Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 15 de 15
Filtrar
1.
J Biol Chem ; 295(15): 5136-5151, 2020 04 10.
Artículo en Inglés | MEDLINE | ID: mdl-32132173

RESUMEN

Increased plasma concentrations of lipoprotein(a) (Lp(a)) are associated with an increased risk for cardiovascular disease. Lp(a) is composed of apolipoprotein(a) (apo(a)) covalently bound to apolipoprotein B of low-density lipoprotein (LDL). Many of apo(a)'s potential pathological properties, such as inhibition of plasmin generation, have been attributed to its main structural domains, the kringles, and have been proposed to be mediated by their lysine-binding sites. However, available small-molecule inhibitors, such as lysine analogs, bind unselectively to kringle domains and are therefore unsuitable for functional characterization of specific kringle domains. Here, we discovered small molecules that specifically bind to the apo(a) kringle domains KIV-7, KIV-10, and KV. Chemical synthesis yielded compound AZ-05, which bound to KIV-10 with a Kd of 0.8 µm and exhibited more than 100-fold selectivity for KIV-10, compared with the other kringle domains tested, including plasminogen kringle 1. To better understand and further improve ligand selectivity, we determined the crystal structures of KIV-7, KIV-10, and KV in complex with small-molecule ligands at 1.6-2.1 Å resolutions. Furthermore, we used these small molecules as chemical probes to characterize the roles of the different apo(a) kringle domains in in vitro assays. These assays revealed the assembly of Lp(a) from apo(a) and LDL, as well as potential pathophysiological mechanisms of Lp(a), including (i) binding to fibrin, (ii) stimulation of smooth-muscle cell proliferation, and (iii) stimulation of LDL uptake into differentiated monocytes. Our results indicate that a small-molecule inhibitor targeting the lysine-binding site of KIV-10 can combat the pathophysiological effects of Lp(a).


Asunto(s)
Apolipoproteínas A/antagonistas & inhibidores , Apolipoproteínas A/metabolismo , Fibrina/metabolismo , Kringles/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Secuencia de Aminoácidos , Ensayos Analíticos de Alto Rendimiento , Humanos , Ligandos , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Homología de Secuencia
2.
Biochem J ; 473(5): 641-9, 2016 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-26656484

RESUMEN

The chemokine receptor CX3CR1 has been implicated as an attractive therapeutic target in several diseases, including atherosclerosis and diabetes. However, there has been a lack of non-peptide CX3CR1 inhibitors to substantiate these findings. A selective small-molecule inhibitor of CX3CR1, AZD8797, was recently reported and we present here an in-depth in vitro characterization of that molecule. In a flow adhesion assay, AZD8797 antagonized the natural ligand, fractalkine (CX3CL1), in both human whole blood (hWB) and in a B-lymphocyte cell line with IC50 values of 300 and 6 nM respectively. AZD8797 also prevented G-protein activation in a [(35)S]GTPγS (guanosine 5'-[γ-thio]triphosphate) accumulation assay. In contrast, dynamic mass redistribution (DMR) experiments revealed a weak Gαi-dependent AZD8797 agonism. Additionally, AZD8797 positively modulated the CX3CL1 response at sub-micromolar concentrations in a ß-arrestin recruitment assay. In equilibrium saturation binding experiments, AZD8797 reduced the maximal binding of (125)I-CX3CL1 without affecting Kd. Kinetic experiments, determining the kon and koff of AZD8797, demonstrated that this was not an artefact of irreversible or insurmountable binding, thus a true non-competitive mechanism. Finally we show that both AZD8797 and GTPγS increase the rate with which CX3CL1 dissociates from CX3CR1 in a similar manner, indicating a connection between AZD8797 and the CX3CR1-bound G-protein. Collectively, these data show that AZD8797 is a non-competitive allosteric modulator of CX3CL1, binding CX3CR1 and effecting G-protein signalling and ß-arrestin recruitment in a biased way.


Asunto(s)
Células Sanguíneas/efectos de los fármacos , Pirimidinas/farmacología , Receptores de Quimiocina/metabolismo , Tiazoles/farmacología , Regulación Alostérica , Animales , Arrestinas/metabolismo , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Células Sanguíneas/metabolismo , Células CHO , Receptor 1 de Quimiocinas CX3C , Quimiocina CX3CL1/farmacología , Cricetulus , Proteínas de Unión al GTP/metabolismo , Humanos , Ensayo de Unión Radioligante , Transducción de Señal , beta-Arrestinas
3.
Arterioscler Thromb Vasc Biol ; 33(12): 2707-14, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24115030

RESUMEN

OBJECTIVE: High circulating levels of group IIA secretory phospholipase A2 (sPLA2-IIA) activity and mass are independent cardiovascular risk factors. Therefore, inhibition of sPLA2-IIA may be a target for the treatment of atherosclerotic cardiovascular disease. The present study evaluated the effects of sPLA2-IIA inhibition with varespladib acid in a novel mouse model, human apolipoprotein B (apoB)/human cholesteryl ester transfer protein (CETP)/human sPLA2-IIA triple transgenic mice (TTT) fed a Western-type diet. APPROACH AND RESULTS: sPLA2-IIA expression increased atherosclerotic lesion formation in TTT compared with human apoB/human CETP double transgenic mice (P<0.01). Varespladib acid effectively inhibited plasma sPLA2-IIA activity. Surprisingly, however, administration of varespladib acid to TTT had no impact on atherosclerosis, which could be attributed to a proatherogenic plasma lipoprotein profile that appears in response to sPLA2-IIA inhibition because of increased plasma CETP activity. In the TTT model, sPLA2-IIA decreased CETP activity by reducing the acceptor properties of sPLA2-IIA-modified very low-density lipoproteins specifically because of a significantly lower apoE content. Increasing very low-density lipoprotein-apoE content by means of adenovirus-mediated gene transfer in sPLA2-IIA transgenic mice restored the acceptor properties for CETP. CONCLUSIONS: These data show that in a humanized triple transgenic mouse model with hypercholesterolemia, sPLA2-IIA inhibition increases CETP activity via increasing the very low-density lipoprotein-apoE content, resulting in a proatherogenic lipoprotein profile.


Asunto(s)
Aorta/enzimología , Enfermedades de la Aorta/enzimología , Aterosclerosis/enzimología , Proteínas de Transferencia de Ésteres de Colesterol/metabolismo , Fosfolipasas A2 Grupo II/metabolismo , Acetatos/farmacología , Animales , Aorta/efectos de los fármacos , Aorta/patología , Enfermedades de la Aorta/sangre , Enfermedades de la Aorta/tratamiento farmacológico , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Apolipoproteína E3/genética , Apolipoproteína E3/metabolismo , Apolipoproteínas B/genética , Apolipoproteínas B/metabolismo , Aterosclerosis/sangre , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/genética , Aterosclerosis/patología , Proteínas de Transferencia de Ésteres de Colesterol/sangre , Proteínas de Transferencia de Ésteres de Colesterol/genética , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Femenino , Fosfolipasas A2 Grupo II/antagonistas & inhibidores , Fosfolipasas A2 Grupo II/sangre , Fosfolipasas A2 Grupo II/genética , Humanos , Hipercolesterolemia/complicaciones , Hipercolesterolemia/enzimología , Hipercolesterolemia/genética , Indoles/farmacología , Cetoácidos , Lipoproteínas VLDL/sangre , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Placa Aterosclerótica
4.
Sci Rep ; 14(1): 11850, 2024 05 24.
Artículo en Inglés | MEDLINE | ID: mdl-38782980

RESUMEN

Natriuretic peptides (NPs) are cardio-derived hormones that have a crucial role in maintaining cardiovascular homeostasis. Physiological effects of NPs are mediated by binding to natriuretic peptide receptors 1 and 2 (NPR1/2), whereas natriuretic peptide receptor 3 (NPR3) acts as a clearance receptor that removes NPs from the circulation. Mouse studies have shown that local NP-signaling in the kidney glomerulus is important for the maintenance of renal homeostasis. In this study we examined the expression of NPR3 in kidney tissue and explored its involvement in renal physiology and disease by generating podocyte-specific knockout mice (NPR3podKO) as well as by using an NPR3 inhibitor (NPR3i) in rodent models of kidney disease. NPR3 was highly expressed by podocytes. NPR3podKO animals showed no renal abnormalities under healthy conditions and responded similarly to nephrotoxic serum (NTS) induced glomerular injury. However, NPR3i showed reno-protective effects in the NTS-induced model evidenced by decreased glomerulosclerosis and reduced podocyte loss. In a ZSF1 rat model of diabetic kidney injury, therapy alone with NPR3i did not have beneficial effects on renal function/histology, but when combined with losartan (angiotensin receptor blocker), NPR3i potentiated its ameliorative effects on albuminuria. In conclusion, these results suggest that NPR3 may contribute to kidney disease progression.


Asunto(s)
Ratones Noqueados , Podocitos , Receptores del Factor Natriurético Atrial , Animales , Receptores del Factor Natriurético Atrial/metabolismo , Receptores del Factor Natriurético Atrial/genética , Ratones , Podocitos/metabolismo , Podocitos/patología , Ratas , Glomérulos Renales/metabolismo , Glomérulos Renales/patología , Masculino , Modelos Animales de Enfermedad , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Losartán/farmacología , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología
5.
Sci Transl Med ; 13(593)2021 05 12.
Artículo en Inglés | MEDLINE | ID: mdl-33980578

RESUMEN

Inhibitors of proprotein convertase subtilisin/kexin type 9 (PCSK9) reduce low-density lipoprotein (LDL) cholesterol and are used for treatment of dyslipidemia. Current PCSK9 inhibitors are administered via subcutaneous injection. We present a highly potent, chemically modified PCSK9 antisense oligonucleotide (ASO) with potential for oral delivery. Past attempts at oral delivery using earlier-generation ASO chemistries and transient permeation enhancers provided encouraging data, suggesting that improving potency of the ASO could make oral delivery a reality. The constrained ethyl chemistry and liver targeting enabled by N-acetylgalactosamine conjugation make this ASO highly potent. A single subcutaneous dose of 90 mg reduced PCSK9 by >90% in humans with elevated LDL cholesterol and a monthly subcutaneous dose of around 25 mg is predicted to reduce PCSK9 by 80% at steady state. To investigate the feasibility of oral administration, the ASO was coformulated in a tablet with sodium caprate as permeation enhancer. Repeated oral daily dosing in dogs resulted in a bioavailability of 7% in the liver (target organ), about fivefold greater than the plasma bioavailability. Target engagement after oral administration was confirmed by intrajejunal administration of a rat-specific surrogate ASO in solution with the enhancer to rats and by plasma PCSK9 and LDL cholesterol lowering in cynomolgus monkey after tablet administration. On the basis of an assumption of 5% liver bioavailability after oral administration in humans, a daily dose of 15 mg is predicted to reduce circulating PCSK9 by 80% at steady state, supporting the development of the compound for oral administration to treat dyslipidemia.


Asunto(s)
Oligonucleótidos Antisentido , Inhibidores de PCSK9 , Animales , Perros , Macaca fascicularis , Ratas , Serina Endopeptidasas
6.
Curr Atheroscler Rep ; 10(3): 252-9, 2008 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-18489854

RESUMEN

Elevated circulating levels of secretory phospholipase A(2) (sPLA(2)) are associated with atherosclerotic cardiovascular disease. sPLA(2) can contribute to atherogenesis by hydrolyzing phospholipids of circulating lipoproteins and lipoproteins entrapped in the arterial wall and/or in cells that reside in the intima and that participate in the inflammatory response to lipoprotein deposition. This article reviews differences and similarities between sPLA(2)-IIA, sPLA(2)-V, and sPLA(2)-X, all of which are members of this family of enzymes with reported potential proatherogenic features. Published data suggest that each of the enzymes has a distinct profile characterized by differences in tissue expression and localization, capacity to act on phospholipids of cell membranes and lipoproteins, and their interaction with arterial proteoglycans. In addition, the article discusses results from the authors' laboratory showing that diet-induced or gene-induced hyperlipidemia in mice enhances the expression of sPLA(2)-V in different tissues, but not sPLA(2)-IIA. Such differences indicate that these enzymes may have different roles in atherosclerotic cardiovascular disease through their distinct profiles.


Asunto(s)
Arterias/enzimología , Aterosclerosis/enzimología , Fosfolipasas A2 Secretoras/metabolismo , Animales , Aorta/enzimología , Aorta/patología , Arterias/patología , Aterosclerosis/etiología , Aterosclerosis/patología , Humanos , Hiperlipidemias/metabolismo , Lipoproteínas/metabolismo , Fosfolípidos/metabolismo , Proteoglicanos/metabolismo
7.
ACS Med Chem Lett ; 9(7): 600-605, 2018 Jul 12.
Artículo en Inglés | MEDLINE | ID: mdl-30034586

RESUMEN

A lead generation campaign identified indole-based sPLA2-X inhibitors with a promising selectivity profile against other sPLA2 isoforms. Further optimization of sPLA2 selectivity and metabolic stability resulted in the design of (-)-17, a novel, potent, and selective sPLA2-X inhibitor with an exquisite pharmacokinetic profile characterized by high absorption and low clearance, and low toxicological risk. Compound (-)-17 was tested in an ApoE-/- murine model of atherosclerosis to evaluate the effect of reversible, pharmacological sPLA2-X inhibition on atherosclerosis development. Despite being well tolerated and achieving adequate systemic exposure of mechanistic relevance, (-)-17 did not significantly affect circulating lipid and lipoprotein biomarkers and had no effect on coronary function or histological markers of atherosclerosis.

8.
ACS Med Chem Lett ; 9(7): 594-599, 2018 Jul 12.
Artículo en Inglés | MEDLINE | ID: mdl-30034585

RESUMEN

In order to assess the potential of sPLA2-X as a therapeutic target for atherosclerosis, novel sPLA2 inhibitors with improved type X selectivity are required. To achieve the objective of identifying such compounds, we embarked on a lead generation effort that resulted in the identification of a novel series of indole-2-carboxamides as selective sPLA2-X inhibitors with excellent potential for further optimization.

9.
Biochim Biophys Acta ; 1761(11): 1301-8, 2006 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-17070102

RESUMEN

Clinical observations strongly support an association of circulating levels of secretory phospholipases A(2) (sPLA(2)) in atherosclerotic cardiovascular disease (ACVD). Two modes of action can provide causal support for these statistical correlations. One is the action of the enzymes on circulating lipoproteins and the other is direct action on the lipoproteins once in the arterial extracellular intima. In this review we discuss results suggesting a distinct profile of characteristics related to localization, action on plasma lipoproteins and interaction with arterial proteoglycans for sPLA(2)-IIA and sPLA(2)-V. The differences observed indicate that these enzymes may contribute to atherosclerosis through dissimilar pathways. Furthermore, we comment on recent animal studies from our laboratory indicating that the expression of type V enzyme is up-regulated by genetically and nutritionally-induced dyslipidemias but not the group type IIA enzyme, which is well known to be up-regulated by acute inflammation. The results suggest that if similar up-regulation occurs in humans in response to hyperlipidemia, it may create a distinctive link between the group V enzyme and the disease.


Asunto(s)
Enfermedad de la Arteria Coronaria/enzimología , Regulación Enzimológica de la Expresión Génica , Lipoproteínas/metabolismo , Fosfolipasas A/metabolismo , Regulación hacia Arriba , Enfermedad Aguda , Animales , Enfermedad de la Arteria Coronaria/patología , Fosfolipasas A2 Grupo II , Fosfolipasas A2 Grupo V , Humanos , Hiperlipidemias/enzimología , Hiperlipidemias/patología , Inflamación/enzimología , Inflamación/patología , Fosfolipasas A2 , Proteoglicanos/metabolismo , Túnica Íntima/enzimología , Túnica Íntima/patología
10.
Arterioscler Thromb Vasc Biol ; 26(7): 1579-85, 2006 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-16601231

RESUMEN

OBJECTIVE: To study the distribution of group V secretory phospholipase A2 (sPLA2) in human and mouse lesions and compare its expression by human vascular cells, its activity toward lipoproteins, and the interaction with arterial proteoglycans (proteoglycans) with those of sPLA2-IIA. In addition, we also investigated the effect of a Western diet and lipopolysaccharide challenge on the aortic expression of these enzymes in mouse models. METHODS AND RESULTS: Immunohistochemistry showed sPLA2-V in human and mouse lesions to be associated with smooth muscle cells and also surrounding foam cells in lipid core areas. mRNA of the enzyme was expressed in human lesions and human vascular cells, supporting the immunohistochemistry data. sPLA2-V but not sPLA2-IIA was active on lipoproteins in human serum. The association with proteoglycans enhanced 2- to 3-fold sPLA2-V activity toward low-density lipoproteins but not that of the group IIA enzyme. Experiments in mouse models showed that treatment with a Western diet induced expression of sPLA2-V but not that of sPLA2-IIA in aorta. On the other hand, lipopolysaccharide-induced acute inflammation augmented the expression of sPLA2-IIA but not that of sPLA2-V. CONCLUSIONS: These results indicate that these phospholipases could have different roles in atherosclerosis.


Asunto(s)
Aorta/enzimología , Arterias/metabolismo , Aterosclerosis/enzimología , Aterosclerosis/patología , Dieta , Fosfolipasas A/metabolismo , Proteoglicanos/metabolismo , Animales , Sangre/efectos de los fármacos , Vasos Sanguíneos/enzimología , Vasos Sanguíneos/patología , Enfermedades de las Arterias Carótidas/enzimología , Enfermedades de las Arterias Carótidas/patología , Interacciones Farmacológicas , Inducción Enzimática , Fosfolipasas A2 Grupo II , Humanos , Inmunohistoquímica/métodos , Isoenzimas/genética , Isoenzimas/metabolismo , Isoenzimas/farmacología , Receptores de Lipopolisacáridos/análisis , Lipoproteínas/efectos de los fármacos , Macrófagos/enzimología , Macrófagos/inmunología , Ratones , Fosfolipasas A/genética , Fosfolipasas A/farmacología , Fosfolipasas A2 , ARN Mensajero/metabolismo , Proteínas Recombinantes/farmacología , Coloración y Etiquetado
11.
Arterioscler Thromb Vasc Biol ; 25(1): 180-5, 2005 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-15539621

RESUMEN

OBJECTIVE: We investigated the potential role of ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin motif type I) in atherogenesis. METHODS AND RESULTS: ADAMTS-1 is expressed at the highest levels in the aorta when compared with other human tissues examined. Immunolocalization studies in human aorta and coronary artery indicate that ADAMTS-1 expression is mainly seen at low levels in the medial layer, but upregulated in the intima when plaque is present. We found that ADAMTS-1 mRNA levels are significantly higher in proliferating/migrating cultured primary aortic vascular smooth muscle cells (VSMCs) compared with resting/confluent cells. Using the mouse carotid artery flow cessation model, we show that there are differences in vessel remodeling in ADAMTS-1 transgenic/apoE-deficient mice compared with apoE deficiency alone, particularly a significant increase in intimal hyperplasia. We show that ADAMTS-1 can cleave the large versican containing proteoglycan population purified from cultured human aortic VSMCs. Finally, using versican peptide substrates, we show data suggesting that ADAMTS-1 cleaves versican at multiple sites. CONCLUSIONS: We hypothesize that ADAMTS-1 may promote atherogenesis by cleaving extracellular matrix proteins such as versican and promoting VSMC migration.


Asunto(s)
Arteriosclerosis/patología , Arteria Carótida Común/patología , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Desintegrinas/fisiología , Inmunohistoquímica/métodos , Metaloendopeptidasas/fisiología , Péptido Hidrolasas/metabolismo , Proteínas ADAM , Proteína ADAMTS1 , Adolescente , Animales , Arteriosclerosis/metabolismo , Arteria Carótida Común/química , Arteria Carótida Común/metabolismo , Arteria Carótida Común/cirugía , Línea Celular , Modelos Animales de Enfermedad , Desintegrinas/biosíntesis , Desintegrinas/inmunología , Desintegrinas/metabolismo , Humanos , Hidrólisis , Lectinas Tipo C , Ligadura/métodos , Masculino , Metaloendopeptidasas/biosíntesis , Metaloendopeptidasas/inmunología , Metaloendopeptidasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Músculo Liso Vascular/química , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/química , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Neovascularización Patológica/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Versicanos
12.
ACS Med Chem Lett ; 7(10): 884-889, 2016 Oct 13.
Artículo en Inglés | MEDLINE | ID: mdl-27774123

RESUMEN

Expedited structure-based optimization of the initial fragment hit 1 led to the design of (R)-7 (AZD2716) a novel, potent secreted phospholipase A2 (sPLA2) inhibitor with excellent preclinical pharmacokinetic properties across species, clear in vivo efficacy, and minimized safety risk. Based on accumulated profiling data, (R)-7 was selected as a clinical candidate for the treatment of coronary artery disease.

13.
Atherosclerosis ; 225(1): 115-20, 2012 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-22999079

RESUMEN

OBJECTIVES: High levels of large HDL (HDL2) reduce cardiovascular disease risks apparently because it mediates reverse cholesterol transport, and it has anti-inflammatory properties. Here we explored the mechanism behind an additional athero-protective HDL effect related to its capacity to interfere with formation of insoluble LDL-proteoglycans associations, a key step in LDL entrapment in the intima and in atherogenesis. METHODS AND RESULTS: We found that HDL2 levels from type 2 diabetes patients and controls are inversely correlated with complex formation between serum LDL and the arterial proteoglycans versican. Reconstitution experiments indicate that HDL2 was more efficacious inhibitor of the LDL-versican association than the smaller HDL3. This may explain why serum from patients with dyslipidemia of insulin resistance, with low levels of HDL2, have a higher capacity to form insoluble LDL-proteoglycan complex. ApoE enrichment of HDL2 and HDL3 or addition of copies of an apoE peptide with the proteoglycan-binding sequence of this apolipoprotein increased their inhibition of LDL-versican associations. CONCLUSIONS: The inhibitory effect of HDL2 and HDL3 on LDL-versican associations was related to formation of apoE-mediated soluble HDL-versican complexes. We speculate that in the intima large, HDL2 subclasses, by forming reversible soluble associations with proteoglycans can compete with formation of irreversible LDL-proteoglycan aggregates. This can contribute to the HDL2 athero-protective effects. In the dyslipidemia of insulin resistance, associated with low levels of HDL2, this athero-protective property may be compromised.


Asunto(s)
Aterosclerosis/prevención & control , Lipoproteínas HDL2/farmacología , Lipoproteínas LDL/metabolismo , Versicanos/metabolismo , Apolipoproteínas E/metabolismo , Diabetes Mellitus Tipo 2 , Humanos , Resistencia a la Insulina/fisiología , Lipoproteínas HDL3/farmacología , Masculino
14.
J Lipid Res ; 49(2): 481-90, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-18025001

RESUMEN

There is much interest in the significance of apolipoproteins and proteins that are noncovalently associated with lipoproteins. It is possible that the high ionic strength used for isolation of lipoproteins with KBr and NaI could alter the pattern of associated exchangeable proteins. Here we describe lipoprotein classes fractionation from up to 0.5 ml of serum or plasma with buffers of physiological ionic strength and pH prepared with deuterium oxide (D(2)O) and sucrose. An advantage of the D(2)O/sucrose procedure was that the lipoproteins could be directly analyzed by the techniques described without need for desalting. We compared the isolated lipoproteins with those obtained using ultracentrifugation in KBr from the same plasma pool. Electrophoretic homogeneity of the lipoproteins was very similar using the two methods, as well as their lipid composition evaluated by HPLC. Two-dimensional electrophoresis and surface-enhanced laser adsorption/ionization time-of-flight mass spectrometry indicated that the patterns of exchangeable proteins of VLDL isolated using with the two procedures were very similar. However, significant differences were found in the profiles of LDL and HDL, indicating that the D(2)O/sucrose method allowed a more complete characterization of its exchangeable apolipoproteins and proteins.


Asunto(s)
Bromuros , Óxido de Deuterio , Lípidos/química , Lipoproteínas/aislamiento & purificación , Compuestos de Potasio , Proteómica/métodos , Cloruro de Sodio , Sacarosa , Apolipoproteínas/sangre , Apolipoproteínas/química , Apolipoproteínas/aislamiento & purificación , Electroforesis en Gel Bidimensional/métodos , Humanos , Lípidos/sangre , Lipoproteínas/sangre , Lipoproteínas/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Ultracentrifugación/métodos
15.
Exp Cell Res ; 300(2): 335-44, 2004 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-15474998

RESUMEN

Macrophages (Mphi) play an important role in the initiation and progress of the atherogenic process. They contribute to the growth of atherosclerotic plaque by affecting lipoprotein metabolism, matrix homeostasis, lipoprotein modification and cholesterol accumulation. Access to in vitro Mphi models is therefore important for understanding the mechanisms involved in the transition of the relatively simple fatty streak to more complex type of lesions. The aim of the present work was to compare the expression profile of macrophages differentiated from the hematopoietic lineage to peritoneal mouse macrophages and two commonly used mouse macrophage cell lines (J774.A1 and RAW264.7). Our results showed that the embryonic stem cell-derived macrophages (ES Mphi) had a more similar expression profile to peritoneal macrophages than the two mouse macrophage cell lines. The ES Mphi had unchanged expression of the majority of cholesterol efflux mediators when compared to mouse peritoneal macrophages, whereas the cell lines showed altered expression of several of these genes. A key gene in this process is apolipoprotein E, which is expressed in ES Mphi but not in macrophage cell lines. In conclusion, ES Mphi are likely to provide a better in vitro model than mouse Mphi cell lines to study macrophage involvement in atherosclerosis.


Asunto(s)
Modelos Animales de Enfermedad , Expresión Génica/fisiología , Macrófagos/fisiología , Enfermedades Vasculares , Animales , Interpretación Estadística de Datos , Perfilación de la Expresión Génica , Inmunohistoquímica , Ratones , Fagocitosis/fisiología
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA