RESUMEN
Intestinal lipid dysregulation is a common feature of insulin-resistant states. The present study investigated alterations in gene expression of key proteins involved in the active absorption of dietary fat and cholesterol in response to development of insulin resistance. Studies were conducted in two diet-induced animal models of insulin resistance: fructose-fed hamster and high-fat-fed mouse. Changes in the mRNA abundance of lipid transporters, adenosine triphosphate cassette (ABC) G5, ABCG8, FA-CoA ligase fatty acid translocase P4, Niemann-Pick C1-Like1 (NPC1L1), fatty acid transport protein 4 (FATP4), and Scavenger Receptor Class B Type I (SR-BI), were assessed in intestinal fragments (duodenum, jejunum, and ileum) using quantitative real-time PCR. Of all the transporters evaluated, SR-B1 showed the most significant changes in both animal models examined. A marked stimulation of SR-B1 expression was observed in all intestinal segments examined in both insulin-resistant animal models. The link between SR-BI expression and intestinal lipoprotein production was then examined in the Caco-2 cell model. SR-B1 overexpression in Caco-2 cells increased apolipoprotein B (apoB) 100 and apoB48 secretion, whereas RNAi knock down of SR-B1 decreased secretion of both apoB100 and apoB48. We also observed changes in subcellular distribution of SR-B1 in response to exogenous lipid and insulin. Confocal microscopy revealed marked changes in SR-BI subcellular distribution in response to both exogenous lipids (oleate) and insulin. In summary, marked stimulation of intestinal SR-BI occurs in vivo in animal models of diet-induced insulin resistance, and modulation of SR-BI in vitro regulates production of apoB-containing lipoprotein particles. We postulate that apical and/or basolateral SR-BI may play an important role in intestinal chylomicron production and may contribute to chylomicron overproduction normally observed in insulin-resistant states.
Asunto(s)
Apolipoproteína B-48/biosíntesis , Antígenos CD36/metabolismo , Antígenos CD36/farmacología , Grasas de la Dieta/farmacología , Fructosa/farmacología , Mucosa Intestinal/metabolismo , ARN Mensajero/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5 , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8 , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Antígenos CD36/genética , Cricetinae , Duodeno/metabolismo , Dislipidemias , Ayuno/fisiología , Proteínas de Transporte de Ácidos Grasos/genética , Proteínas de Transporte de Ácidos Grasos/metabolismo , Expresión Génica , Íleon/metabolismo , Insulina/fisiología , Resistencia a la Insulina , Yeyuno/metabolismo , Lipoproteínas/genética , Lipoproteínas/metabolismo , Masculino , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Mesocricetus , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Periodo Posprandial/fisiología , Regulación hacia ArribaRESUMEN
Scavenger receptor, class B, type I (SR-BI) mediates binding and internalization of a variety of lipoprotein and nonlipoprotein ligands, including HDL. Studies in genetically engineered mice revealed that SR-BI plays an important role in HDL reverse cholesterol transport and protection against atherosclerosis. Understanding how SR-BI's function is regulated may reveal new approaches to therapeutic intervention in atherosclerosis and heart disease. We utilized a model cell system to explore pathways involved in SR-BI-mediated lipid uptake from and signaling in response to distinct lipoprotein ligands: the physiological ligand, HDL, and a model ligand, acetyl LDL (AcLDL). In Chinese hamster ovary-derived cells, murine SR-BI (mSR-BI) mediates lipid uptake via distinct pathways that are dependent on the lipoprotein ligand. Furthermore, HDL and AcLDL activate distinct signaling pathways. Finally, mSR-BI-mediated selective lipid uptake versus endocytic uptake are differentially regulated by protein kinase signaling pathways. The protein kinase C (PKC) activator PMA and the phosphatidyl inositol 3-kinase inhibitor wortmannin increase the degree of mSR-BI-mediated selective lipid uptake, whereas a PKC inhibitor has the opposite effect. These data demonstrate that SR-BI's selective lipid uptake activity can be acutely regulated by intracellular signaling cascades, some of which can originate from HDL binding to murine SR-BI itself.
Asunto(s)
Antígenos CD36/metabolismo , Metabolismo de los Lípidos , Proteína Quinasa C/metabolismo , Transducción de Señal , Androstadienos/farmacología , Animales , Transporte Biológico , Antígenos CD36/farmacología , Células CHO , Línea Celular , Cricetinae , Cricetulus , Femenino , Indoles/farmacología , Lipoproteínas HDL/metabolismo , Lipoproteínas HDL/farmacología , Lipoproteínas LDL/metabolismo , Lipoproteínas LDL/farmacología , Ratones , Modelos Biológicos , Receptores de Lipoproteína , WortmaninaRESUMEN
A key event in atherosclerosis is the interaction between monocytes and endothelial cells. Binding of oxidized low-density lipoprotein (oxLDL) to CD36 on endothelial cells results in activation and subsequent monocyte adhesion. In this study, a recombinant soluble CD36 molecule was expressed to delineate its ability to block the adhesion of monocytes. To construct soluble CD36, the extra-cellular domain of CD36 was fused to the Fc domain of human IgG1. The N-terminal sequence of CD36 was replaced with N-terminal signal peptide sequence of CD59, a type I membrane protein. The resulting chimeric sCD36-Ig cDNA (sCD36-Ig) was transfected into COS-7 and CHOK1 cells and supernatants were analyzed for secretion of this molecule. Sandwich ELISA and oxLDL binding analyses showed that recombinant sCD36-Ig is secreted in a functionally active form. Western blot analysis of the purified sCD36-Ig using three different anti-CD36 monoclonal antibodies and anti-human IgG showed that the chimeric sCD36-Ig is a dimer of 220kDa. Further, the sCD36-Ig inhibited the adhesion of monocytes to oxLDL. Interestingly, sCD36-Ig blocked the oxLDL-induced adhesion of monocytes to the endothelial cell specific protein, ICAM-1. Our results indicate that the chimeric sCD36-Ig protein is folded correctly and can effectively compete for the binding of oxLDL to membrane-expressed CD36. These results suggest that oxLDL-induced monocyte adhesion can be blocked using sCD36-Ig and this may be useful in blocking the cell-cell interaction leading to atherogenesis.
Asunto(s)
Antígenos CD36/farmacología , Molécula 1 de Adhesión Intercelular/inmunología , Lipoproteínas LDL/farmacología , Monocitos/inmunología , Animales , Aterosclerosis/inmunología , Antígenos CD36/genética , Células COS , Adhesión Celular/efectos de los fármacos , Adhesión Celular/inmunología , Comunicación Celular/efectos de los fármacos , Comunicación Celular/inmunología , Chlorocebus aethiops , Células Endoteliales/inmunología , Humanos , Regiones Constantes de Inmunoglobulina/genética , Regiones Constantes de Inmunoglobulina/farmacología , Inmunoglobulina G/genética , Inmunoglobulina G/farmacología , Lipoproteínas LDL/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Células U937RESUMEN
Oxidized low-density lipoprotein plays a critical role in the pathogenesis of atherosclerosis and exerts pleiotropic effects on various cellular functions. The present study was designed to evaluate the effects of mildly oxidized LDL (mLDL) on the induction and regulation of an in vitro specific antibody response. We found that mLDL significantly inhibited the induction of the anti-Candida albicans antibody response by human peripheral blood mononuclear cells (PBMC). mLDL-induced down-regulation of antibody production was abrogated by blocking the major receptors that bind and internalize modified LDL. In the mLDL-treated C. albicans-stimulated PBMC cultures an early increase in IL-1beta production was observed and the addition of anti-IL-1beta antibody abrogated the mLDL-induced inhibitory effect. Moreover, the addition of IL-1beta to the cultures inhibited the induction of the specific antibody response, similar to mLDL. On the other hand, mLDL up-regulated PWM-induced polyclonal immunoglobulin (Ig) production. In the same cultures IgM anti-mLDL was found. These results indicate that the up-regulation of IL-1beta production induced by mLDL may be involved in the hindering of B cell function, i.e., specific antibody production. This could be relevant in the pathogenesis of inflammatory diseases such as atherosclerosis.
Asunto(s)
Anticuerpos Antifúngicos/biosíntesis , Linfocitos B/inmunología , Candida albicans/inmunología , Leucocitos Mononucleares/inmunología , Lipoproteínas LDL/farmacología , Formación de Anticuerpos/efectos de los fármacos , Células Productoras de Anticuerpos/efectos de los fármacos , Células Productoras de Anticuerpos/inmunología , Linfocitos B/citología , Antígenos CD36/farmacología , Regulación hacia Abajo , Humanos , Inmunoglobulina G/biosíntesis , Inmunoglobulina M/biosíntesis , Interleucina-1/biosíntesis , Interleucina-1/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Oxidación-Reducción , Mitógenos de Phytolacca americana/inmunología , Proteínas Recombinantes/farmacología , Regulación hacia ArribaRESUMEN
Thrombospondins-1 and -2 (TSP-1, TSP-2) are matricellular glycoproteins with potent antiangiogenic activity. We have previously shown that the antiangiogenic activity of TSP-1 is mediated by the interaction of the type I repeats (TSR) with the receptor CD36, although other domains of TSP-1 have also been implicated. We now show that the antiangiogenic activity of TSP-2, which contains three TSRs but, unlike TSP-1, lacks the capacity to activate TGF-beta, is similarly dependent on CD36. Using the corneal pocket assay we found that TSP-2 did not inhibit bFGF-induced angiogenesis in CD36 null mice. We then demonstrated that (125)[I]-TSP-2 bound to murine macrophages and that binding was diminished by 70% by anti-CD36 antibody or by using cells from CD36 null animals. Solid-phase binding studies revealed that (125)[I]-TSP-2 bound to CD36/glutathione-S-transferase (GST) fusion proteins encoding the region spanning amino acids 93-120, but not amino acids 298-439. This 93-120 amino acid region, previously identified as the TSP-1 binding site, is homologous to domains on other TSP binding proteins, such as LIMP-2 and histidine-rich glycoprotein (HRGP). Finally, we showed with an immunoabsorbent binding assay that TSP-2 bound HRGP with high affinity and that HRGP blocked the antiangiogenic activity of TSP-2, acting like a "decoy" receptor. These data suggest that modulation of the TSR/CD36 system may play an important role in the regulation of the angiogenic "switch," and may provide a target for therapeutic interventions.
Asunto(s)
Antígenos CD36/farmacología , Neovascularización Patológica , Proteínas/química , Trombospondinas/farmacología , Inhibidores de la Angiogénesis/farmacología , Animales , Antígenos CD36/química , Moléculas de Adhesión Celular/metabolismo , Relación Dosis-Respuesta a Droga , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Glutatión Transferasa/metabolismo , Sustancias Macromoleculares/química , Macrófagos/metabolismo , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos C57BL , Péptidos/química , Glicoproteínas de Membrana Plaquetaria/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Temperatura , Trombospondinas/química , Factores de TiempoRESUMEN
Monocyte scavenger receptor, CD36 has been implicated in the pathogenesis of atherosclerosis as a major oxidised LDL receptor mediating lipid accumulation and foam cell formation. Previously, we found that treatment of monocyte cultures with the carboxyl terminal fragment of alpha1-antitrypsin (C-36) increases lipid binding and uptake, induces LDL receptor mRNA and CD36 receptor protein expression, and also significantly increases production of pro-inflammatory molecules. To assess the role of the CD36 receptor in proatherogenic monocyte activation by the C-36 fragment, we tested whether specific anti-CD36 receptor antibodies would block the effects of C-36 on monocyte activation. We find that pre-incubation of cells with anti-LDL and anti-CD36 receptor antibodies (10 microg/ml) blocks binding of 125I-C-36 by about 50%. Similarly, cells pre-incubated with oxidised LDL or native LDL at concentrations from 2.5 to 10 microg/ml showed a loss of 125I-C-36 binding (up to 49 and 57%) and uptake (up to 47 and 59.8%), respectively. In parallel experiments, monocytes were first incubated for 1 or 6 h with anti-CD36 antibodies (10 microg/ml) prior to adding C-36 peptide. Anti-CD36 antibodies suppressed C-36-induced production of gelatinase B, monocyte chemoattractant protein-1, interleukin-6 and cellular oxygen consumption to control levels, whereas levels of TNFalpha were unaffected. In contrast, saturation of LDL receptors with excess of anti-LDL (20 microg/ml) significantly inhibited C-36 induced TNFalpha levels. Results indicate that the C-36 peptide binds to both LDL and CD36 scavenger receptors which involves selective upregulation of pro-inflammatory molecules and activation of the respiratory burst in human monocytes. This also supports important roles for CD36 and LDL receptors in atherogenesis and suggests that blockade of CD36 receptor can be protective in pro-inflammatory activation of human monocytes.
Asunto(s)
Antígenos CD36/fisiología , Mediadores de Inflamación/metabolismo , Proteínas de la Membrana , Monocitos/fisiología , Fragmentos de Péptidos/fisiología , Receptores Inmunológicos , Receptores de LDL/fisiología , Receptores de Lipoproteína , alfa 1-Antitripsina/fisiología , Arteriosclerosis/fisiopatología , Antígenos CD36/metabolismo , Antígenos CD36/farmacología , Células Cultivadas , Quimiocina CCL2/metabolismo , Humanos , Interleucina-6/metabolismo , Lipoproteínas LDL/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Monocitos/metabolismo , Oxidación-Reducción , Consumo de Oxígeno , Fragmentos de Péptidos/farmacología , Receptores de LDL/metabolismo , Receptores Depuradores , Receptores Depuradores de Clase B , Factor de Necrosis Tumoral alfa/metabolismo , alfa 1-Antitripsina/farmacologíaRESUMEN
Transforming growth factor (TGF)-beta1 is an important regulator of inflammation and fibrosis. TGF-beta1 is usually secreted as a biologically latent protein called latent TGF-beta1 (L-TGF-beta1). L-TGF-beta1 has no biologic effect unless L-TGF-beta1 is converted to its active form. Using a well-recognized model of lung injury induced by the antineoplastic antibiotic bleomycin (Blm), we demonstrated that 7 d after intratracheal Blm administration, total lung TGF-beta was maximally increased. This induction was due to TGF-beta1 production by alveolar macrophages that, when explanted, generated increased quantities of L-TGF-beta1 complexed with the glycoprotein thrombospondin (TSP)-1. The TSP-1/L-TGF-beta1 complex was associated with CD36, a receptor for TSP-1. The association of TSP-1/L-TGF-beta1 to CD36 was critical for plasmin-mediated release of mature TGF-beta1. In this paper we show that, compared with administration of Blm by itself, when a synthetic peptide of CD36 between amino acids 93 and 110 is given concomitantly with Blm to rats, alveolar macrophages generate markedly less active TGF-beta1, the rats gain weight more rapidly, and there is less inflammation, collagen I and III, and fibronectin synthesis. These findings demonstrate a novel in vivo mechanism of activation of L-TGF-beta1 in lung injury and the importance of alveolar macrophage- derived active TGF-beta1 in the pathogenesis of pulmonary inflammation and fibrosis.
Asunto(s)
Bleomicina/efectos adversos , Antígenos CD36/farmacología , Tejido Conectivo/efectos de los fármacos , Proteínas de la Matriz Extracelular/efectos de los fármacos , Inflamación/prevención & control , Enfermedades Pulmonares/prevención & control , Animales , Peso Corporal/efectos de los fármacos , Líquido del Lavado Bronquioalveolar/citología , Antígenos CD36/química , Recuento de Células/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Colágeno/efectos de los fármacos , Colágeno/metabolismo , Tejido Conectivo/química , Tejido Conectivo/metabolismo , Decorina , Proteínas de la Matriz Extracelular/biosíntesis , Femenino , Fibronectinas/efectos de los fármacos , Fibronectinas/metabolismo , Inflamación/inducido químicamente , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Enfermedades Pulmonares/inducido químicamente , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Oligopéptidos/farmacología , Proteoglicanos/efectos de los fármacos , Proteoglicanos/metabolismo , Ratas , Ratas Sprague-Dawley , Factor de Crecimiento Transformador beta/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismo , Células Tumorales CultivadasRESUMEN
BACKGROUND AND OBJECTIVE: The effect of local and circulating thrombin on platelet adhesion onto vascular surfaces was explored in the absence of plasma adhesive proteins using flow conditions. DESIGN AND METHODS: To study the local effects of thrombin, denuded rabbit aorta segments were incubated with thrombin concentrations of 0.001, 0.01 and 0.1 U/mL. To evaluate the effects of circulating thrombin, the same concentrations were added to perfusates consisting of washed platelets and washed red blood cells suspended in a human albumin solution (5%). In some experiments, purified von Willebrand's factor (vWF) (Haemate-P) was added to the perfusates (0. 8 U/mL of vWF, final concentration). A humanized chimeric antibody to the GPIIb-IIIa complex (Reopro) was used to determine the role of this glycoprotein on platelet adhesion under the conditions described. The effect of blocking GPIb was also assessed. Perfusions were carried out at 800 s(-1) for 10 min. The interaction of platelet with the vessel surface was morphometrically evaluated and expressed as percentage of surface coverage (%SC). Changes in the surface expression of the major platelet antigens were also analyzed by flow cytometry. RESULTS: Incubation of subendothelial surfaces with thrombin enhanced platelet deposition with respect to control levels (increases in SC of 64%, 79% and 86% with 0.001, 0.01 and 0.1 U/mL of thrombin, respectively). Low concentrations of thrombin (0.001 and 0.01 U/mL) incorporated in the perfusates resulted in a similar pro-adhesive effect (increases in SC of 64% and 71%, respectively) while the highest concentration (0.1 U/mL) failed to produce a pro-adhesive effect due to the augmented formation of platelet aggregates with subsequent thrombocytopenia (15+/-1 vs. 160+/-5x10(9) plt/L in the perfusates). Similar results were obtained when VWF was present in the perfusate. Reduction of platelet deposition by blockade of GPIIb-IIIa (to 5.3+/-0.7%) was partially restored by thrombin. Blockade of GPIb prevented platelets from adhering even when thrombin was present (%SC of 2.0+/-0.8%). No significant changes in the distribution of platelet membrane glycoproteins during perfusion experiments were detected. INTERPRETATION AND CONCLUSIONS: Our results suggest that thrombin facilitates primary platelet adhesion onto vascular surfaces even in the absence of plasma adhesive proteins. This effect seems to be mainly dependent on the GPIb/vWF axis.
Asunto(s)
Endotelio Vascular/fisiología , Adhesividad Plaquetaria/efectos de los fármacos , Glicoproteínas de Membrana Plaquetaria/farmacología , Trombina/farmacología , Animales , Antígenos de Plaqueta Humana/metabolismo , Antígenos de Plaqueta Humana/farmacología , Aorta , Bioensayo , Biomarcadores/sangre , Antígenos CD36/metabolismo , Antígenos CD36/farmacología , Endotelio Vascular/metabolismo , Factor VIII/farmacología , Citometría de Flujo , Humanos , Perfusión , Activación Plaquetaria/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/farmacología , Glicoproteínas de Membrana Plaquetaria/metabolismo , Conejos , Factor de von Willebrand/farmacologíaRESUMEN
Lactoferrin (LF), a human serum protein, strongly inhibited the adherence of Plasmodium falciparum-infected erythrocytes (PE) to immobilized chondroitin sulfate A (CSA)-conjugated albumin at a concentration of 100 microg/mL and blocked the PE binding to CD36-expressing Chinese hamster ovary (CHO) cells, as well as immobilized CD36 at concentrations of 5 microg/mL and 100 microg/mL, respectively. Biotinylated LF bound to CD36 in a saturable manner, and such binding was inhibited by unlabeled LF and the anti-CD36 monoclonal antibody, 8A6, suggesting specificity of binding. Additionally, LF inhibited PE binding to immobilized thrombospondin (TSP) at a concentration of 100 microg/mL, and specific binding of LF to TSP was confirmed using biotinylated LF. LF inhibited PE binding to C32 amelanotic melanoma cells in a dose-dependent manner. A peptide of LF, Arg-Asn-Met Arg-Lys-Val Arg-Gly-Pro-Pro-Val-Ser-Cys (amino acid residues 25-37 of LF), which has been suggested to contribute to LF binding to various materials, including CSA, inhibited PE binding to immobilized CSA-conjugated albumin, immobilized CD36, CD36-expressing CHO cells, immobilized TSP, and C32 amelanotic melanoma cells, as well as LF itself. These results suggest that LF peptide may provide the basis for developing agents that are able to inhibit CSA-, CD36-, and TSP-mediated cytoadherence of PE.