCPE2 is atheroprotective and an important component of the reverse cholesterol transport HDL system.
Asunto(s)
Aterosclerosis/metabolismo , Aterosclerosis/prevención & control , Ésteres del Colesterol/metabolismo , Glicoproteínas/metabolismo , Lipoproteínas HDL/metabolismo , Receptores Depuradores de Clase B/metabolismo , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/genética , Antígenos de Diferenciación Mielomonocítica/metabolismo , Aorta/metabolismo , Aorta/patología , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Aterosclerosis/inducido químicamente , Aterosclerosis/genética , Aterosclerosis/patología , Transporte Biológico Activo/genética , Células CHO , Ésteres del Colesterol/genética , Cricetulus , Glicoproteínas/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Lipoproteínas HDL/genética , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Ratones Noqueados , Receptores de LDL/genética , Receptores de LDL/metabolismo , Receptores Depuradores de Clase B/genéticaRESUMEN
1. 5-(N-(4-((4-ethylbenzyl)thio)phenyl)sulfamoyl)-2-methyl benzoic acid (CP-778875), an agonist of the peroxisome proliferator-activated receptor alpha, has been evaluated in the clinic to treat dyslipidemia and type 2 diabetes mellitus. Herein, we investigate the effect of CP-778875 on the pharmacokinetics of atorvastatin acid and its metabolites in humans. 2. The study incorporated a fixed-sequence design conducted in two groups. Group A was designed to estimate the effects of multiple doses of CP-778875 on the single dose pharmacokinetics of atorvastatin. Subjects in group A (n = 26) received atorvastatin (40 mg) on days 1 and 9 and CP-778875 (1.0 mg QD) on days 5-12. Group B was designed to examine the effects of multiple doses of atorvastatin on the single dose pharmacokinetics of CP-778875. Subjects in group B (n = 29) received CP-778875 (0.3 mg) on days 1 and 9 and atorvastatin (40 mg QD) on days 5-12. 3. Mean maximum serum concentration (Cmax) and area under the curve of atorvastatin were increased by 45% and 20%, respectively, upon co-administration with CP-778875. Statistically significant increases in the systemic exposure of ortho- and para-hydroxyatorvastatin were also observed upon concomitant dosing with CP-778875. CP-778875 pharmacokinetics, however, were not impacted upon concomitant dosing with atorvastatin. 4. Inhibition of organic anion transporting polypeptide 1B1 by CP-778875 (IC50 = 2.14 ± 0.40 µM) could be the dominant cause of the pharmacokinetic interaction as CP-778875 did not exhibit significant inhibition of cytochrome P450 3A4/3A5, multidrug resistant protein 1 or breast cancer resistant protein, which are also involved in the hepatobiliary disposition of atorvastatin.
Asunto(s)
Benzoatos/farmacología , Ácido Benzoico/farmacología , Ácidos Heptanoicos/farmacología , PPAR alfa/agonistas , Pirroles/farmacología , Sulfonamidas/farmacología , Animales , Atorvastatina , Benzoatos/química , Ácido Benzoico/química , Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/metabolismo , Perros , Interacciones Farmacológicas , Células HEK293 , Ácidos Heptanoicos/sangre , Ácidos Heptanoicos/farmacocinética , Humanos , Hidroxilación/efectos de los fármacos , Células de Riñón Canino Madin Darby , Proteínas de Transporte de Membrana/metabolismo , Oxidación-Reducción/efectos de los fármacos , Pirroles/sangre , Pirroles/farmacocinética , Sulfonamidas/química , Factores de TiempoRESUMEN
Dysregulation of ceramide synthesis has been associated with metabolic disorders such as atherosclerosis and diabetes. We examined the changes in lipid homeostasis and gene expression in Huh7 hepatocytes when the synthesis of ceramide is perturbed by knocking down serine pal mitoyltransferase subunits 1, 2, and 3 (SPTLC123) or dihydroceramide desaturase 1 (DEGS1). Although knocking down all SPTLC subunits is necessary to reduce total ceramides significantly, depleting DEGS1 is sufficient to produce a similar outcome. Lipidomic analysis of distribution and speciation of multiple lipid classes indicates an increase in phospholipids in SPTLC123-silenced cells, whereas DEGS1 depletion leads to the accumulation of sphingolipid intermediates, free fatty acids, and diacylglycerol. When cer amide synthesis is disrupted, the transcriptional profiles indicate inhibition in biosynthetic processes, downregulation of genes involved in general endomembrane trafficking, and upregulation of endocytosis and endosomal recycling. SPTLC123 silencing strongly affects the expression of genes involved with lipid metabolism. Changes in amino acid, sugar, and nucleotide metabolism, as well as vesicle trafficking between organelles, are more prominent in DEGS1-silenced cells. These studies are the first to provide a direct and comprehensive understanding at the lipidomic and transcriptomic levels of how Huh7 hepatocytes respond to changes in the inhibition of ceramide synthesis.
Asunto(s)
Ceramidas/biosíntesis , Ceramidas/metabolismo , Silenciador del Gen , Homeostasis/genética , Oxidorreductasas/genética , Serina C-Palmitoiltransferasa/genética , Transcriptoma/genética , Línea Celular , Línea Celular Tumoral , Regulación Enzimológica de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Humanos , Oxidorreductasas/deficiencia , Serina C-Palmitoiltransferasa/deficiencia , Transcripción Genética/genéticaRESUMEN
Weak peroxisome proliferator-activated receptor (PPAR) α agonists (fibrates) are used to treat dyslipidemia. This study compared the effects of the potent and selective PPARα agonist CP-778875 on peroxisomal ß-oxidation and cardiac and/or skeletal muscle injury with those of the weak PPARα agonist fenofibrate. We hypothesized that these muscle effects are mediated through the PPARα receptor, leading to increased ß-oxidation and consequent oxidative stress. CP-778875 (5 or 500 mg/kg) and fenofibrate (600 or 2,000â1,200 mg/kg, dose lowered because of intolerance) were administered to rats for six weeks. Standard end points, serum troponin I, heart and skeletal muscle ß-oxidation of palmitoyl-CoA, and acyl co-oxidase (AOX) mRNA were assessed. Both compounds dose-dependently increased the incidence and/or severity of cardiomyocyte degeneration and necrosis, heart weight, troponin I, and skeletal muscle degeneration. Mean heart ß-oxidation (3.4- to 5.1-fold control) and AOX mRNA (2.4- to 3.2-fold control) were increased with CP-778875 500 mg/kg and both doses of fenofibrate. ß-Oxidation of skeletal muscle was not affected by either compound; however, a significant increase in AOX mRNA (1.6- to 2.1-fold control) was observed with CP-778875 500 mg/kg and both doses of fenofibrate. Taken together, these findings were consistent with PPARα agonism and support the link between increased cardiac and skeletal muscle ß-oxidation and resultant muscle injury in the rat.
Asunto(s)
Fenofibrato/toxicidad , Corazón/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , PPAR alfa/agonistas , Animales , Análisis Químico de la Sangre , Peso Corporal , Relación Dosis-Respuesta a Droga , Femenino , Fenofibrato/farmacocinética , Hígado/química , Hígado/efectos de los fármacos , Hígado/enzimología , Hígado/patología , Masculino , Proteínas Musculares/metabolismo , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Enfermedades Musculares/inducido químicamente , Enfermedades Musculares/metabolismo , Miocardio/química , Miocardio/metabolismo , Miocardio/patología , NAD/metabolismo , Peroxisomas/metabolismo , Ratas , Ratas Sprague-Dawley , Pruebas de Toxicidad , Troponina I/sangre , Troponina I/metabolismoRESUMEN
Adeno-associated viruses derived from human hematopoietic stem cells (AAVHSCs) are naturally occurring AAVs. Fifteen AAVHSCs have demonstrated broad biodistribution while displaying differences in transduction. We examine the structure-function relationships of these natural amino acid variations on cellular binding. We demonstrate that AAVHSC16 is the only AAVHSC that does not preferentially bind to terminal galactose. AAVHSC16 contains two unique amino acids, 501I and 706C, compared with other AAVHSCs. Through mutagenesis, we determined that residue 501 contributes to the lack of galactose binding. Structural analysis revealed that residue 501 is in proximity to the galactose binding pocket, hence confirming its functional role in galactose binding. Biodistribution analysis of AAVHSC16 indicated significantly less liver tropism in mice and non-human primates compared with other clade F members, likely associated with overall binding differences observed in vitro. AAVHSC16 maintained robust tropism to other key tissues in the peripheral and central nervous systems after intravenous injection, including to the brain, heart, and gastrocnemius. Importantly, AAVHSC16 did not induce elevated liver enzyme levels in non-human primates after intravenous injection at high doses. The unique glycan binding and tropism of AAVHSC16 makes this naturally occurring capsid an attractive candidate for therapies requiring less liver tropism while maintaining broad biodistribution.
RESUMEN
Given the increased prevalence of cardiovascular disease in the world, the search for genetic variations that impact risk factors associated with the development of this disease continues. Multiple genetic association studies demonstrate that procollagen C-proteinase enhancer 2 (PCPE2) modulates HDL levels. Recent studies revealed an unexpected role for this protein in the proteolytic processing of pro-apolipoprotein (apo) A-I by enhancing the cleavage of the hexapeptide extension present at the N-terminus of apoA-I. To investigate the role of the PCPE2 protein in an in vivo model, PCPE2-deficient (PCPE2 KO) mice were examined, and a detailed characterization of plasma lipid profiles, apoA-I, HDL speciation, and function was done. Results of isoelectric focusing (IEF) electrophoresis together with the identification of the amino terminal peptides DEPQSQWDK and WHVWQQDEPQSQWDVK, representing mature apoA-I and pro-apoA-I, respectively, in serum from PCPE2 KO mice confirmed that PCPE2 has a role in apoA-I maturation. Lipid profiles showed a marked increase in plasma apoA-I and HDL-cholesterol (HDL-C) levels in PCPE2 KO mice compared with wild-type littermates, regardless of gender or diet. Changes in HDL particle size and electrophoretic mobility observed in PCPE2 KO mice suggest that the presence of pro-apoA-I impairs the maturation of HDL. ABCA1-dependent cholesterol efflux is defective in PCPE2 KO mice, suggesting that the functionality of HDL is altered.
Asunto(s)
Apolipoproteína A-I/sangre , Glicoproteínas/deficiencia , Glicoproteínas/genética , Lipoproteínas HDL/sangre , Secuencia de Aminoácidos , Animales , Apolipoproteína A-I/química , Colesterol/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Glicoproteínas/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Lipoproteínas HDL/química , Lipoproteínas HDL/metabolismo , Masculino , Ratones , Datos de Secuencia Molecular , Tamaño de la PartículaRESUMEN
Here we demonstrate that the ABC transporter ABCG1 plays a critical role in lipid homeostasis by controlling both tissue lipid levels and the efflux of cellular cholesterol to HDL. Targeted disruption of Abcg1 in mice has no effect on plasma lipids but results in massive accumulation of both neutral lipids and phospholipids in hepatocytes and in macrophages within multiple tissues following administration of a high-fat and -cholesterol diet. In contrast, overexpression of human ABCG1 protects murine tissues from dietary fat-induced lipid accumulation. Finally, we show that cholesterol efflux to HDL specifically requires ABCG1, whereas efflux to apoA1 requires ABCA1. These studies identify Abcg1 as a key gene involved in both cholesterol efflux to HDL and in tissue lipid homeostasis.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Colesterol/metabolismo , Regulación de la Expresión Génica , Metabolismo de los Lípidos , Lipoproteínas HDL/metabolismo , Lipoproteínas/fisiología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1 , Animales , Compuestos Azo/farmacología , Colorantes/farmacología , Femenino , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Hibridación in Situ , Operón Lac , Hígado/metabolismo , Pulmón/metabolismo , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , ARN/metabolismo , ARN Mensajero/metabolismoRESUMEN
The CETP inhibitor, torcetrapib, was prematurely terminated from phase 3 clinical trials due to an increase in cardiovascular and noncardiovascular mortality. Because nearly half of the latter deaths involved patients with infection, we have tested torcetrapib and other CETPIs to see if they interfere with lipopolysaccharide binding protein (LBP) or bactericidal/permeability increasing protein (BPI). No effect of these potent CETPIs on LPS binding to either protein was detected. Purified CETP itself bound weakly to LPS with a Kd >or= 25 microM compared with 0.8 and 0.5 nM for LBP and BPI, respectively, and this binding was not blocked by torcetrapib. In whole blood, LPS induced tumor necrosis factor-alpha normally in the presence of torcetrapib. Furthermore, LPS had no effect on CETP activity. We conclude that the sepsis-related mortality of the ILLUMINATE trial was unlikely due to a direct effect of torcetrapib on LBP or BPI function, nor to inhibition of an interaction of CETP with LPS. Instead, we speculate that the negative outcome seen for patients with infections might be related to the changes in plasma lipoprotein composition and metabolism, or alternatively to the known off-target effects of torcetrapib, such as aldosterone elevation, which may have aggravated the effects of sepsis.
Asunto(s)
Proteínas de Transferencia de Ésteres de Colesterol/antagonistas & inhibidores , Infecciones/inmunología , Quinolinas/farmacología , Proteínas de Fase Aguda/inmunología , Proteínas de Fase Aguda/metabolismo , Péptidos Catiónicos Antimicrobianos/inmunología , Péptidos Catiónicos Antimicrobianos/metabolismo , Proteínas Sanguíneas/inmunología , Proteínas Sanguíneas/metabolismo , Proteínas Portadoras/inmunología , Proteínas Portadoras/metabolismo , Humanos , Lipopolisacáridos/metabolismo , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Unión Proteica/efectos de los fármacos , Resonancia por Plasmón de SuperficieRESUMEN
Peroxisome proliferator-activated receptors (PPARs) are involved in the regulation of lipid and glucose metabolism. PPARgamma agonists improve insulin sensitivity and hyperglycemia and are effective in treating type 2 diabetes mellitus (T2DM), whereas PPARalpha agonists are used to treat dyslipidemia and atherosclerosis. The goal here was to examine the efficacy of a selective PPARalpha agonist {(S)-3-[3-(1-carboxy-1-methyl-ethoxy)-phenyl]-piperidine-1-carboxylic acid 4-trifluoromethyl-benzyl ester; CP-900691} on lipid, glycemic, and inflammation indices in 14 cynomolgus monkeys with spontaneous T2DM maintained on daily insulin therapy. Monkeys were dosed orally with either vehicle (n = 7) or CP-900691 (3 mg/kg, n = 7) daily for 6 weeks. CP-900691 treatment increased plasma high-density lipoprotein cholesterol (HDLC) (33 +/- 3 to 60 +/- 4 mg/dL, p < 0.001) and apolipoprotein A1 (96 +/- 5 to 157 +/- 5 mg/dL, p < 0.001), reduced plasma triglycerides (547 +/- 102 to 356 +/- 90 mg/dL, p < 0.01), and apolipoprotein B (62 +/- 3 to 45 +/- 3 mg/dL, p < 0.01), improved the lipoprotein index (HDL to non-HDLC ratio; 0.28 +/- 0.06 to 0.79 +/- 0.16, p < 0.001), decreased body weight (p < 0.01) and C-reactive protein (CRP) (1700 +/- 382 to 304 +/- 102 ng/ml, p < 0.01), and increased adiponectin (1697 +/- 542 to 4242 +/- 1070 ng/ml, p < 0.001) compared with baseline. CP-900691 treatment reduced exogenous insulin requirements by approximately 25% (p < 0.04) while lowering plasma fructosamine from 2.87 +/- 0.09 to 2.22 +/- 0.17 mM (p < 0.05), indicative of improved glycemic control. There were no changes in any of the aforementioned parameters in the vehicle group. Because low HDLC and high triglycerides are well established risk factors for cardiovascular disease, the marked improvements in these parameters, and in glycemic control, body weight, and CRP, suggest that CP-900691 may be of benefit in diabetic and obese or hyperlipidemic populations.
Asunto(s)
Glucemia/metabolismo , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hipoglucemiantes/farmacología , Hipolipemiantes , Lípidos/sangre , Lipoproteínas/sangre , PPAR alfa/agonistas , Piperidinas/farmacología , Propionatos/farmacología , Adiponectina/sangre , Animales , Área Bajo la Curva , Proteína C-Reactiva/metabolismo , Diabetes Mellitus Tipo 2/genética , Relación Dosis-Respuesta a Droga , Prueba de Tolerancia a la Glucosa , Resistencia a la Insulina , Macaca fascicularis , Pérdida de Peso/efectos de los fármacosRESUMEN
Phenylketonuria is an inborn error of metabolism caused by loss of function of the liver-expressed enzyme phenylalanine hydroxylase and is characterized by elevated systemic phenylalanine levels that are neurotoxic. Current therapies do not address the underlying genetic disease or restore the natural metabolic pathway resulting in the conversion of phenylalanine to tyrosine. A family of hepatotropic clade F adeno-associated viruses (AAVs) was isolated from human CD34+ hematopoietic stem cells (HSCs) and one (AAVHSC15) was utilized to deliver a vector to correct the phenylketonuria phenotype in Pahenu2 mice. The AAVHSC15 vector containing a codon-optimized form of the human phenylalanine hydroxylase cDNA was administered as a single intravenous dose to Pahenu2 mice maintained on a phenylalanine-containing normal chow diet. Optimization of the transgene resulted in a vector that produced a sustained reduction in serum phenylalanine and normalized tyrosine levels for the lifespan of Pahenu2 mice. Brain levels of phenylalanine and the downstream serotonin metabolite 5-hydroxyindoleacetic acid were restored. In addition, the coat color of treated mice darkened following treatment, indicating restoration of the phenylalanine metabolic pathway. Taken together, these data support the potential of an AAVHSC15-based gene therapy as an investigational therapeutic for phenylketonuria patients.
RESUMEN
Given the increased prevalence of cardiovascular disease in the world, the search for genetic variations controlling the levels of risk factors associated with the development of the disease continues. Multiple genetic association studies suggest the involvement of procollagen C-proteinase enhancer-2 (PCPE2) in modulating HDL-C levels. Therefore biochemical and mechanistic studies were undertaken to determine whether there might be a basis for a role of PCPE2 in HDL biogenesis. Our studies indicate that PCPE2 accelerates the proteolytic processing of pro-apolipoprotein (apo) AI by enhancing the cleavage of the hexapeptide extension present at the N terminus of apoAI. Surface Plasmon Resonance and immunoprecipitation studies indicate that PCPE2 interacts with BMP-1 and pro-apoAI to form a ternary pro-apoAI/BMP-1/PCPE2 complex. The most favorable interaction among these proteins begins with the association of BMP-1 to pro-apoAI followed by the binding of PCPE2 which further stabilizes the complex. PCPE2 resides, along with apoAI, on the HDL fraction of lipoproteins in human plasma supporting a relationship between HDL and PCPE2. Taken together, the findings from our studies identify a new player in the regulation of apoAI post-translational processing and open a new avenue to the study of mechanisms involved in the regulation of apoAI synthesis, HDL levels, and potentially, cardiovascular disease.
Asunto(s)
Apolipoproteína A-I/metabolismo , Proteína Morfogenética Ósea 1/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Precursores de Proteínas/metabolismo , Animales , Apolipoproteína A-I/genética , Proteína Morfogenética Ósea 1/genética , Células CHO , Línea Celular Tumoral , HDL-Colesterol/sangre , Cricetinae , Cricetulus , Proteínas de la Matriz Extracelular/genética , Glicoproteínas/genética , Humanos , Polimorfismo Genético , Precursores de Proteínas/genéticaRESUMEN
The nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha) is recognized as the primary target of the fibrate class of hypolipidemic drugs and mediates lipid lowering in part by activating a transcriptional cascade that induces genes involved in the catabolism of lipids. We report here the characterization of three novel PPARalpha agonists with therapeutic potential for treating dyslipidemia. These structurally related compounds display potent and selective binding to human PPARalpha and support robust recruitment of coactivator peptides in vitro. These compounds markedly potentiate chimeric transcription systems in cell-based assays and strikingly lower serum triglycerides in vivo. The transcription networks induced by these selective PPARalpha agonists were assessed by transcriptional profiling of mouse liver after short- and long-term treatment. The induction of several known PPARalpha target genes involved with fatty acid metabolism were observed, reflecting the expected pharmacology associated with PPARalpha activation. We also noted the down-regulation of a number of genes related to immune cell function, the acute phase response, and glucose metabolism, suggesting that these compounds may have anti-inflammatory action in the mammalian liver. Whereas these compounds are efficacious in acute preclinical models, extended safety studies and further clinical testing will be required before the full therapeutic promise of a selective PPARalpha agonist is realized.
Asunto(s)
Metabolismo de los Lípidos/fisiología , PPAR alfa/agonistas , Piperidinas/farmacología , Animales , Perfilación de la Expresión Génica , Humanos , Hipolipemiantes/farmacología , Metabolismo de los Lípidos/genética , Hígado , Ratones , Ratones Transgénicos , PPAR alfa/genética , PPAR alfa/metabolismo , Piperidinas/uso terapéuticoRESUMEN
The weak peroxisome proliferator activated receptor-alpha (PPAR-alpha) agonists gemfibrozil and fenofibrate achieve only small increases in high-density lipoprotein (HDL) cholesterol. CP-778,875 is a more potent PPAR-alpha agonist developed to produce greater HDL cholesterol increases. This randomized, multicenter, double-blinded, placebo-controlled study evaluated the efficacy and safety of CP-778,875 in subjects with mixed dyslipidemia and type 2 diabetes. Eight-six subjects with low HDL cholesterol (< or =45 mg/dl for men and < or =55 mg/dl for women) and increased triglycerides (150 to 500 mg/dl) who had coexisting type 2 diabetes were randomized. Subjects received CP-778,875 doses of 0.5, 2, or 6 mg/day or placebo for 6 weeks. Any other lipid-altering therapy was stopped at screening. The primary end point was percent change in HDL cholesterol from baseline. The 2-mg/day dose of CP-778,875 significantly increased HDL cholesterol by 14%. The 2-mg dose also increased concentrations of apolipoprotein (apo) A-I, HDL(2) cholesterol, and HDL(3) cholesterol by 13%, 12%, and 19%, respectively. An unusual dose-response pattern was observed in that at 6 mg/day CP-778,875 only increased HDL cholesterol by 3% and decreased HDL(2) cholesterol by 24%. Fasting triglyceride levels were significantly decreased to a similar extent (26%) by all 3 doses of CP-778,875. CP-778,875 significantly increased homocysteine levels. There was no significant relation between change in homocysteine and change in apoA-I or HDL cholesterol. No subjects developed myopathy. In conclusion, CP-778,875 2 mg/day significantly increased HDL cholesterol, significantly lowered fasting triglycerides, and increased apoA-I and HDL subfractions. The clinical relevance of the increase in homocysteine levels is unknown.
Asunto(s)
Diabetes Mellitus Tipo 2/complicaciones , Dislipidemias/tratamiento farmacológico , Fenofibrato/uso terapéutico , Gemfibrozilo/uso terapéutico , Hipolipemiantes/uso terapéutico , PPAR alfa/agonistas , Adulto , Anciano , Apolipoproteína A-I/efectos de los fármacos , Apolipoproteína B-100/efectos de los fármacos , Proteína C-Reactiva/efectos de los fármacos , HDL-Colesterol/efectos de los fármacos , Diabetes Mellitus Tipo 2/fisiopatología , Método Doble Ciego , Dislipidemias/fisiopatología , Femenino , Fenofibrato/efectos adversos , Gemfibrozilo/efectos adversos , Homocisteína/efectos de los fármacos , Humanos , Hipolipemiantes/efectos adversos , Masculino , Persona de Mediana EdadRESUMEN
Peroxisome proliferator-activated receptor alpha (PPARalpha) is a member of the nuclear receptor family of transcription factors and is recognized as the molecular target of the hypolipidemic fibrate drugs. Fibrates promote lipid catabolism by inducing genes involved in fatty acid beta-oxidation. In rodents this is accompanied by peroxisome proliferation, and after chronic dosing hepatocarcinoma, whereas epidemiological studies suggest these adverse events are lacking in humans. Rodents such as rats and mice appear particularly sensitive to PPARalpha-induced peroxisome proliferation while humans are resistant. These findings question the utility of rodent models for safety monitoring of experimental PPARalpha agonists and highlight the need for additional preclinical models that display greater physiological relevance for human response. Thus we have focused on elucidating the molecular mechanism of species-dependent peroxisome proliferation by understanding the PPARalpha-dependent regulation of the acyl-CoA oxidase (AOX) promoter, the rate-limiting step of peroxisomal beta-oxidation. We have chosen the cynomolgus monkey as a model that is modestly responsive to peroxisome proliferation and functionally characterized it against the highly responsive rat and non-responsive human species. We report the identification of a putative peroxisome proliferator response element (PPRE) within the 2.3 kb proximal promoter of the cynomolgus monkey AOX gene. Characterization of these promoters using a series of constitutively active, PPARalpha constructs demonstrate that the PPREs within the proximal cynomolgus and human AOX promoters are non-responsive to PPARalpha whereas the rat PPRE is highly responsive. These findings were verified in vivo using a small molecule PPARalpha agonist. Taken together, we demonstrate concordant regulation of the AOX promoter by PPARalpha in cynomolgus monkeys and humans and suggest that this model is superior to rodent models with respect to preclinical evaluation of PPARalpha agonists.
Asunto(s)
Acil-CoA Oxidasa/metabolismo , Regulación Enzimológica de la Expresión Génica , PPAR alfa/metabolismo , Regiones Promotoras Genéticas , Activación Transcripcional , Animales , Secuencia de Bases , Células Cultivadas , Clonación Molecular , Humanos , Macaca fascicularis , Datos de Secuencia Molecular , PPAR alfa/genética , Ratas , Elementos de Respuesta/genética , TransfecciónRESUMEN
OBJECTIVE: Studies in bone marrow transplanted from ATP-binding cassette transporter A1 (ABCA1)-deficient mice into normal mice provides direct evidence that the absence of leukocyte ABCA1 exerts a marked proatherogenic effect independent of changes in plasma lipids, suggesting that ABCA1 plays a key role in the regulation of cholesterol homeostasis and function of macrophages. Therefore, we examined whether the absence of ABCA1 affects the morphology, properties, and functional activities of macrophages that could be related to the development of atherosclerosis. METHODS AND RESULTS: We conducted a series of experiments in macrophages isolated from Abca1-deficient and wild-type mice and compared several of their properties that are thought to be related to the development of atherosclerosis. Macrophages isolated from Abca1-deficient mice have an increase in cholesterol content, expression of scavenger receptors, and secretion of chemokines, growth factors, and cytokines, resulting in an increased ability to respond to a variety of chemotactic factors. CONCLUSIONS: Our studies indicate that the absence of ABCA1 leads to significant changes in the morphology, properties, and functional activities of macrophages. These changes, together with the proinflammatory condition present in ABCA1-deficient mice and increased reactivity of macrophages to chemotactic factors, play a key role in the development and progression of atherosclerosis.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Aterosclerosis/fisiopatología , Colesterol/metabolismo , Macrófagos Peritoneales/metabolismo , Receptores Depuradores/genética , Transportador 1 de Casete de Unión a ATP , Animales , Aterosclerosis/inmunología , Aterosclerosis/patología , Células Cultivadas , Quimiocinas/metabolismo , Quimiotaxis de Leucocito/fisiología , Femenino , Lipopolisacáridos/farmacología , Macrófagos Peritoneales/patología , Macrófagos Peritoneales/ultraestructura , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Mutantes , Microscopía Electrónica , Choque Séptico/inmunología , Choque Séptico/patología , Choque Séptico/fisiopatología , Factor de Necrosis Tumoral alfa/metabolismo , Vacuolas/metabolismo , Vacuolas/ultraestructuraRESUMEN
Studies with ATP-binding cassette transporter (ABCA1)-deficient mice have been critical in demonstrating the relation between ABCA1 expression, cellular lipid efflux, and HDL metabolism. The phenotype of the ABCA1-deficient mouse parallels the phenotype observed in human Tangier disease, including substantial reductions in both apolipoprotein B and apolipoprotein AI with confounding affects on atherosclerosis.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Animales , Apolipoproteínas B/metabolismo , Arteriosclerosis/sangre , Arteriosclerosis/genética , HDL-Colesterol/metabolismo , Modelos Animales de Enfermedad , Femenino , Células Espumosas/patología , Humanos , Infertilidad/genética , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Fenotipo , Relación Estructura-Actividad , Enfermedad de Tangier/genética , Enfermedad de Tangier/metabolismoRESUMEN
The ATP-binding cassette transporter A1 (ABCA1) encodes a membrane protein that promotes cholesterol and phospholipid efflux from cells. Mutations in ABCA1 lead to HDL deficiency and tissue accumulation of macrophages in patients with homozygous Tangier disease. In this study, we examined whether the complete absence of ABCA1 or selected inactivation in macrophages is accompanied by an increase in atherosclerotic lesion progression in hypercholesterolemic apolipoprotein E-deficient (apoE(-/-)) mice and LDLR receptor-deficient (LDLr(-/-)) mice. The absence of ABCA1 led to reduced plasma cholesterol levels in both the apoE(-/-) and LDLr(-/-) mice, along with severe skin xanthomatosis characterized by marked foamy macrophages and cholesterol ester accumulation. However, the complete absence of ABCA1 did not affect the development, progression, or composition of atherosclerotic lesions in either the LDLr(-/-) or the apoE(-/-) mice fed a chow or atherogenic diet. In contrast, bone marrow transplantation studies demonstrated that the selective inactivation of ABCA1 in macrophages markedly increased atherosclerosis and foam cell accumulation in apoE(-/-). Taken together, these findings demonstrate that the complete absence of ABCA1 has a major impact on plasma lipoprotein homeostasis, and the proposed antiatherogenic effect resulting from ABCA1 deficiency is compensated by a less atherogenic profile. ABCA1 deficiency in macrophages, however, demonstrates the antiatherogenic properties of ABCA1 independent of plasma lipids and HDL levels.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Apolipoproteínas E/deficiencia , Arteriosclerosis/etiología , Colesterol/sangre , Hiperlipidemias/complicaciones , Macrófagos/metabolismo , Receptores de LDL/deficiencia , Transportador 1 de Casete de Unión a ATP , Animales , Arteriosclerosis/metabolismo , Arteriosclerosis/patología , Trasplante de Médula Ósea , Cruzamientos Genéticos , Femenino , Hiperlipidemias/metabolismo , Lípidos/sangre , Lipoproteínas/sangre , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL/genética , Ratones Endogámicos DBA/genética , Xantomatosis/patologíaRESUMEN
Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited disorder characterized by skin fragility, blistering, and multiple skin wounds with no currently approved or consistently effective treatment. It is due to mutations in the gene encoding type VII collagen (C7). Using recombinant human C7 (rhC7) purified from human dermal fibroblasts (FB-rhC7), we showed previously that intravenously injected rhC7 distributed to engrafted RDEB skin, incorporated into its dermal-epidermal junction (DEJ), and reversed the RDEB disease phenotype. Human dermal fibroblasts, however, are not used for commercial production of therapeutic proteins. Therefore, we generated rhC7 from Chinese hamster ovary (CHO) cells. The CHO-derived recombinant type VII collagen (CHO-rhC7), similar to FB-rhC7, was secreted as a correctly folded, disulfide-bonded, helical trimer resistant to protease degradation. CHO-rhC7 bound to fibronectin and promoted human keratinocyte migration in vitro. A single dose of CHO-rhC7, administered intravenously into new-born C7-null RDEB mice, incorporated into the DEJ of multiple skin sites, tongue and esophagus, restored anchoring fibrils, improved dermal-epidermal adherence, and increased the animals' life span. Furthermore, no circulating or tissue-bound anti-C7 antibodies were observed in the mice. These data demonstrate the efficacy of CHO-rhC7 in a preclinical murine model of RDEB.