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1.
Biomed Res Int ; 2015: 607120, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26504816

RESUMEN

The role of scavenger receptor class B, type I (SR-BI) in endothelial cells (EC) was examined in several novel transgenic mouse models expressing SR-BI in endothelium of mice with normal C57Bl6/N, apoE-KO, or Scarb1-KO backgrounds. Mice were also created expressing SR-BI exclusively in endothelium and liver. Endothelial expression of the Tie2-Scarb1 transgene had no significant effect on plasma lipoprotein levels in mice on a normal chow diet but on an atherogenic diet, significantly decreased plasma cholesterol levels, increased plasma HDL cholesterol (HDL-C) levels, and protected mice against atherosclerosis. In 8-month-old apoE-KO mice fed a normal chow diet, the Tie2-Scarb1 transgene decreased aortic lesions by 24%. Mice expressing SR-BI only in EC and liver had a 1.5 ± 0.1-fold increase in plasma cholesterol compared to mice synthesizing SR-BI only in liver. This elevation was due mostly to increased HDL-C. In EC culture studies, SR-BI was found to be present in both basolateral and apical membranes but greater cellular uptake of cholesterol from HDL was found in the basolateral compartment. In summary, enhanced expression of SR-BI in EC resulted in a less atherogenic lipoprotein profile and decreased atherosclerosis, suggesting a possible role for endothelial SR-BI in the flux of cholesterol across EC.


Asunto(s)
Aterosclerosis/metabolismo , Endotelio Vascular/metabolismo , Receptores Depuradores de Clase B/metabolismo , Animales , Aorta/química , Aorta/citología , Aorta/metabolismo , Aterosclerosis/prevención & control , Colesterol/sangre , Endotelio Vascular/química , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Biológicos , Receptores Depuradores de Clase B/análisis , Receptores Depuradores de Clase B/genética
2.
J Lipid Res ; 56(9): 1727-37, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26117661

RESUMEN

The goal of this study was to understand how the reconstituted HDL (rHDL) phospholipid (PL) composition affects its cholesterol efflux and anti-inflammatory properties. An ApoA-I mimetic peptide, 5A, was combined with either SM or POPC. Both lipid formulations exhibited similar in vitro cholesterol efflux by ABCA1, but 5A-SM exhibited higher ABCG1- and SR-BI-mediated efflux relative to 5A-POPC (P < 0.05). Injection of both rHDLs in rats resulted in mobilization of plasma cholesterol, although the relative potency was 3-fold higher for the same doses of 5A-SM than for 5A-POPC. Formation of preß HDL was observed following incubation of rHDLs with both human and rat plasma in vitro, with 5A-SM inducing a higher extent of preß formation relative to 5A-POPC. Both rHDLs exhibited anti-inflammatory properties, but 5A-SM showed higher inhibition of TNF-α, IL-6, and IL-1ß release than did 5A-POPC (P < 0.05). Both 5A-SM and 5A-POPC showed reduction in total plaque area in ApoE(-/-) mice, but only 5A-SM showed a statistically significant reduction over placebo control and baseline (P < 0.01). The type of PL used to reconstitute peptide has significant influence on rHDL's anti-inflammatory and anti-atherosclerosis properties.


Asunto(s)
Aterosclerosis/metabolismo , Colesterol/metabolismo , Inflamación/metabolismo , Esfingomielinas/metabolismo , Animales , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/patología , Humanos , Inflamación/tratamiento farmacológico , Inflamación/patología , Lipoproteínas HDL/metabolismo , Ratones , Péptidos/administración & dosificación , Fosfatidilcolinas/administración & dosificación , Fosfolípidos/metabolismo , Ratas
3.
J Lipid Res ; 56(7): 1282-95, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-25964513

RESUMEN

LCAT, a plasma enzyme that esterifies cholesterol, has been proposed to play an antiatherogenic role, but animal and epidemiologic studies have yielded conflicting results. To gain insight into LCAT and the role of free cholesterol (FC) in atherosclerosis, we examined the effect of LCAT over- and underexpression in diet-induced atherosclerosis in scavenger receptor class B member I-deficient [Scarab(-/-)] mice, which have a secondary defect in cholesterol esterification. Scarab(-/-)×LCAT-null [Lcat(-/-)] mice had a decrease in HDL-cholesterol and a high plasma ratio of FC/total cholesterol (TC) (0.88 ± 0.033) and a marked increase in VLDL-cholesterol (VLDL-C) on a high-fat diet. Scarab(-/-)×LCAT-transgenic (Tg) mice had lower levels of VLDL-C and a normal plasma FC/TC ratio (0.28 ± 0.005). Plasma from Scarab(-/-)×LCAT-Tg mice also showed an increase in cholesterol esterification during in vitro cholesterol efflux, but increased esterification did not appear to affect the overall rate of cholesterol efflux or hepatic uptake of cholesterol. Scarab(-/-)×LCAT-Tg mice also displayed a 51% decrease in aortic sinus atherosclerosis compared with Scarab(-/-) mice (P < 0.05). In summary, we demonstrate that increased cholesterol esterification by LCAT is atheroprotective, most likely through its ability to increase HDL levels and decrease pro-atherogenic apoB-containing lipoprotein particles.


Asunto(s)
Aterosclerosis/sangre , Aterosclerosis/enzimología , Antígenos CD36/deficiencia , Antígenos CD36/genética , Colesterol/metabolismo , Dieta Alta en Grasa/efectos adversos , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Animales , Aterosclerosis/etiología , Aterosclerosis/metabolismo , Transporte Biológico , Plaquetas/metabolismo , Plaquetas/patología , Colesterol/sangre , Recuento de Eritrocitos , Eritrocitos/metabolismo , Eritrocitos/patología , Esterificación , Femenino , Regulación Enzimológica de la Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Lipoproteínas VLDL/biosíntesis , Lipoproteínas VLDL/sangre , Lipoproteínas VLDL/química , Hígado/metabolismo , Ratones , Ratones Transgénicos , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Recuento de Plaquetas
4.
Biology (Basel) ; 3(4): 866-91, 2014 Dec 04.
Artículo en Inglés | MEDLINE | ID: mdl-25485894

RESUMEN

We have previously shown that GFP-tagged human ABCG1 on the plasma membrane (PM) and in late endosomes (LE) mobilizes sterol on both sides of the membrane lipid bilayer, thereby increasing cellular cholesterol efflux to lipid surfaces. In the present study, we examined ABCG1-induced changes in membrane cholesterol distribution, organization, and mobility. ABCG1-GFP expression increased the amount of mobile, non-sphingomyelin(SM)-associated cholesterol at the PM and LE, but not the amount of SM-associated-cholesterol or SM. ABCG1-mobilized non-SM-associated-cholesterol rapidly cycled between the PM and LE and effluxed from the PM to extracellular acceptors, or, relocated to intracellular sites of esterification. ABCG1 increased detergent-soluble pools of PM and LE cholesterol, generated detergent-resistant, non-SM-associated PM cholesterol, and increased resistance to both amphotericin B-induced (cholesterol-mediated) and lysenin-induced (SM-mediated) cytolysis, consistent with altered organization of both PM cholesterol and SM. ABCG1 itself resided in detergent-soluble membrane domains. We propose that PM and LE ABCG1 residing at the phase boundary between ordered (Lo) and disordered (Ld) membrane lipid domains alters SM and cholesterol organization thereby increasing cholesterol flux between Lo and Ld, and hence, the amount of cholesterol available for removal by acceptors on either side of the membrane bilayer for either efflux or esterification.

5.
Biology (Basel) ; 3(4): 781-800, 2014 Nov 14.
Artículo en Inglés | MEDLINE | ID: mdl-25405320

RESUMEN

We have developed a suitable heterologous cell expression system to study the localization, trafficking, and site(s) of function of the human ABCG1 transporter. Increased plasma membrane (PM) and late endosomal (LE) cholesterol generated by ABCG1 was removed by lipoproteins and liposomes, but not apoA-I. Delivery of ABCG1 to the PM and LE was required for ABCG1-mediated cellular cholesterol efflux. ABCG1 LEs frequently contacted the PM, providing a collisional mechanism for transfer of ABCG1-mobilized cholesterol, similar to ABCG1-mediated PM cholesterol efflux to lipoproteins. ABCG1-mobilized LE cholesterol also trafficked to the PM by a non-vesicular pathway. Transfer of ABCG1-mobilized cholesterol from the cytoplasmic face of LEs to the PM and concomitant removal of cholesterol from the outer leaflet of the PM bilayer by extracellular acceptors suggests that ABCG1 mobilizes cholesterol on both sides of the lipid bilayer for removal by acceptors. ABCG1 increased uptake of HDL into LEs, consistent with a potential ABCG1-mediated cholesterol efflux pathway involving HDL resecretion. Thus, ABCG1 at the PM mobilizes PM cholesterol and ABCG1 in LE/LYS generates mobile pools of cholesterol that can traffic by both vesicular and non-vesicular pathways to the PM where it can also be transferred to extracellular acceptors with a lipid surface.

6.
J Pharmacol Exp Ther ; 344(1): 50-8, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23042953

RESUMEN

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.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Aminoácidos/química , Apolipoproteínas A/farmacología , Colesterol/metabolismo , Transportador 1 de Casete de Unión a ATP , Secuencia de Aminoácidos , Animales , Línea Celular , Dicroismo Circular , Cricetinae , Femenino , Lípidos/sangre , Lipoproteínas/sangre , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Imitación Molecular , Datos de Secuencia Molecular , Fosfolípidos/metabolismo
7.
Obesity (Silver Spring) ; 20(10): 2057-62, 2012 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22402736

RESUMEN

Diet-induced weight loss in women may be associated with decreases not only in plasma levels of low-density lipoprotein cholesterol (LDL-C), but also in high-density lipoprotein cholesterol (HDL-C). Whether a decrease in HDL-C is associated with altered HDL function is unknown. One hundred overweight or obese women (age 46 ± 11 years, 60 black; 12 diabetic) were enrolled in the 6-month program of reduced fat and total energy diet and low-intensity exercise. Serum cholesterol efflux capacity was measured in (3)H-cholesterol-labeled BHK cells expressing ABCA1, ABCG1, or SR-B1 transporters and incubated with 1% apolipoprotein B (apoB)-depleted serum. Antioxidant properties of HDL were estimated by paraoxonase-1 (PON1) activity and oxygen radical absorbance capacity (ORAC). Endothelial nitric oxide synthase (eNOS) activation was measured by conversion of L-arginine to L-citrulline in endothelial cells incubated with HDL from 49 subjects. Participants achieved an average weight loss of 2.2 ± 3.9 kg (P < 0.001), associated with reductions in both LDL-C (-6 ± 21 mg/dl, P = 0.004) and HDL-C (-3 ± 9 mg/dl, P = 0.016). Cholesterol efflux capacity by the ABCA1 transporter decreased by 10% (P = 0.006); efflux capacities by the ABCG1 and SR-B1 transporters were not significantly altered. ORAC decreased by 15% (P = 0.018); neither PON1 activity nor eNOS activation was significantly altered by reduction in HDL-C. Findings were similar for diabetic and nondiabetic subjects. Diet-induced weight loss in overweight or obese women is associated with a decrease in HDL-C levels, but overall HDL function is relatively spared, suggesting that decrease in HDL-C in this setting is not deleterious to cardiovascular risk.


Asunto(s)
HDL-Colesterol/sangre , Enfermedad de la Arteria Coronaria/sangre , Dieta con Restricción de Grasas , Endotelio Vascular/metabolismo , Ejercicio Físico , Obesidad/sangre , Pérdida de Peso , Absorciometría de Fotón , Adiposidad , Adulto , Arildialquilfosfatasa/sangre , Índice de Masa Corporal , LDL-Colesterol/sangre , Enfermedad de la Arteria Coronaria/epidemiología , Enfermedad de la Arteria Coronaria/fisiopatología , Endotelio Vascular/fisiopatología , Femenino , Humanos , Resistencia a la Insulina , Persona de Mediana Edad , Óxido Nítrico Sintasa de Tipo III/sangre , Obesidad/epidemiología , Obesidad/fisiopatología , Medición de Riesgo , Estados Unidos/epidemiología
8.
J Lipid Res ; 53(1): 158-67, 2012 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-22039582

RESUMEN

The role of endothelial ABCA1 expression in reverse cholesterol transport (RCT) was examined in transgenic mice, using the endothelial-specific Tie2 promoter. Human ABCA1 (hABCA1) was significantly expressed in endothelial cells (EC) of most tissues except the liver. Increased expression of ABCA1 was not observed in resident peritoneal macrophages. ApoA-I-mediated cholesterol efflux from aortic EC was 2.6-fold higher (P < 0.0001) for cells from transgenic versus control mice. On normal chow diet, Tie2 hABCA1 transgenic mice had a 25% (P < 0.0001) increase in HDL-cholesterol (HDL-C) and more than a 2-fold increase of eNOS mRNA in the aorta (P < 0.04). After 6 months on a high-fat, high-cholesterol (HFHC) diet, transgenic mice compared with controls had a 40% increase in plasma HDL-C (P < 0.003) and close to 40% decrease in aortic lesions (P < 0.02). Aortas from HFHC-fed transgenic mice also showed gene expression changes consistent with decreased inflammation and apoptosis. Beneficial effects of the ABCA1 transgene on HDL-C levels or on atherosclerosis were absent when the transgene was transferred onto ApoE or Abca1 knockout mice. In summary, expression of hABCA1 in EC appears to play a role in decreasing diet-induced atherosclerosis in mice and is associated with increased plasma HDL-C levels and beneficial gene expression changes in EC.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/biosíntesis , Aterosclerosis/prevención & control , HDL-Colesterol/sangre , Endotelio Vascular/metabolismo , Transportador 1 de Casete de Unión a ATP , Animales , Aorta/metabolismo , Apolipoproteína A-I/metabolismo , Colesterol en la Dieta/administración & dosificación , Grasas de la Dieta/administración & dosificación , Femenino , Humanos , Ratones , Ratones Transgénicos
9.
Mol Cancer Res ; 8(9): 1284-94, 2010 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-20671065

RESUMEN

Androgen deprivation therapy for prostate cancer leads to a significant increase of high-density lipoprotein (HDL), which is generally viewed as beneficial, particularly for cardiovascular disease, but the effect of HDL on prostate cancer is unknown. In this study, we investigated the effect of HDL on prostate cancer cell proliferation, migration, intracellular cholesterol levels, and the role of cholesterol transporters, namely ABCA1, ABCG1, and SR-BI in these processes. HDL induced cell proliferation and migration of the androgen-independent PC-3 and DU145 cells by a mechanism involving extracellular signal-regulated kinase (ERK) 1/2 and Akt, but had no effect on the androgen-dependent LNCaP cell, which did not express ABCA1 unlike the other cell lines. Treatment with HDL did not significantly alter the cholesterol content of the cell lines. Knockdown of ABCA1 but not ABCG1 or SR-BI by small interfering RNA (siRNA) inhibited HDL-induced cell proliferation, migration, and ERK1/2 and Akt signal transduction in PC-3 cells. Moreover, after treatment of LNCaP cells with charcoal-stripped fetal bovine serum, ABCA1 was induced ∼10-fold, enabling HDL to induce ERK1/2 activation, whereas small interfering RNA knockdown of ABCA1 inhibited HDL-induced ERK1/2 activation. Simvastatin, which inhibited ABCA1 expression in PC-3 and DU145 cells, attenuated HDL-induced PC-3 and DU145 cell proliferation, migration, and ERK1/2 and Akt phosphorylation. In human prostate biopsy samples, ABCA1 mRNA expression was ∼2-fold higher in the androgen deprivation therapy group than in subjects with benign prostatic hyperplasia or pretreatment prostate cancer groups. In summary, these results suggest that HDL by an ABCA1-dependent mechanism can mediate signal transduction, leading to increased proliferation and migration of prostate cancer cells.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Movimiento Celular/efectos de los fármacos , Lipoproteínas HDL/farmacología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/genética , Andrógenos/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Colesterol/metabolismo , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Masculino , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Simvastatina/farmacología
10.
J Pharmacol Exp Ther ; 335(1): 140-8, 2010 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-20605907

RESUMEN

Lecithin cholesterol acyl transferase (LCAT) deficiency is associated with low high-density lipoprotein (HDL) and the presence of an abnormal lipoprotein called lipoprotein X (Lp-X) that contributes to end-stage renal disease. We examined the possibility of using LCAT an as enzyme replacement therapy agent by testing the infusion of human recombinant (r)LCAT into several mouse models of LCAT deficiency. Infusion of plasma from human LCAT transgenic mice into LCAT-knockout (KO) mice rapidly increased HDL-cholesterol (C) and lowered cholesterol in fractions containing very-low-density lipoprotein (VLDL) and Lp-X. rLCAT was produced in a stably transfected human embryonic kidney 293f cell line and purified to homogeneity, with a specific activity of 1850 nmol/mg/h. Infusion of rLCAT intravenously, subcutaneously, or intramuscularly into human apoA-I transgenic mice showed a nearly identical effect in increasing HDL-C approximately 2-fold. When rLCAT was intravenously injected into LCAT-KO mice, it showed a similar effect as plasma from human LCAT transgenic mice in correcting the abnormal lipoprotein profile, but it had a considerably shorter half-life of approximately 1.23 ± 0.63 versus 8.29 ± 1.82 h for the plasma infusion. rLCAT intravenously injected in LCAT-KO mice crossed with human apolipoprotein (apo)A-I transgenic mice had a half-life of 7.39 ± 2.1 h and increased HDL-C more than 8-fold. rLCAT treatment of LCAT-KO mice was found to increase cholesterol efflux to HDL isolated from mice when added to cells transfected with either ATP-binding cassette (ABC) transporter A1 or ABCG1. In summary, rLCAT treatment rapidly restored the normal lipoprotein phenotype in LCAT-KO mice and increased cholesterol efflux, suggesting the possibility of using rLCAT as an enzyme replacement therapy agent for LCAT deficiency.


Asunto(s)
Lipoproteínas/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferasa/farmacología , Animales , Apolipoproteínas E/metabolismo , Colesterol/metabolismo , Ésteres del Colesterol/metabolismo , Cricetinae , Humanos , Infusiones Intravenosas , Metabolismo de los Lípidos/efectos de los fármacos , Lipoproteínas VLDL/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfatidilcolina-Esterol O-Aciltransferasa/administración & dosificación , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Proteínas Recombinantes
11.
Circ Res ; 107(2): 217-27, 2010 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-20508181

RESUMEN

RATIONALE: Apolipoprotein (apoA)-I mimetic peptides are a promising type of anti-atherosclerosis therapy, but how the structural features of these peptides relate to the multiple antiatherogenic functions of HDL is poorly understood. OBJECTIVE: To establish structure/function relationships of apoA-I mimetic peptides with their antiatherogenic functions. METHODS AND RESULTS: Twenty-two bihelical apoA-I mimetic peptides were investigated in vitro for the capacity and specificity of cholesterol efflux, inhibition of inflammatory response of monocytes and endothelial cells, and inhibition of low-density lipoprotein (LDL) oxidation. It was found that mean hydrophobicity, charge, size of hydrophobic face, and angle of the link between the helices are the major factors determining the efficiency and specificity of cholesterol efflux. The peptide with optimal parameters was more effective and specific toward cholesterol efflux than human apoA-I. Charge and size of hydrophobic face were also the major factors affecting antiinflammatory properties, and the presence of cysteine and histidine residues was the main factor determining antioxidant properties. There was no significant correlation between capacities of the peptides to support individual functions; each function had its own optimal set of features. CONCLUSIONS: None of the peptides was equally effective in all the antiatherogenic functions tested, suggesting that different functions of HDL may have different mechanisms and different structural requirements. The results do suggest, however, that rationalizing the design of apoA-I mimetic peptides may improve their therapeutic value and may lead to a better understanding of mechanisms of various antiatherogenic functions of HDL.


Asunto(s)
Antiinflamatorios/farmacología , Antioxidantes/farmacología , Apolipoproteína A-I/metabolismo , Aterosclerosis/prevención & control , Fármacos Cardiovasculares/farmacología , Células Endoteliales/efectos de los fármacos , Monocitos/efectos de los fármacos , Péptidos/farmacología , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Antiinflamatorios/química , Antioxidantes/química , Aterosclerosis/inmunología , Aterosclerosis/metabolismo , Transporte Biológico , Fármacos Cardiovasculares/química , Línea Celular , Colesterol/metabolismo , Diseño de Fármacos , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Mediadores de Inflamación/metabolismo , Lipoproteínas LDL/metabolismo , Ratones , Imitación Molecular , Monocitos/inmunología , Monocitos/metabolismo , Péptidos/química , Conformación Proteica , Relación Estructura-Actividad , Propiedades de Superficie
12.
J Pharmacol Exp Ther ; 334(2): 634-41, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20484557

RESUMEN

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.


Asunto(s)
Apolipoproteína A-I/fisiología , Aterosclerosis/tratamiento farmacológico , Colesterol/metabolismo , Péptidos , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1 , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Aorta/efectos de los fármacos , Aorta/patología , Apolipoproteína A-I/química , Apolipoproteínas E/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Transporte Biológico , Femenino , Humanos , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intercelular , Lipoproteínas/metabolismo , Lipoproteínas HDL/sangre , Lipoproteínas LDL/sangre , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Imitación Molecular , Péptidos/química , Fosfatidilcolinas/química , Ratas , Ratas Sprague-Dawley
13.
J Biol Chem ; 283(47): 32273-82, 2008 Nov 21.
Artículo en Inglés | MEDLINE | ID: mdl-18805791

RESUMEN

ApoA-I contains a tandem array of amphipathic helices with varying lipid affinity, which are critical in its ability to bind and remove lipids from cells by the ABCA1 transporter. In this study, the effect of asymmetry in the lipid affinity of amphipathic helices in a bihelical apoA-I mimetic peptide, 37pA, on lipid efflux by the ABCA1 transporter was examined. Seven peptide variants of 37pA were produced by substituting a varying number of hydrophobic amino acids for alanine on either one or both helices. The 5A peptide with five alanine substitutions in the second helix had decreased helical content compared with 37pA (5A, 12+/-1% helicity; 37pA, 28+/-2% helicity) and showed less self-association but, similar to the parent peptide, was able to readily solubilize phospholipid vesicles. Furthermore, 5A, unlike the parent peptide 37pA, was not hemolytic (37pA, 27+/-2% RBC lysis, 2 h, 18 microm). Finally, the 5A peptide stimulated cholesterol and phospholipid efflux by the ABCA1 transporter with higher specificity (ABCA1-transfected versus untransfected cells) than 37pA (5A, 9.7+/-0.77%, 18 h, 18 microm versus 1.5+/-0.27%, 18 h, 18 microm (p<0.0001); 37pA, 7.4+/-0.85%, 18 h, 18 microm versus 5.8+/-0.20%, 18 h, 18 microm (p=0.03)). In summary, we describe a novel bihelical peptide with asymmetry in the lipid affinity of its helices and properties similar to apoA-I in terms of specificity for cholesterol efflux by the ABCA1 transporter and low cytotoxicity.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Colesterol/metabolismo , Lípidos/química , Transportador 1 de Casete de Unión a ATP , Alanina/química , Secuencia de Aminoácidos , Transporte Biológico , Dicroismo Circular , Eritrocitos/citología , Guanidina/química , Humanos , Datos de Secuencia Molecular , Péptidos/química , Fosfolípidos/química , Conformación Proteica , Factores de Tiempo
14.
J Pharmacol Exp Ther ; 324(2): 776-83, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-18042829

RESUMEN

Apolipoprotein A-I (apoA-I) mimetic peptides may represent an alternative to apoA-I for large-scale production of synthetic high-density lipoproteins (sHDL) as a therapeutic agent. In this study, the cardioprotective activity of sHDL made with either L37pA peptide or its d-stereoisomer, D37pA, was compared to sHDL made with apoA-I. The peptides were reconstituted with palmitoyl-oleoyl-phosphatidylcholine, which yielded sHDL particles comparable to apoA-I sHDL in diameter, molecular weight, and alpha-helical content. Pretreatment of endothelial cells with either peptide sHDL reduced tumor necrosis factor alpha-stimulated vascular cell adhesion molecule-1 expression to the same extent as apoA-I sHDL. In an isolated rat heart model of ischemia/reperfusion (I/R) injury, L37pA and D37pA sHDL significantly reduced postischemic cardiac contractile dysfunction compared to the saline control, as indicated by a 49.7 +/- 6.4% (L37pA; P < 0.001) and 53.0 +/- 9.1% (D37pA; P < 0.001) increase of left ventricular-developed pressure (LVDP) after reperfusion and by a 45.4 +/- 3.4% (L37pA; P < 0.001) and 49.6 +/- 2.6% (D37pA; P < 0.001) decrease of creatine kinase (CK) release. These effects were similar to the 51.3 +/- 3.0% (P < 0.001) increase of LVDP and 51.3 +/- 3.0 (P < 0.001) reduction of CK release induced by apoA-I sHDL. Consistent with their cardioprotective effects, all three types of sHDL particles mediated an approximate 20% (P < 0.001) reduction of cardiac tumor necrosis factor alpha (TNFalpha) content and stimulated an approximate 35% (P < 0.05) increase in postischemic release of prostacyclin. In summary, L37pA and D37pA peptides can form sHDL particles that retain a similar level of protective activity as apoA-I sHDL on the endothelium and the heart; thus, apoA-I mimetic peptides may be useful therapeutic agents for the prevention of cardiac I/R injury.


Asunto(s)
Antiinflamatorios/uso terapéutico , Apolipoproteína A-I/uso terapéutico , Materiales Biomiméticos/uso terapéutico , Cardiotónicos/uso terapéutico , Péptidos/uso terapéutico , Secuencia de Aminoácidos/fisiología , Animales , Antiinflamatorios/síntesis química , Antiinflamatorios/farmacología , Apolipoproteína A-I/síntesis química , Apolipoproteína A-I/farmacología , Materiales Biomiméticos/síntesis química , Materiales Biomiméticos/farmacología , Cardiotónicos/síntesis química , Cardiotónicos/farmacología , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/prevención & control , Humanos , Lipoproteínas HDL/síntesis química , Lipoproteínas HDL/farmacología , Lipoproteínas HDL/uso terapéutico , Masculino , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/farmacología , Ratas , Ratas Sprague-Dawley , Molécula 1 de Adhesión Celular Vascular/metabolismo
15.
Biochem Biophys Res Commun ; 321(4): 936-41, 2004 Sep 03.
Artículo en Inglés | MEDLINE | ID: mdl-15358117

RESUMEN

Serum amyloid A (SAA) is an acute phase protein that associates with HDL. In order to examine the role of SAA in reverse-cholesterol transport, lipid efflux was tested to SAA from HeLa cells before and after transfection with the ABCA1 transporter. ABCA1 expression increased efflux of cholesterol and phospholipid to SAA by 3-fold and 2-fold, respectively. In contrast to apoA-I, SAA also removed lipid without ABCA1; cholesterol efflux from control cells to SAA was 10-fold higher than for apoA-I. Furthermore, SAA effluxed cholesterol from Tangier disease fibroblasts and from cells after inhibition of ABCA1 by fixation with paraformaldehyde. In summary, SAA can act as a lipid acceptor for ABCA1, but unlike apoA-I, it can also efflux lipid without ABCA1, by most likely a detergent-like extraction process. These results suggest that SAA may play a unique role as an auxiliary lipid acceptor in the removal of lipid from sites of inflammation.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Apolipoproteínas/metabolismo , Metabolismo de los Lípidos , Proteína Amiloide A Sérica/metabolismo , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/genética , Apolipoproteínas/genética , Transporte Biológico Activo , Línea Celular , Colesterol/metabolismo , Células HeLa , Humanos , Fosfolípidos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína Amiloide A Sérica/genética , Enfermedad de Tangier/genética , Enfermedad de Tangier/metabolismo , Transfección
16.
J Biol Chem ; 279(15): 15571-8, 2004 Apr 09.
Artículo en Inglés | MEDLINE | ID: mdl-14747463

RESUMEN

We have previously established that the ABCA1 transporter, which plays a critical role in the lipidation of extracellular apolipoprotein acceptors, traffics between late endocytic vesicles and the cell surface (Neufeld, E. B., Remaley, A. T., Demosky, S. J., Jr., Stonik, J. A., Cooney, A. M., Comly, M., Dwyer, N. K., Zhang, M., Blanchette-Mackie, J., Santamarina-Fojo, S., and Brewer, H. B., Jr. (2001) J. Biol. Chem. 276, 27584-27590). The present study provides evidence that ABCA1 in late endocytic vesicles plays a role in cellular lipid efflux. Late endocytic trafficking was defective in Tangier disease fibroblasts that lack functional ABCA1. Consistent with a late endocytic protein trafficking defect, the hydrophobic amine U18666A retained NPC1 in abnormally tubulated, cholesterol-poor, Tangier disease late endosomes, rather than cholesterol-laden lysosomes, as in wild type fibroblasts. Consistent with a lipid trafficking defect, Tangier disease late endocytic vesicles accumulated both cholesterol and sphingomyelin and were immobilized in a perinuclear localization. The excess cholesterol in Tangier disease late endocytic vesicles retained massive amounts of NPC1, which traffics lysosomal cholesterol to other cellular sites. Exogenous apoA-I abrogated the cholesterol-induced retention of NPC1 in wild type but not in Tangier disease late endosomes. Adenovirally mediated ABCA1-GFP expression in Tangier disease fibroblasts corrected the late endocytic trafficking defects and restored apoA-I-mediated cholesterol efflux. ABCA1-GFP expression in wild type fibroblasts also reduced late endosome-associated NPC1, induced a marked uptake of fluorescent apoA-I into ABCA1-GFP-containing endosomes (that shuttled between late endosomes and the cell surface), and enhanced apoA-I-mediated cholesterol efflux. The combined results of this study suggest that ABCA1 converts pools of late endocytic lipids that retain NPC1 to pools that can associate with endocytosed apoA-I, and be released from the cell as nascent high density lipoprotein.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Enfermedad de Tangier/genética , Enfermedad de Tangier/terapia , Transportador 1 de Casete de Unión a ATP , Androstenos/farmacología , Anticolesterolemiantes/farmacología , Apolipoproteína A-I/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Colesterol/metabolismo , Detergentes/farmacología , Endocitosis , Endosomas/metabolismo , Fibroblastos/metabolismo , Proteínas Fluorescentes Verdes , Humanos , Inmunohistoquímica , Metabolismo de los Lípidos , Lipoproteínas HDL/metabolismo , Proteínas Luminiscentes/metabolismo , Lisosomas/metabolismo , Microscopía Confocal , Modelos Biológicos , Esfingomielinas/metabolismo
17.
J Lipid Res ; 44(4): 828-36, 2003 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-12562845

RESUMEN

In order to examine the necessary structural features for a protein to promote lipid efflux by the ABCA1 transporter, synthetic peptides were tested on ABCA1-transfected cells (ABCA1 cells) and on control cells. L-37pA, an l amino acid peptide that contains two class-A amphipathic helices linked by proline, showed a 4-fold increase in cholesterol and phospholipid efflux from ABCA1 cells compared to control cells. The same peptide synthesized with a mixture of l and d amino acids was less effective than L-37pA in solubilizing dimyristoyl phosphatidyl choline vesicles and in effluxing lipids. In contrast, the 37pA peptide synthesized with all d amino acids (D-37pA) was as effective as L-37pA. Unlike apoA-I, L-37pA and D-37pA were also capable, although at a reduced rate, of causing lipid efflux independent of ABCA1 from control cells, Tangier disease cells, and paraformaldehyde fixed ABCA1 cells. The ability of peptides to bind to cells correlated with their lipid affinity. In summary, the amphipathic helix was found to be a key structural motif for peptide-mediated lipid efflux from ABCA1, but there was no stereoselective requirement. In addition, unlike apoA-I, synthetic peptides can also efflux lipid by a passive, energy-independent pathway that does not involve ABCA1 but does depend upon their lipid affinity.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Metabolismo de los Lípidos , Péptidos/farmacología , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/genética , Colesterol/metabolismo , Células HeLa , Humanos , Cinética , Modelos Biológicos , Péptidos/síntesis química , Fosfolípidos/metabolismo , Estructura Secundaria de Proteína , Solubilidad/efectos de los fármacos , Estereoisomerismo , Tensoactivos/síntesis química , Tensoactivos/farmacología , Transfección
18.
J Lipid Res ; 44(2): 296-302, 2003 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-12576511

RESUMEN

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.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , HDL-Colesterol/sangre , Colesterol/metabolismo , Hígado/metabolismo , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/genética , Adenoviridae/genética , Adenoviridae/metabolismo , Animales , Células Cultivadas , Hepatocitos/citología , Hepatocitos/metabolismo , Lípidos/sangre , Lipoproteínas/sangre , Hígado/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo
19.
Biochem Biophys Res Commun ; 297(4): 974-9, 2002 Oct 04.
Artículo en Inglés | MEDLINE | ID: mdl-12359250

RESUMEN

ABCA1 on the cell surface and in endosomes plays an essential role in the cell-mediated lipidation of apoA-I to form nascent HDL. Our previous studies of transgenic mice overexpressing ABCA1 suggested that ABCA1 in the liver plays a major role in regulating plasma HDL levels. The site of function of ABCA1 in the polarized hepatocyte was currently assessed by expression of an adenoviral construct encoding a human ABCA1-GFP fusion protein in the polarized hepatocyte-like WIF-B cell line. Consistent with localization of ABCA1 at the basolateral (vascular) cell surface, expression of ABCA1-GFP stimulated apoA-I mediated efflux of WIF-B cell cholesterol into the culture medium. Confocal fluorescence microscopy revealed that ABCA1-GFP was expressed solely on the basolateral surface and associated endocytic vesicles. These findings suggest an important role for hepatocyte basolateral membrane ABCA1 in the regulation of the levels of intracellular hepatic cholesterol, as well as plasma HDL.


Asunto(s)
Apolipoproteína A-I/metabolismo , Hepatocitos/metabolismo , Apolipoproteína A-I/sangre , Membrana Celular/metabolismo , Colesterol/metabolismo , Endocitosis , Genes Reporteros , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transfección
20.
Infect Immun ; 70(6): 2995-3003, 2002 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-12010990

RESUMEN

Lipopolysaccharide (LPS) has recently been shown to facilitate macrophage foam cell formation and has been suggested to be a proatherogenic factor. The mechanism of LPS induced cholesterol accumulation, however, is unclear. In this report, using the macrophage-like RAW 264.7 cell line, we provide experimental evidence that LPS's proatherogenic effects may at least in part reflect altered cholesterol metabolism. Data presented demonstrate that in a dose-dependent manner, LPS is able to down regulate the mRNA expression of the two primary high-density lipoprotein (HDL) receptors, scavenger receptor B1 (SR-B1) and ATP binding cassette A1 (ABCA1), with a 50% inhibitory concentration of less than 0.2 ng/ml, as well as to decrease SR-B1 protein expression by 80%. We also found that LPS treatment resulted in a significant decrease (to 20% of the control level) of the specific (125)I-HDL binding as well as in 50% inhibition of the HDL-mediated cholesterol efflux compared to untreated cells. In addition, we compared the potencies of various modified LPS preparations and demonstrated that the phosphorylated lipid A portion of LPS, which is highly conserved among gram-negative microorganisms, including Chlamydia, is primarily responsible for the effects of LPS on SR-B1 and ABCA1 expression. Inhibitors of NF-kappaB activation were observed to efficiently block the suppressive effect of LPS on SR-B1 and ABCA1, suggesting a mechanism involving NF-kappaB. These data indicate that the LPS effects on cholesterol metabolism may contribute to the proatherogenic properties of LPS.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Antígenos CD36/genética , Regulación hacia Abajo , Lipopolisacáridos/inmunología , Proteínas de la Membrana , Receptores Inmunológicos , Receptores de Lipoproteína , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/biosíntesis , Animales , Antígenos CD36/biosíntesis , Línea Celular , Colesterol/metabolismo , Relación Dosis-Respuesta a Droga , Interleucina-1/biosíntesis , Interleucina-1/genética , Radioisótopos de Yodo , Marcaje Isotópico , Lipopolisacáridos/farmacología , Lipoproteínas HDL/metabolismo , Macrófagos/citología , Ratones , FN-kappa B/antagonistas & inhibidores , ARN Mensajero/biosíntesis , Receptores Depuradores , Receptores Depuradores de Clase B , Factores de Tiempo , Clorometilcetona de Tosilfenilalanila/farmacología
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