RESUMEN
Plaque vascularity has been implicated in its growth and stability. However, there is a paucity of information regarding the origin of plaque vasculature and the role of vasa vasorum in plaque growth. To inhibit growth of vasa vasorum in atherogenic mice and assess its effect on plaque growth, we used a truncated plasminogen activator inhibitor (PAI)-1 protein, rPAI-1(23), that has significant antiangiogenic activity. Female LDLR(-/-)ApoB-48-deficient mice fed Paigen's diet without cholate for 20 weeks received rPAI-1(23) treatment (n=21) for the last 6 weeks. Plaque size and vasa vasorum density were compared to 2 controls: mice fed Paigen's diet and treated with saline for the last 6 weeks (n=16) and mice fed Paigen's diet until the onset of treatment (n=14). The rPAI-1(23) treatment significantly reduced plaque area and plaque cholesterol in the descending aorta and plaque area in the innominate artery. Measurements of reconstructed confocal microscopy images of vasa vasorum demonstrate that rPAI-1(23) treatment decreased vasa vasorum area and length, which was supported by microCT images. Confocal images provide evidence for vascularized plaque in the saline-treated group but not in rPAI-1(23)-treated mice. The increased vessel density in saline-treated mice is attributable, in part, to upregulated fibroblast growth factor-2 expression, which is inhibited by rPAI-1(23). In conclusion, rPAI-1(23) inhibits growth of vasa vasorum, as well as vessels within the adjacent plaque and vessel wall, through inhibition of fibroblast growth factor-2, leading to reduced plaque growth in atherogenic female LDLR(-/-)ApoB-48-deficient mice.
Asunto(s)
Inhibidores de la Angiogénesis/fisiología , Aterosclerosis/prevención & control , Inhibidor 1 de Activador Plasminogénico/fisiología , Vasa Vasorum/efectos de los fármacos , Inhibidores de la Angiogénesis/farmacología , Animales , Apolipoproteína B-48/genética , Arterias/patología , Aterosclerosis/patología , Femenino , Factor 2 de Crecimiento de Fibroblastos/genética , Ratones , Ratones Endogámicos , Ratones Mutantes , Microscopía Confocal , Fragmentos de Péptidos/farmacología , Fragmentos de Péptidos/fisiología , Inhibidor 1 de Activador Plasminogénico/farmacología , Receptores de LDL/genética , Proteínas Recombinantes/farmacología , Vasa Vasorum/crecimiento & desarrollo , Vasa Vasorum/patología , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
CD163, a monocyte and macrophage-specific surface glycoprotein, which is increased by interleukin-10 and glucocorticoids, is a scavenger receptor for hemoglobin/haptoglobin complexes. We report a rapid and highly reproducible rise in soluble CD163 in the plasma of human volunteers given intravenous lipopolysaccharide (LPS). We also show that LPS induces shedding of CD163 from the surface of isolated monocytes, identifying shedding from monocytes and macrophages as a likely mechanism for the endotoxemia-associated rise in plasma CD163 in vivo. Studies using the inhibitor TAPI-0 indicate that a metalloproteinase is responsible for LPS-mediated shedding of CD163. Finally, we demonstrate a marked increase in surface CD163 expression on circulating monocytes 24 h following experimental endotoxemia. These findings show that CD163 is rapidly mobilized in response to bacterial endotoxin. As hemoglobin can bind LPS and enhance its toxicity, it will be important to determine how cell surface and soluble CD163 influence inflammatory processes during sepsis.
Asunto(s)
Endotoxemia/sangre , Glicoproteínas de Membrana/sangre , Metaloendopeptidasas/antagonistas & inhibidores , Monocitos/inmunología , Receptores Inmunológicos/sangre , Regulación hacia Arriba , Membrana Celular/inmunología , Dipéptidos/farmacología , Endotoxemia/inmunología , Ácidos Hidroxámicos/farmacología , Inyecciones Intravenosas , Lipopolisacáridos/administración & dosificación , Lipopolisacáridos/efectos adversos , Lipopolisacáridos/farmacología , Monocitos/efectos de los fármacos , Receptores Depuradores , Acetato de Tetradecanoilforbol/farmacología , Factores de TiempoRESUMEN
Vascular endothelial growth factor (VEGF) is a regulator of vascularization in development and is a key growth factor in tissue repair. In disease, VEGF contributes to vascularization of solid tumors and arthritic joints. This study examines the role of the mRNA-binding protein AUF1/heterogeneous nuclear ribonucleoprotein D (AUF1) in VEGF gene expression. We show that overexpression of AUF1 in mouse macrophage-like RAW-264.7 cells suppresses endogenous VEGF protein levels. To study 3' untranslated region (UTR)-mediated regulation, we introduced the 3' UTR of VEGF mRNA into a luciferase reporter gene. Coexpression of AUF1 represses VEGF-3' UTR reporter expression in RAW-264.7 cells and in mouse bone marrow-derived macrophages. The C-terminus of AUF1 contains arginine-glycine-glycine (RGG) repeat motifs that are dimethylated. Deletion of the RGG domain of AUF1 eliminated the repressive effects of AUF1. Surprisingly, expression of an AUF1-RGG peptide reduced endogenous VEGF protein levels and repressed VEGF-3' UTR reporter activity in RAW-264.7 cells. These findings demonstrate that AUF1 regulates VEGF expression, and this study identifies an RGG peptide that suppresses VEGF gene expression.
Asunto(s)
Regiones no Traducidas 3' , Ribonucleoproteína Heterogénea-Nuclear Grupo D/metabolismo , Macrófagos/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Células de la Médula Ósea , Regulación de la Expresión Génica , Ribonucleoproteína Nuclear Heterogénea D0 , Ribonucleoproteína Heterogénea-Nuclear Grupo D/biosíntesis , Metilación , Ratones , Péptidos/metabolismo , Unión Proteica/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Eliminación de Secuencia , Factor A de Crecimiento Endotelial Vascular/biosíntesisRESUMEN
Many types of cancer cells require a supply of fatty acids (FA) for growth and survival, and interrupting de novo FA synthesis in model systems causes potent anticancer effects. We hypothesized that, in addition to synthesis, cancer cells may obtain preformed, diet-derived FA by uptake from the bloodstream. This would require hydrolytic release of FA from triglyceride in circulating lipoprotein particles by the secreted enzyme lipoprotein lipase (LPL), and the expression of CD36, the channel for cellular FA uptake. We find that selected breast cancer and sarcoma cells express and secrete active LPL, and all express CD36. We further show that LPL, in the presence of triglyceride-rich lipoproteins, accelerates the growth of these cells. Providing LPL to prostate cancer cells, which express low levels of the enzyme, did not augment growth, but did prevent the cytotoxic effect of FA synthesis inhibition. Moreover, LPL knockdown inhibited HeLa cell growth. In contrast to the cell lines, immunohistochemical analysis confirmed the presence of LPL and CD36 in the majority of breast, liposarcoma, and prostate tumor tissues examined (n = 181). These findings suggest that, in addition to de novo lipogenesis, cancer cells can use LPL and CD36 to acquire FA from the circulation by lipolysis, and this can fuel their growth. Interfering with dietary fat intake, lipolysis, and/or FA uptake will be necessary to target the requirement of cancer cells for FA.
Asunto(s)
Proliferación Celular , Grasas de la Dieta/metabolismo , Ácidos Grasos/metabolismo , Lipoproteína Lipasa/metabolismo , Neoplasias/metabolismo , Animales , Antígenos CD36/genética , Línea Celular Tumoral , Inhibidores de la Síntesis de Ácidos Grasos/farmacología , Ácidos Grasos/farmacología , Femenino , Humanos , Lipólisis , Liposarcoma/genética , Liposarcoma/metabolismo , Masculino , Ratones , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Interferente Pequeño/genéticaRESUMEN
Liposarcomas (LS) are mesenchymal tumors that can recur after surgical resection and often do not respond to presently available medical therapies. This study demonstrates the dependence of LS on de novo long-chain fatty acid synthesis for growth. Lipogenesis can be impaired by inhibiting the activities of lipogenic enzymes, including acetyl CoA-carboxylase (ACC) and fatty acid synthase (FASN), or by suppressing the expression of key genes involved in the pathway and its regulation. The FASN inhibitors cerulenin and orlistat reduced the growth of two LS cell lines (LiSa2, SW872), as did inhibition of ACC with soraphen A. CDDO-Me, a synthetic triterpenoid, suppressed expression of Spot 14 and FASN genes and likewise inhibited LS cell growth. Importantly, the anti-proliferative effect of each agent was prevented by the co-administration of palmitate, the major product of cellular long-chain fatty acid synthesis. In stark contrast to LS cells, these compounds had no effect on the growth of fibroblasts. Four biochemically distinct agents that target critical points in the fatty acid synthetic pathway exert anti-proliferative effects on LS cells, and rescue of cell growth by palmitic acid suggests that reduced tumor cell lipogenesis mediates the growth inhibition. These findings warrant further studies aimed at the clinical exploitation of the dependence of LS cell growth on fatty acids.
Asunto(s)
Ácidos Grasos/química , Regulación Neoplásica de la Expresión Génica , Liposarcoma/metabolismo , Acetil-CoA Carboxilasa/metabolismo , Línea Celular Tumoral , Proliferación Celular , Relación Dosis-Respuesta a Droga , Ácido Graso Sintasas/metabolismo , Fibroblastos/metabolismo , Humanos , Lactonas/farmacología , Lípidos/química , Orlistat , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
AIMS: Given the importance of IgG Fc receptors in immune regulation, we hypothesized that Fcg receptor type III (FcgRIII, CD16) plays an important role in atherogenesis. We therefore analysed the formation of arterial lesions in LDL receptor-deficient (LDLR(-/-)) and FcgRIII(-/-)xLDLR(-/-) double knockout mice at three different points up to 24 weeks of exposure to a high-fat diet. METHODS AND RESULTS: Analysis of Oil Red-O-stained sections revealed no difference in lesion formation between strains after 6 weeks of a high-fat diet, and a modest decrease after 14 weeks in double knockouts relative to LDLR(-/-) controls. After 24 weeks, lesion formation was decreased in the aortic root (30%) and innominate artery (50%) in FcgRIII double knockouts relative to LDLR(-/-) controls. Analysis of peripheral CD4+ T-cells by intracellular flow cytometry from double knockouts after 24 weeks of a high-fat diet revealed statistically significant increases in the percentages of cells producing interferon-gamma, interleukin (IL)-10, and IL-4 relative to controls, differences that were also observed by analyses of whole aortas for cytokine mRNA levels. As determined by flow cytometry, FcgRIII deficiency resulted in an expansion of CD4+ cells and an increase in the CD4 to CD8 ratio. Analysis of plasma anti-oxidized LDL (OxLDL) antibodies by chemiluminescent assay revealed that IgG1 and IgG2c titers to OxLDL were increased in FcgRIII (-/-)xLDLR(-/-) double knockouts relative to LDLR(-/-) controls, while total IgG levels were similar. CONCLUSION: These results reveal altered immunity in FcgRIII(-/-)xLDLR(-/-) mice and a reduction in lesion formation associated with increased production of IL-10 by an expansion of CD4+ T-cells. The reduction in lesion formation was manifest well after evidence of an immune response to OxLDL, suggesting that FcgRIII contributes to lesion progression in murine atherosclerosis.
Asunto(s)
Arterias/patología , Interleucina-10/fisiología , Receptores de IgG/fisiología , Animales , Aterosclerosis/patología , Autoanticuerpos/sangre , Linfocitos T CD4-Positivos/inmunología , Citocinas/biosíntesis , Femenino , Inmunidad , Interferón gamma/fisiología , Lípidos/sangre , Lipoproteínas LDL/inmunología , Masculino , Ratones , Receptores de LDL/deficiencia , Receptores de LDL/fisiologíaRESUMEN
Most breast cancers are "lipogenic", defined by high fatty acid synthase (FAS) content and dependence on fatty acid synthesis for growth and survival. S14 (Spot 14; THRSP) is a nuclear protein that activates genes required for fatty acid synthesis. The S14 gene is amplified in approximately 15% of breast cancers, but clinical correlates of its expression were unknown. We analyzed 131 breast cancers by immunohistochemistry for S14 and FAS. Staining was graded 0, 1, or 2+, and scores were correlated with traditional tumor markers, histological features, and outcome. S14 and FAS staining were related to tumor size (p=0.05 for S14, p=0.038 for FAS), but not to stage. S14 but not FAS scores correlated with tumor grade in both DCIS (p=0.003) and invasive cases (p<0.001). Invasive cases (pooled node - and +) with weak S14 staining (n=21) showed no recurrence over 3000 d follow-up, including 10 cases with lymph node involvement, whereas 32% of 67 strongly-staining tumors recurred (log rank p<0.0001). S14 scores did not cosegregate with sex steroid receptors, Her2/neu, or cyclin D1. Low level S14 expression is associated with prolonged disease-free survival in invasive cases, including those with nodal metastasis. High-level expression of S14 identifies a subset of high-risk breast cancers that is not specified by analysis of sex steroid receptors, Her2/neu, or cyclin D1, and provides a molecular correlate to histologic features that predict recurrence.
Asunto(s)
Biomarcadores de Tumor/análisis , Neoplasias de la Mama/química , Proteínas Nucleares/análisis , Factores de Transcripción/análisis , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Ciclina D1/análisis , Grasas de la Dieta/administración & dosificación , Femenino , Humanos , Inmunohistoquímica , Persona de Mediana EdadRESUMEN
Most breast cancers exhibit brisk lipogenesis, and require it for growth. S14 is a lipogenesis-related nuclear protein that is overexpressed in most breast cancers. Sterol response element-binding protein-1c (SREBP-1c) is required for induction of lipogenesis-related genes, including S14 and fatty acid synthase (FAS), in hepatocytes, and correlation of SREBP-1c and FAS expression suggested that SREBP-1c drives lipogenesis in tumors as well. We directly tested the hypothesis that SREBP-1c drives S14 expression and mediates lipogenic effects of progestin in T47D breast cancer cells. Dominant-negative SREBP-1c inhibited induction of S14 and FAS mRNAs by progestin, while active SREBP-1c induced without hormone and superinduced in its presence. Changes in S14 mRNA were reflected in protein levels. A lag time and lack of progestin response elements indicated that S14 and FAS gene activation by progestin is indirect. Knockdown of S14 reduced, whereas overexpression stimulated, T47D cell growth, while nonlipogenic MCF10a mammary epithelial cells were not growth-inhibited. These data directly demonstrate that SREBP-1c drives S14 gene expression in breast cancer cells, and progestin magnifies that effect via an indirect mechanism. This supports the prediction, based on S14 gene amplification and overexpression in breast tumors, that S14 augments breast cancer cell growth and survival.
Asunto(s)
Neoplasias de la Mama/genética , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Nucleares/genética , Progestinas/farmacología , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/farmacología , Factores de Transcripción/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Mama/metabolismo , Neoplasias de la Mama/metabolismo , Proliferación Celular , Células Cultivadas , Células Epiteliales/metabolismo , Ácido Graso Sintasas , Amplificación de Genes , Genes Dominantes , Humanos , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Elementos de Respuesta , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Activación Transcripcional , Receptor fas/genética , Receptor fas/metabolismoRESUMEN
The cardiac ganglion in the lobster Homarus americanus was examined with a transmission electron microscope. Nerve terminals often existed in large aggregations surrounded by glial and connective tissue elements. Axo-axonic and axo-dendritic synapses were present. Six ultrastructurally different types of nerve terminal, each containing an abundance of vesicles, were distinguished: three formed discrete chemical synapses as indicated by typical release site morphology; three did not. The latter appear to be neurosecretory axon terminals of extrinsic neurons. More than one morphologically distinct type of synaptic vesicle occurred commonly in a given terminal, suggesting the presence of coexisting neurotransmitters and/or neuroregulatory factors. Symmetrical chemical synapses and electrotonic junctions between axons were present.
RESUMEN
To test the possibility that acyl-coenzyme A:cholesterol acyltransferase 2 (ACAT2) may be expressed in human macrophages under pathologic conditions, we employed specific anti-ACAT2 antibodies and found clear ACAT2 signals in lipid-laden as well as lipid-free macrophages under various disease conditions, including atherosclerosis. However, no ACAT2 signal was detectable in macrophages under normal physiologic conditions. Using cultured human macrophages derived from blood-borne monocytes, immunoblot and RT-PCR analyses demonstrated that immature macrophages expressed only ACAT1, but the fully differentiated macrophages expressed both ACAT1 and ACAT2. Furthermore, RT-PCR clearly revealed the presence of both ACAT1 and ACAT2 mRNAs in human atherosclerotic aorta. Double immunohistochemical staining indicated that in human atherosclerotic aorta, all macrophages expressed ACAT1, while approximately 70% to 80% of macrophages also expressed ACAT2. In congenital hyperlipidemic mice, immunohistochemistry and RT-PCR demonstrated that ACAT2 was also present in lipid-laden cells of the atheromatous plaques. Our results suggest that in atherosclerotic plaque, the ability of macrophage foam cell transformation may be augmented by the dual expressions of ACAT1 and ACAT2. Additional immunoblot and RT-PCR experiments showed that the ACAT2 signal was clearly detectable in thioglycollate-elicited exudate mouse macrophages but not in peritoneal resident macrophages. We conclude that under various pathologic conditions, fully differentiated macrophages express ACAT2 in addition to ACAT1.