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1.
J Lipid Res ; 63(1): 100160, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-34902367

RESUMEN

A significant proportion of patients with elevated LDL and a clinical presentation of familial hypercholesterolemia do not carry known genetic mutations associated with hypercholesterolemia, such as defects in the LDL receptor. To identify new genes involved in the cellular uptake of LDL, we developed a novel whole-genome clustered regularly interspaced short palindromic repeat-Cas9 KO screen in HepG2 cells. We identified transgelin (TAGLN), an actin-binding protein, as a potentially new gene involved in LDL endocytosis. In silico validation demonstrated that genetically predicted differences in expression of TAGLN in human populations were significantly associated with elevated plasma lipids (triglycerides, total cholesterol, and LDL-C) in the Global Lipids Genetics Consortium and lipid-related phenotypes in the UK Biobank. In biochemical studies, TAGLN-KO HepG2 cells showed a reduction in cellular LDL uptake, as measured by flow cytometry. In confocal microscopy imaging, TAGLN-KO cells had disrupted actin filaments as well as an accumulation of LDL receptor on their surface because of decreased receptor internalization. Furthermore, TAGLN-KO cells exhibited a reduction in total and free cholesterol content, activation of SREBP2, and a compensatory increase in cholesterol biosynthesis. TAGLN deficiency also disrupted the uptake of VLDL and transferrin, other known cargoes for receptors that depend upon clathrin-mediated endocytosis. Our data suggest that TAGLN is a novel factor involved in the actin-dependent phase of clathrin-mediated endocytosis of LDL. The identification of novel genes involved in the endocytic uptake of LDL may improve the diagnosis of hypercholesterolemia and provide future therapeutic targets for the prevention of cardiovascular disease.


Asunto(s)
Proteínas de Microfilamentos , Proteínas Musculares
2.
J Lipid Res ; 61(12): 1577-1588, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-32907987

RESUMEN

Lipoproteins play a key role in transport of cholesterol to and from tissues. Recent studies have also demonstrated that red blood cells (RBCs), which carry large quantities of free cholesterol in their membrane, play an important role in reverse cholesterol transport. However, the exact role of RBCs in systemic cholesterol metabolism is poorly understood. RBCs were incubated with autologous plasma or isolated lipoproteins resulting in a significant net amount of cholesterol moved from RBCs to HDL, while cholesterol from LDL moved in the opposite direction. Furthermore, the bi-directional cholesterol transport between RBCs and plasma lipoproteins was saturable and temperature-, energy-, and time-dependent, consistent with an active process. We did not find LDLR, ABCG1, or scavenger receptor class B type 1 in RBCs but found a substantial amount of ABCA1 mRNA and protein. However, specific cholesterol efflux from RBCs to isolated apoA-I was negligible, and ABCA1 silencing with siRNA or inhibition with vanadate and Probucol did not inhibit the efflux to apoA-I, HDL, or plasma. Cholesterol efflux from and cholesterol uptake by RBCs from Abca1+/+ and Abca1-/- mice were similar, arguing against the role of ABCA1 in cholesterol flux between RBCs and lipoproteins. Bioinformatics analysis identified ABCA7, ABCG5, lipoprotein lipase, and mitochondrial translocator protein as possible candidates that may mediate the cholesterol flux. Together, these results suggest that RBCs actively participate in cholesterol transport in the blood, but the role of cholesterol transporters in RBCs remains uncertain.


Asunto(s)
Colesterol/metabolismo , Eritrocitos/metabolismo , Lipoproteínas/metabolismo , Transporte Biológico , Biología Computacional , Humanos
3.
J Pharmacol Exp Ther ; 368(3): 423-434, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30563940

RESUMEN

Familial LCAT deficiency (FLD) is due to mutations in lecithin:cholesterol acyltransferase (LCAT), a plasma enzyme that esterifies cholesterol on lipoproteins. FLD is associated with markedly reduced levels of plasma high-density lipoprotein and cholesteryl ester and the formation of a nephrotoxic lipoprotein called LpX. We used a mouse model in which the LCAT gene is deleted and a truncated version of the SREBP1a gene is expressed in the liver under the control of a protein-rich/carbohydrate-low (PRCL) diet-regulated PEPCK promoter. This mouse was found to form abundant amounts of LpX in the plasma and was used to determine whether treatment with recombinant human LCAT (rhLCAT) could prevent LpX formation and renal injury. After 9 days on the PRCL diet, plasma total and free cholesterol, as well as phospholipids, increased 6.1 ± 0.6-, 9.6 ± 0.9-, and 6.7 ± 0.7-fold, respectively, and liver cholesterol and triglyceride concentrations increased 1.7 ± 0.4- and 2.8 ±0.9-fold, respectively, compared with chow-fed animals. Transmission electron microscopy revealed robust accumulation of lipid droplets in hepatocytes and the appearance of multilamellar LpX particles in liver sinusoids and bile canaliculi. In the kidney, LpX was found in glomerular endothelial cells, podocytes, the glomerular basement membrane, and the mesangium. The urine albumin/creatinine ratio increased 30-fold on the PRCL diet compared with chow-fed controls. Treatment of these mice with intravenous rhLCAT restored the normal lipoprotein profile, eliminated LpX in plasma and kidneys, and markedly decreased proteinuria. The combined results suggest that rhLCAT infusion could be an effective therapy for the prevention of renal disease in patients with FLD.


Asunto(s)
Modelos Animales de Enfermedad , Riñón/metabolismo , Deficiencia de la Lecitina Colesterol Aciltransferasa/tratamiento farmacológico , Deficiencia de la Lecitina Colesterol Aciltransferasa/metabolismo , Lipoproteína X/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferasa/administración & dosificación , Animales , Dieta Baja en Carbohidratos/efectos adversos , Proteínas en la Dieta/efectos adversos , Femenino , Riñón/efectos de los fármacos , Riñón/patología , Deficiencia de la Lecitina Colesterol Aciltransferasa/patología , Lipoproteína X/antagonistas & inhibidores , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos
4.
Circulation ; 136(5): 464-475, 2017 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-28473446

RESUMEN

BACKGROUND: Thioredoxin (TRX)-1, a ubiquitous 12-kDa protein, exerts antioxidant and anti-inflammatory effects. In contrast, the truncated form, called TRX80, produced by macrophages induces upregulation of proinflammatory cytokines. TRX80 also promotes the differentiation of mouse peritoneal and human macrophages toward a proinflammatory M1 phenotype. METHODS: TRX1 and TRX80 plasma levels were determined with a specific ELISA. A disintegrin and metalloproteinase domain-containing protein (ADAM)-10, ADAM-17, and ADAM-10 activities were measured with SensoLyte 520 ADAM10 Activity Assay Kit, Fluorimetric, and InnoZyme TACE Activity Kit, respectively. Western immunoblots were performed with specific antibodies to ADAM-10 or ADAM-17. Angiogenesis study was evaluated in vitro with human microvascular endothelial cells-1 and in vivo with the Matrigel plug angiogenesis assay in mice. The expression of macrophage phenotype markers was investigated with real-time polymerase chain reaction. Phosphorylation of Akt, mechanistic target of rapamycin, and 70S6K was determined with specific antibodies. The effect of TRX80 on NLRP3 inflammasome activity was evaluated by measuring the level of interleukin-1ß and -18 in the supernatants of activated macrophages with ELISA. Hearts were used for lesion surface evaluation and immunohistochemical studies, and whole descending aorta were stained with Oil Red O. For transgenic mice generation, the human scavenger receptor (SR-A) promoter/enhancer was used to drive macrophage-specific expression of human TRX80 in mice. RESULTS: In this study, we observed a significant increase of plasma levels of TRX80 in old subjects compared with healthy young subjects. In parallel, an increase in expression and activity of ADAM-10 and ADAM-17 in old peripheral blood mononuclear cells compared with those of young subjects was observed. Furthermore, TRX80 was found to colocalize with tumor necrosis factor-α, a macrophage M1 marker, in human atherosclerotic plaque. In addition, TRX80 induced the expression of murine M1 macrophage markers through Akt2/mechanistic target of rapamycin-C1/70S6K pathway and activated the inflammasome NLRP3, leading to the release of interleukin-1ß and -18, potent atherogenic cytokines. Moreover, TRX80 exerts a powerful angiogenic effect in both in vitro and in vivo mouse studies. Finally, transgenic mice that overexpress human TRX80 specifically in macrophages of apoE-/- mice have a significant increase of aortic atherosclerotic lesions. CONCLUSIONS: TRX80 showed an age-dependent increase in human plasma. In mouse models, TRX80 was associated with a proinflammatory status and increased atherosclerosis.


Asunto(s)
Envejecimiento , Aterosclerosis/patología , Fragmentos de Péptidos/sangre , Tiorredoxinas/sangre , Proteína ADAM10/metabolismo , Proteína ADAM17/metabolismo , Adulto , Anciano , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/metabolismo , Biomarcadores/sangre , Biomarcadores/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunohistoquímica , Inflamación , Interleucina-18/sangre , Interleucina-1beta/sangre , Leucocitos Mononucleares/metabolismo , Lipopolisacáridos/toxicidad , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microscopía Confocal , Complejos Multiproteicos/antagonistas & inhibidores , Complejos Multiproteicos/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Tiorredoxinas/farmacología
5.
J Immunol ; 196(7): 3135-47, 2016 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-26936883

RESUMEN

The class B scavenger receptors BI (SR-BI) and BII (SR-BII) are high-density lipoprotein receptors that recognize various pathogens, including bacteria and their products. It has been reported that SR-BI/II null mice are more sensitive than normal mice to endotoxin-induced inflammation and sepsis. Because the SR-BI/II knockout model demonstrates multiple immune and metabolic disorders, we investigated the role of each receptor in the LPS-induced inflammatory response and tissue damage using transgenic mice with pLiv-11-directed expression of human SR-BI (hSR-BI) or human SR-BII (hSR-BII). At 6 h after i.p. LPS injection, transgenic hSR-BI and hSR-BII mice demonstrated markedly higher serum levels of proinflammatory cytokines and 2- to 3-fold increased expression levels of inflammatory mediators in the liver and kidney, compared with wild-type (WT) mice. LPS-stimulated inducible NO synthase expression was 3- to 6-fold higher in the liver and kidney of both transgenic strains, although serum NO levels were similar in all mice. Despite the lower high-density lipoprotein plasma levels, both transgenic strains responded to LPS by a 5-fold increase of plasma corticosterone levels, which were only moderately lower than in WT animals. LPS treatment resulted in MAPK activation in tissues of all mice; however, the strongest response was detected for hepatic extracellular signal-regulated protein kinase 1 and 2 and kidney JNK of both transgenic mice. Histological examination of hepatic and renal tissue from LPS-challenged mice revealed more injury in hSR-BII, but not hSR-BI, transgenic mice versus WT controls. Our findings demonstrate that hSR-BII, and to a lesser extent hSR-BI, significantly increase LPS-induced inflammation and contribute to LPS-induced tissue injury in the liver and kidney, two major organs susceptible to LPS toxicity.


Asunto(s)
Lesión Renal Aguda/genética , Lesión Renal Aguda/inmunología , Antígenos CD36/genética , Lipopolisacáridos/inmunología , Hepatopatías/genética , Hepatopatías/inmunología , Proteínas de Membrana de los Lisosomas/genética , Receptores Depuradores/genética , Lesión Renal Aguda/patología , Animales , Antígenos CD36/metabolismo , Línea Celular , Citocinas/sangre , Citocinas/metabolismo , Modelos Animales de Enfermedad , Expresión Génica , Humanos , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Mediadores de Inflamación/sangre , Mediadores de Inflamación/metabolismo , Hepatopatías/patología , Proteínas de Membrana de los Lisosomas/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Transgénicos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Especificidad de Órganos/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Depuradores/metabolismo
6.
J Pharmacol Exp Ther ; 362(2): 306-318, 2017 08.
Artículo en Inglés | MEDLINE | ID: mdl-28576974

RESUMEN

Lecithin:cholesterol acyltransferase (LCAT) catalyzes plasma cholesteryl ester formation and is defective in familial lecithin:cholesterol acyltransferase deficiency (FLD), an autosomal recessive disorder characterized by low high-density lipoprotein, anemia, and renal disease. This study aimed to investigate the mechanism by which compound A [3-(5-(ethylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile], a small heterocyclic amine, activates LCAT. The effect of compound A on LCAT was tested in human plasma and with recombinant LCAT. Mass spectrometry and nuclear magnetic resonance were used to determine compound A adduct formation with LCAT. Molecular modeling was performed to gain insight into the effects of compound A on LCAT structure and activity. Compound A increased LCAT activity in a subset (three of nine) of LCAT mutations to levels comparable to FLD heterozygotes. The site-directed mutation LCAT-Cys31Gly prevented activation by compound A. Substitution of Cys31 with charged residues (Glu, Arg, and Lys) decreased LCAT activity, whereas bulky hydrophobic groups (Trp, Leu, Phe, and Met) increased activity up to 3-fold (P < 0.005). Mass spectrometry of a tryptic digestion of LCAT incubated with compound A revealed a +103.017 m/z adduct on Cys31, consistent with the addition of a single hydrophobic cyanopyrazine ring. Molecular modeling identified potential interactions of compound A near Cys31 and structural changes correlating with enhanced activity. Functional groups important for LCAT activation by compound A were identified by testing compound A derivatives. Finally, sulfhydryl-reactive ß-lactams were developed as a new class of LCAT activators. In conclusion, compound A activates LCAT, including some FLD mutations, by forming a hydrophobic adduct with Cys31, thus providing a mechanistic rationale for the design of future LCAT activators.


Asunto(s)
Cisteína/fisiología , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Compuestos de Sulfhidrilo/farmacología , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Activadores de Enzimas/química , Activadores de Enzimas/metabolismo , Activadores de Enzimas/farmacología , Células HEK293 , Humanos , Deficiencia de la Lecitina Colesterol Aciltransferasa/metabolismo , Modelos Moleculares , Fosfatidilcolina-Esterol O-Aciltransferasa/química , Compuestos de Sulfhidrilo/química
7.
Arterioscler Thromb Vasc Biol ; 36(12): 2283-2291, 2016 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-27758769

RESUMEN

OBJECTIVE: We examined the function of ABCA1 (ATP-binding cassette transporter A1) in ApoA-I (apolipoprotein A-I) mobilization of cholesterol microdomains deposited into the extracellular matrix by cholesterol-enriched macrophages. We have also determined whether an ApoA-I mimetic peptide without and with complexing to sphingomyelin can mobilize macrophage-deposited cholesterol microdomains. APPROACH AND RESULTS: Extracellular cholesterol microdomains deposited by cholesterol-enriched macrophages were detected with a monoclonal antibody, 58B1. ApoA-I and an ApoA-I mimetic peptide 5A mobilized cholesterol microdomains deposited by ABCA1+/+ macrophages but not by ABCA1-/- macrophages. In contrast, ApoA-I mimetic peptide 5A complexed with sphingomyelin could mobilize cholesterol microdomains deposited by ABCA1-/- macrophages. CONCLUSIONS: Our findings show that a unique pool of extracellular cholesterol microdomains deposited by macrophages can be mobilized by both ApoA-I and an ApoA-I mimetic peptide but that mobilization depends on macrophage ABCA1. It is known that ABCA1 complexes ApoA-I and ApoA-I mimetic peptide with phospholipid, a cholesterol-solubilizing agent, explaining the requirement for ABCA1 in extracellular cholesterol microdomain mobilization. Importantly, ApoA-I mimetic peptide already complexed with phospholipid can mobilize macrophage-deposited extracellular cholesterol microdomains even in the absence of ABCA1.


Asunto(s)
Transportador 1 de Casete de Unión a ATP/metabolismo , Apolipoproteína A-I/metabolismo , Macrófagos/efectos de los fármacos , Microdominios de Membrana/efectos de los fármacos , Imitación Molecular , Péptidos/farmacología , Transportador 1 de Casete de Unión a ATP/deficiencia , Transportador 1 de Casete de Unión a ATP/genética , Animales , Células Cultivadas , Colesterol/metabolismo , Femenino , Genotipo , Humanos , Péptidos y Proteínas de Señalización Intercelular , Macrófagos/metabolismo , Microdominios de Membrana/metabolismo , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Péptidos/metabolismo , Fenotipo , Esfingomielinas/metabolismo
8.
Biochem J ; 473(2): 211-9, 2016 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-26556891

RESUMEN

HDL (high-density lipoproteins) exert anti-thrombotic activities by preventing platelet adhesion and activation and by stimulating the protein C pathway and fibrinolysis. The aim of the present study was to assess the effect of plasma-derived and synthetic HDL on endothelial and monocyte expression of TF (tissue factor), the primary initiator of coagulation. HDL inhibited TF expression and activity in stimulated endothelial cells and monocytes in a dose-dependent way. Synthetic HDL fully retain the ability to inhibit TF expression in a dose-dependent manner; lipid-free apoA-I (apolipoprotein A-I) was not effective and neither was sphingosine 1-phosphate involved. HDL-mediated TF inhibition was due to a modulation of cellular cholesterol content through the interaction with SR-BI (scavenger receptor BI); downstream, HDL inhibited the activation of p38 MAPK (mitogen-activated protein kinase) and the repression of the PI3K (phosphoinositide 3-kinase) pathway responsible for TF expression. In vivo, human apoA-I-transgenic mice displayed a reduced aortic TF expression compared with wild-type animals and TF plasma levels were increased in subjects with low HDL-C (HDL-cholesterol) levels compared with high HDL-C subjects. Thus the anti-thrombotic activity of HDL could also be mediated by the inhibition of TF expression and activity in endothelial cells and monocytes; synthetic HDL retain the inhibitory activity of plasma-derived HDL, supporting the hypothesis that synthetic HDL infusion may be beneficial in the setting of acute coronary syndrome.


Asunto(s)
Células Endoteliales/metabolismo , Lipoproteínas HDL/metabolismo , Monocitos/metabolismo , Tromboplastina/antagonistas & inhibidores , Tromboplastina/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Células Endoteliales/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Lipoproteínas HDL/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Monocitos/efectos de los fármacos
9.
J Pharmacol Exp Ther ; 356(2): 341-53, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26574515

RESUMEN

Apolipoprotein C-II (apoC-II) is a cofactor for lipoprotein lipase, a plasma enzyme that hydrolyzes triglycerides (TGs). ApoC-II deficiency in humans results in hypertriglyceridemia. We used zinc finger nucleases to create Apoc2 mutant mice to investigate the use of C-II-a, a short apoC-II mimetic peptide, as a therapy for apoC-II deficiency. Mutant mice produced a form of apoC-II with an uncleaved signal peptide that preferentially binds high-density lipoproteins (HDLs) due to a 3-amino acid deletion at the signal peptide cleavage site. Homozygous Apoc2 mutant mice had increased plasma TG (757.5 ± 281.2 mg/dl) and low HDL cholesterol (31.4 ± 14.7 mg/dl) compared with wild-type mice (TG, 55.9 ± 13.3 mg/dl; HDL cholesterol, 55.9 ± 14.3 mg/dl). TGs were found in light (density < 1.063 g/ml) lipoproteins in the size range of very-low-density lipoprotein and chylomicron remnants (40-200 nm). Intravenous injection of C-II-a (0.2, 1, and 5 µmol/kg) reduced plasma TG in a dose-dependent manner, with a maximum decrease of 90% occurring 30 minutes after the high dose. Plasma TG did not return to baseline until 48 hours later. Similar results were found with subcutaneous or intramuscular injections. Plasma half-life of C-II-a is 1.33 ± 0.72 hours, indicating that C-II-a only acutely activates lipolysis, and the sustained TG reduction is due to the relatively slow rate of new TG-rich lipoprotein synthesis. In summary, we describe a novel mouse model of apoC-II deficiency and show that an apoC-II mimetic peptide can reverse the hypertriglyceridemia in these mice, and thus could be a potential new therapy for apoC-II deficiency.


Asunto(s)
Apolipoproteína C-II/genética , Materiales Biomiméticos/metabolismo , Hiperlipoproteinemia Tipo I/genética , Hipertrigliceridemia/genética , Mutación/genética , Fragmentos de Péptidos/genética , Secuencia de Aminoácidos , Animales , Femenino , Hiperlipoproteinemia Tipo I/sangre , Hipertrigliceridemia/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Embarazo , Triglicéridos/sangre
10.
J Lipid Res ; 56(9): 1720-6, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26203076

RESUMEN

We previously reported that cholesterol-enriched macrophages excrete cholesterol into the extracellular matrix. A monoclonal antibody that detects cholesterol microdomains labels the deposited extracellular particles. Macro-phage deposition of extracellular cholesterol depends, in part, on ABCG1, and this cholesterol can be mobilized by HDL components of the reverse cholesterol transport process. The objective of the current study was to determine whether ABCA1 also contributes to macrophage deposition of extracellular cholesterol. ABCA1 functioned in extracellular cholesterol deposition. The liver X receptor agonist, TO901317 (TO9), an ABCA1-inducing factor, restored cholesterol deposition that was absent in cholesterol-enriched ABCG1(-/-) mouse macrophages. In addition, the ABCA1 inhibitor, probucol, blocked the increment in cholesterol deposited by TO9-treated wild-type macrophages, and completely inhibited deposition from TO9-treated ABCG1(-/-) macrophages. Lastly, ABCA1(-/-) macrophages deposited much less extracellular cholesterol than wild-type macrophages. These findings demonstrate a novel function of ABCA1 in contributing to macrophage export of cholesterol into the extracellular matrix.


Asunto(s)
Transportador 1 de Casete de Unión a ATP/genética , Aterosclerosis/genética , Colesterol/metabolismo , Transportador 1 de Casete de Unión a ATP/antagonistas & inhibidores , Transportador 1 de Casete de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1 , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Aterosclerosis/metabolismo , Aterosclerosis/patología , Colesterol/genética , Matriz Extracelular/metabolismo , Humanos , Hidrocarburos Fluorados/administración & dosificación , Lipoproteínas/genética , Lipoproteínas/metabolismo , Lipoproteínas HDL/genética , Lipoproteínas HDL/metabolismo , Receptores X del Hígado , Macrófagos/metabolismo , Ratones , Receptores Nucleares Huérfanos/agonistas , Receptores Nucleares Huérfanos/genética , Probucol/administración & dosificación , Sulfonamidas/administración & dosificación
11.
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
12.
Am J Respir Cell Mol Biol ; 51(5): 626-36, 2014 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-24813055

RESUMEN

Apolipoprotein A-I (apoA-I) is an important component of high-density lipoprotein particles that mediates reverse cholesterol transport out of cells by interacting with the ATP-binding cassette transporter 1 (ABCA1). apoA-I has also been shown to attenuate neutrophilic airway inflammation in experimental ovalbumin (OVA)-induced asthma by reducing the expression of granulocyte colony-stimulating factor (G-CSF). Here, we hypothesized that overexpression of the ABCA1 transporter might similarly attenuate OVA-induced neutrophilic airway inflammation. Tie2-human ABCA1 (hABCA1) mice expressing human ABCA1 under the control of the Tie2 promoter, which is primarily expressed by vascular endothelial cells, but can also be expressed by macrophages, received daily intranasal OVA challenges, 5 d/wk for 5 weeks. OVA-challenged Tie2-hABCA1 mice had significant reductions in total bronchoalveolar lavage fluid (BALF) cells that reflected a decrease in neutrophils, as well as reductions in peribronchial inflammation, OVA-specific IgE levels, and airway epithelial thickness. The reduced airway neutrophilia in OVA-challenged Tie2-hABCA1 mice was associated with significant decreases in G-CSF protein levels in pulmonary vascular endothelial cells, alveolar macrophages, and BALF. Intranasal administration of recombinant murine G-CSF to OVA-challenged Tie2-hABCA1 mice for 5 days increased BALF neutrophils to a level comparable to that of OVA-challenged wild-type mice. We conclude that ABCA1 suppresses OVA-induced airway neutrophilia by reducing G-CSF production by vascular endothelial cells and alveolar macrophages. These findings suggest that ABCA1 expressed by vascular endothelial cells and alveolar macrophages may play important roles in attenuating the severity of neutrophilic airway inflammation in asthma.


Asunto(s)
Transportador 1 de Casete de Unión a ATP/genética , Transportador 1 de Casete de Unión a ATP/inmunología , Neutrófilos/inmunología , Neumonía/inmunología , Animales , Asma/inmunología , Líquido del Lavado Bronquioalveolar/inmunología , Colesterol/inmunología , Células Endoteliales/inmunología , Factor Estimulante de Colonias de Granulocitos/genética , Humanos , Macrófagos Alveolares/inmunología , Ratones Endogámicos C57BL , Ratones Transgénicos , Ovalbúmina/inmunología , Ovalbúmina/farmacología , Neumonía/inducido químicamente , Regiones Promotoras Genéticas/genética , Receptor TIE-2/genética
13.
J Lipid Res ; 55(8): 1721-9, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-24950691

RESUMEN

A key step in plasma HDL maturation from discoidal to spherical particles is the esterification of cholesterol to cholesteryl ester, which is catalyzed by LCAT. HDL-like lipoproteins in cerebrospinal fluid (CSF) are also spherical, whereas nascent lipoprotein particles secreted from astrocytes are discoidal, suggesting that LCAT may play a similar role in the CNS. In plasma, apoA-I is the main LCAT activator, while in the CNS, it is believed to be apoE. apoE is directly involved in the pathological progression of Alzheimer's disease, including facilitating ß-amyloid (Aß) clearance from the brain, a function that requires its lipidation by ABCA1. However, whether apoE particle maturation by LCAT is also required for Aß clearance is unknown. Here we characterized the impact of LCAT deficiency on CNS lipoprotein metabolism and amyloid pathology. Deletion of LCAT from APP/PS1 mice resulted in a pronounced decrease of apoA-I in plasma that was paralleled by decreased apoA-I levels in CSF and brain tissue, whereas apoE levels were unaffected. Furthermore, LCAT deficiency did not increase Aß or amyloid in APP/PS1 LCAT(-/-) mice. Finally, LCAT expression and plasma activity were unaffected by age or the onset of Alzheimer's-like pathology in APP/PS1 mice. Taken together, these results suggest that apoE-containing discoidal HDLs do not require LCAT-dependent maturation to mediate efficient Aß clearance.


Asunto(s)
Péptidos beta-Amiloides/metabolismo , Apolipoproteína A-I/metabolismo , Deficiencia de la Lecitina Colesterol Aciltransferasa/metabolismo , Animales , Apolipoproteína A-I/genética , Deficiencia de la Lecitina Colesterol Aciltransferasa/genética , Deficiencia de la Lecitina Colesterol Aciltransferasa/patología , Ratones , Ratones Noqueados , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo
14.
J Immunol ; 188(6): 2749-58, 2012 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-22327076

RESUMEN

Class B scavenger receptors (SR-Bs), such as SR-BI/II or CD36, bind lipoproteins but also mediate bacterial recognition and phagocytosis. In evaluating whether blocking receptors can prevent intracellular bacterial proliferation, phagocyte cytotoxicity, and proinflammatory signaling in bacterial infection/sepsis, we found that SR-BI/II- or CD36-deficient phagocytes are characterized by a reduced intracellular bacterial survival and a lower cytokine response and were protected from bacterial cytotoxicity in the presence of antibiotics. Mice deficient in either SR-BI/II or CD36 are protected from antibiotic-treated cecal ligation and puncture (CLP)-induced sepsis, with greatly increased peritoneal granulocytic phagocyte survival (8-fold), a drastic diminution in peritoneal bacteria counts, and a 50-70% reduction in systemic inflammation (serum levels of IL-6, TNF-α, and IL-10) and organ damage relative to CLP in wild-type mice. The survival rate of CD36-deficient mice after CLP was 58% compared with 17% in control mice. When compensated for mineralocorticoid and glucocorticoid deficiency, SR-BI/II-deficient mice had nearly a 50% survival rate versus 5% in mineralo-/glucocorticoid-treated controls. Targeting SR-B receptors with L-37pA, a peptide that functions as an antagonist of SR-BI/II and CD36 receptors, also increased peritoneal granulocyte counts, as well as reduced peritoneal bacteria and bacterium-induced cytokine secretion. In the CLP mouse sepsis model, L-37pA improved survival from 6 to 27%, reduced multiple organ damage, and improved kidney function. These results demonstrate that the reduction of both SR-BI/II- and CD36-dependent bacterial invasion and inflammatory response in the presence of antibiotic treatment results in granulocyte survival and local bacterial containment, as well as reduces systemic inflammation and organ damage and improves animal survival during severe infections.


Asunto(s)
Antígenos CD36/inmunología , Receptores Depuradores de Clase B/inmunología , Sepsis/inmunología , Animales , Antígenos CD36/metabolismo , Modelos Animales de Enfermedad , Granulocitos/inmunología , Granulocitos/metabolismo , Inflamación/inmunología , Inflamación/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Fagocitosis/inmunología , Receptores Depuradores de Clase B/antagonistas & inhibidores , Sepsis/patología
15.
J Lipid Res ; 54(9): 2450-7, 2013 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-23812625

RESUMEN

Scavenger receptor class B type I (SR-BI) is a multi-ligand receptor that binds a variety of lipoproteins, including high density lipoprotein (HDL) and low density lipoprotein (LDL), but lipoprotein(a) [Lp(a)] has not been investigated as a possible ligand. Stable cell lines (HEK293 and HeLa) expressing human SR-BI were incubated with protein- or lipid-labeled Lp(a) to investigate SR-BI-dependent Lp(a) cell association. SR-BI expression enhanced the association of both (125)I- and Alexa Fluor-labeled protein from Lp(a). By confocal microscopy, SR-BI was also found to promote the internalization of fluorescent lipids (BODIPY-cholesteryl ester (CE)- and DiI-labeled) from Lp(a), and by immunocytochemistry the cellular internalization of apolipoprotein(a) and apolipoprotein B. When dual-labeled ((3)H-cholesteryl ether,(125)I-protein) Lp(a) was added to cells expressing SR-BI, there was a greater relative increase in lipid uptake over protein, indicating that SR-BI mediates selective lipid uptake from Lp(a). Compared with C57BL/6 control mice, transgenic mice overexpressing human SR-BI in liver were found to have increased plasma clearance of (3)H-CE-Lp(a), whereas mouse scavenger receptor class B type I knockout (Sr-b1-KO) mice had decreased plasma clearance (fractional catabolic rate: 0.63 ± 0.08/day, 1.64 ± 0.62/day, and 4.64 ± 0.40/day for Sr-b1-KO, C57BL/6, and human scavenger receptor class B type I transgenic mice, respectively). We conclude that Lp(a) is a novel ligand for SR-BI and that SR-BI mediates selective uptake of Lp(a)-associated lipids.


Asunto(s)
Antígenos CD36/metabolismo , Lipoproteína(a)/metabolismo , Animales , Células HEK293 , Humanos , Lipoproteína(a)/sangre , Ratones , Transporte de Proteínas
16.
J Biol Chem ; 287(21): 17483-17492, 2012 May 18.
Artículo en Inglés | MEDLINE | ID: mdl-22474282

RESUMEN

Pregnenolone (PREG) can be converted to PREG esters (PE) by the plasma enzyme lecithin: cholesterol acyltransferase (LCAT), and by other enzyme(s) with unknown identity. Acyl-CoA:cholesterol acyltransferase 1 and 2 (ACAT1 and ACAT2) convert various sterols to steryl esters; their activities are activated by cholesterol. PREG is a sterol-like molecule, with 3-ß-hydroxy moiety at steroid ring A, but with much shorter side chain at steroid ring D. Here we show that without cholesterol, PREG is a poor ACAT substrate; with cholesterol, the V(max) for PREG esterification increases by 100-fold. The binding affinity of ACAT1 for PREG is 30-50-fold stronger than that for cholesterol; however, PREG is only a substrate but not an activator, while cholesterol is both a substrate and an activator. These results indicate that the sterol substrate site in ACAT1 does not involve significant sterol-phospholipid interaction, while the sterol activator site does. Studies utilizing small molecule ACAT inhibitors show that ACAT plays a key role in PREG esterification in various cell types examined. Mice lacking ACAT1 or ACAT2 do not have decreased PREG ester contents in adrenals, nor do they have altered levels of the three major secreted adrenal steroids in serum. Mice lacking LCAT have decreased levels of PREG esters in the adrenals. These results suggest LCAT along with ACAT1/ACAT2 contribute to control pregnenolone ester content in different cell types and tissues.


Asunto(s)
Acetil-CoA C-Acetiltransferasa/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Pregnenolona/metabolismo , Esterol O-Aciltransferasa/metabolismo , Acetil-CoA C-Acetiltransferasa/genética , Glándulas Suprarrenales/metabolismo , Animales , Línea Celular Tumoral , Colesterol/genética , Colesterol/metabolismo , Humanos , Ratones , Ratones Noqueados , Especificidad de Órganos/fisiología , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Pregnenolona/genética , Esterol O-Aciltransferasa/genética , Esterol O-Aciltransferasa 2
17.
Biomacromolecules ; 14(5): 1465-73, 2013 May 13.
Artículo en Inglés | MEDLINE | ID: mdl-23530926

RESUMEN

The in vivo degradation and elimination after subcutaneous implantation of injectable p(SA-RA) 3:7 copolymer in rats, followed by characterization of the polymer matrix composition during hydrolysis and erosion, is reported. Major chemical changes were observed during the first few days post implantation, the anhydride bonds hydrolyzed along with about 45% weight loss and a significant decrease in the molecular weight. 1H NMR spectral analysis was used to determine the structures and content of ricinoleic acid containing oligomeric chains present in the degraded polymer. The polymer degrades into ester oligomers of 2-4 ricinoleic acid units which further degrade to ricinoleic acid, a natural fatty acid. The polymer hydrolytic degradation process fit the in vitro degradation process.


Asunto(s)
Materiales Biocompatibles/metabolismo , Ácidos Decanoicos/metabolismo , Polímeros/metabolismo , Ácidos Ricinoleicos/metabolismo , Animales , Materiales Biocompatibles/química , Biotransformación , Ácidos Decanoicos/química , Femenino , Hidrólisis , Inyecciones Subcutáneas , Peso Molecular , Polímeros/química , Ratas , Ratas Sprague-Dawley , Ácidos Ricinoleicos/química
18.
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
19.
Curr Atheroscler Rep ; 13(3): 249-56, 2011 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-21331766

RESUMEN

Lecithin cholesterol acyl transferase (LCAT) is a plasma enzyme that esterifies cholesterol and raises high-density lipoprotein cholesterol, but its role in atherosclerosis is not clearly established. Studies of various animal models have yielded conflicting results, but studies done in rabbits and non-human primates, which more closely simulate human lipoprotein metabolism, indicate that LCAT is likely atheroprotective. Although suggestive, there are also no biomarker studies that mechanistically link LCAT with cardiovascular disease. Imaging studies of patients with LCAT deficiency have also not yielded a clear answer to the role of LCAT in atherosclerosis. Recombinant LCAT, however, is currently being developed as a therapeutic product for enzyme replacement therapy of patients with genetic disorders of LCAT for the prevention and/or treatment of renal disease, but it may also have value for the treatment of acute coronary syndrome.


Asunto(s)
Aterosclerosis , HDL-Colesterol/metabolismo , Deficiencia de la Lecitina Colesterol Aciltransferasa/enzimología , Metabolismo de los Lípidos/genética , Fosfatidilcolina-Esterol O-Aciltransferasa , Animales , Aterosclerosis/enzimología , Aterosclerosis/genética , Transporte Biológico/genética , Ésteres del Colesterol/metabolismo , HDL-Colesterol/genética , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Ácidos Grasos/metabolismo , Humanos , Deficiencia de la Lecitina Colesterol Aciltransferasa/genética , Ratones , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferasa/uso terapéutico , Conejos , Saimiri
20.
FEBS Lett ; 595(6): 773-788, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33020907

RESUMEN

Apolipoprotein (apo) A-I, the major structural protein of high-density lipoprotein (HDL), is present in human and mouse cerebrospinal fluid (CSF) despite its lack of expression in brain cells. To identify the origin of apoA-I in CSF, we generated intestine-specific and liver-specific Apoa1 knockout mice (Apoa1ΔInt and Apoa1Δliv mice, respectively). Lipoprotein profiles of Apoa1ΔInt and Apoa1ΔLiv mice resembled those of control littermates, whereas knockout of Apoa1 in both intestine and liver (Apoa1ΔIntΔLiv ) resulted in a 60-percent decrease in HDL-cholesterol levels, thus strongly mimicking the Apoa1-/- mice. Immunoassays revealed that mouse apoA-I was not present in the CSF of the Apoa1ΔIntΔLiv mice. Furthermore, apoA-I levels in CSF were highly correlated with plasma spherical HDL levels, which were regulated by ABCA1 and LCAT. Collectively, these results suggest that apoA-I protein in CSF originates in liver and small intestine and is taken up from the plasma.


Asunto(s)
Transportador 1 de Casete de Unión a ATP/metabolismo , Apolipoproteína A-I/sangre , Apolipoproteína A-I/líquido cefalorraquídeo , Mucosa Intestinal/metabolismo , Lipoproteínas HDL/metabolismo , Hígado/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Transportador 1 de Casete de Unión a ATP/genética , Animales , Lipoproteínas HDL/genética , Ratones , Ratones Noqueados , Fosfatidilcolina-Esterol O-Aciltransferasa/genética
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