RESUMEN
OBJECTIVES: To establish a carbofuran intragastric administration death model in rabbits, and to observe the postmortem distribution and postmortem redistribution of carbofuran-7-phenyl glucuronic acid (Glu-7PH) in rabbits. METHODS: The postmortem distribution: Rabbits were given an administration of 1/2LD50, LD50, 2LD50 carbofuran. Dead rabbits were dissected immediately. Rabbits that had remained alive 2 hours were sacrificed by carbon dioxide (CO2) inhalation and dissected immediately. The myocardium, cardiac blood, liver, spleen, lung, kidney, brain and right hindlimb muscle were collected. The postmortem redistribution: After giving an administration of 4LD50 carbofuran, the myocardium, cardiac blood, liver, spleen, lung, kidney, brain, and right hindlimb muscle were collected at 0, 12, 24, 48, and 72 h postmortem in supine position at 15 â room temperature. The quantity of Glu-7PH was determined by LC-MS/MS. RESULTS: The postmortem distribution: Among the three dose groups, there were significant differences in the quantities of Glu-7PH in different tissues. The postmortem redistribution: There was no significant difference in the Glu-7PH quantities in cardiac blood, mycardium, spleen, kidney, brain and right hindlimb muscle, but there was a significant difference in the Glu-7PH quantities in the liver and lung. CONCLUSIONS: The mycardium, cardiac blood, liver, lung, kidney, brain and hindlimb muscle of rabbits can be used as appropriate samples for Glu-7PH detection. However, it should be noted that Glu-7PH was redistributed postmortem in rabbit liver and lung.
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Carbofurano , Animales , Conejos , Cromatografía Liquida , Cambios Post Mortem , Espectrometría de Masas en Tándem , AutopsiaRESUMEN
OBJECTIVE: Our previous study showed that Coiled-Coil Domain Containing 80 (CCDC80) accelerates the development of atherosclerosis by decreasing lipoprotein lipase (LPL) expression and activity in apoE knockout mice. However, the regulatory mechanism for CCDC80 expression is unclear. This study was designed to evaluate whether noncoding RNAs involved the regulation of CCDC80 expression in vascular smooth muscle cells. METHODS AND RESULTS: Bioinformatics prediction and luciferase reporter gene results showed that miR-141-3p/200a-3p bound to the 3'UTR of CCDC80. Furthermore, miR-141-3p/200a-3p mimics decreased the expression of CCDC80 but increased LPL expression. Opposite results were observed with miR-141-3p/200a-3p inhibitors. We also found that lncRNA metastasis-associated lung adenocarcinoma transcript-1 (MALAT1) interacted with the sequences of miR-141-3p/200a-3p and decreased their expression. RT-qPCR and western blotting results showed that MALAT1 overexpression increased CCDC80 expression and decreased LPL expression, while MALAT1 knockdown displayed an opposite phenotype. The effects of both MALAT1 overexpression and knockdown were blocked by miR-141-3p/200a-3p mimics or inhibitors. CONCLUSIONS: Thus, we demonstrated that lncRNA MALAT1 regulates CCDC80 and LPL expression through miR-141-3p/200a-3p.
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Proteínas de la Matriz Extracelular/metabolismo , MicroARNs/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , ARN Largo no Codificante/metabolismo , Sitios de Unión , Células Cultivadas , Proteínas de la Matriz Extracelular/genética , Regulación de la Expresión Génica , Humanos , Lipoproteína Lipasa/genética , Lipoproteína Lipasa/metabolismo , MicroARNs/genética , Regiones Promotoras Genéticas , ARN Largo no Codificante/genéticaRESUMEN
BACKGROUND AND AIMS: Krüppel-like factor 14 (KLF14) is known to play a role in atherosclerosis, but the underlying mechanisms are still largely unknown. The aim of our study was to explore the effects of KLF14 on lipid metabolism and inflammatory response, providing a potential target for lowering the risk of atherosclerosis-causing disease. METHODS AND RESULTS: mRNA and protein levels of KLF14 were significantly decreased in oxidized low-density lipoprotein (oxLDL)-treated macrophages and in the atherosclerotic lesion area. Chromatin immunoprecipitation (ChIP) and luciferase reporter gene assays were used to confirm that KLF14 positively regulated miR-27a expression by binding to its promoter. We also found that KLF14 could restored appropriate cellular lipid homeostasis and inflammatory responses via negatively regulating lipoprotein lipase (LPL) expression in THP1-derived macrophages through miR-27a. In addition, gypenosides (GP), a KLF14 activator, delayed the development of atherosclerosis in apolipoprotein E deficient (apoE-/-) mice. CONCLUSIONS: KLF14 plays an antiatherogenic role via the miR-27a-dependent down-regulation of LPL and subsequent inhibition of proinflammatory cytokine secretion and lipid accumulation.
Asunto(s)
Aterosclerosis/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Lipoproteína Lipasa/metabolismo , MicroARNs/metabolismo , Animales , Aterosclerosis/patología , Regulación hacia Abajo , Regulación Enzimológica de la Expresión Génica , Gynostemma , Homeostasis , Metabolismo de los Lípidos , Lípidos/química , Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , Masculino , Ratones , Ratones Noqueados para ApoE , Extractos Vegetales/farmacología , Células RAW 264.7 , TransfecciónRESUMEN
BACKGROUND: Tanshinone IIA (Tan IIA) and Omentin-1 have a protective role in the cardiovascular system. However, if and how Tan IIA and Omentin-1 regulate cholesterol metabolism in macrophages has not been fully elucidated. OBJECTIVE: To investigate the possible mechanisms of Tan IIA and Omentin-1 on preventing macrophage cholesterol accumulation and atherosclerosis development. METHODS: The effect of Tan IIA on the protein and mRNA levels of Omentin-1 and ATP-binding cassette transporter A1 (ABCA1) in macrophages was examined by Western blot and qRT-PCR assay, respectively. Cholesterol efflux was assessed by liquid scintillation counting (LSC). Cellular lipid droplet was measured by Oil Red O staining, and intracellular lipid content was detected by high performance liquid chromatography (HPLC). In addition, the serum lipid profile of apoE-/- mice was measured by enzymatic method. The size of atherosclerotic lesion areas and content of lipids and collagen in the aortic of apoE-/- mice were examined by Sudan IV, Oil-red O, and Masson staining, respectively. RESULTS: Tan IIA up-regulated expression of Omentin-1 and ABCA1 in THP-1 macrophages, promoting ABCA1-mediated cholesterol efflux and consequently decreasing cellular lipid content. Consistently, Tan IIA increased reverse cholesterol transport in apoE-/- mice. Plasma levels of high-density lipoprotein cholesterol (HDL-C), ABCA1 expression and atherosclerotic plaque collagen content were increased while plasma levels of low-density lipoprotein cholesterol (LDL-C) and atherosclerotic plaque sizes were reduced in Tan IIA-treated apoE-/- mice. These beneficial effects were, however, essentially blocked by knockdown of Omentin-1. CONCLUSION: Our results revealed that Tan IIA promotes cholesterol efflux and ameliorates lipid accumulation in macrophages most likely via the Omentin-1/ABCA1 pathway, reducing the development of aortic atherosclerosis.
Asunto(s)
Transportador 1 de Casete de Unión a ATP/metabolismo , Abietanos/farmacología , Aterosclerosis/tratamiento farmacológico , Colesterol/metabolismo , Citocinas/metabolismo , Proteínas Ligadas a GPI/metabolismo , Lectinas/metabolismo , Macrófagos/efectos de los fármacos , Abietanos/uso terapéutico , Animales , Apolipoproteínas E/genética , Aterosclerosis/metabolismo , Transporte Biológico , Línea Celular Tumoral , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal , Regulación hacia ArribaRESUMEN
Recent studies showed that coiled-coil domain-containing 80 (CCDC80) has a positive link with atherosclerosis and that plasma CCDC80 levels are positively correlated with the levels of fasting plasma triglycerides (TG) in obese individuals. The underlying mechanisms, however, are unclear. Using Hematoxylin-eosin (H&E) and Oil Red O staining, we found that CCDC80 overexpression in vivo significantly increased plasma lipid contents, decreased the expression and activity of lipoprotein lipase (LPL), and accelerated the development of atherosclerosis. Conversely, knockdown of CCDC80 decreased plaque lesions area. In vitro, qRT-PCR and western blot results showed that CCDC80 overexpression significantly decreased, while CCDC80 knockdown increased, LPL expression in cultured vascular smooth muscle cells (VSMCs). Further, we found that CCDC80 reduced LPL expression via inhibiting the phosphorylation of extracellular regulated protein kinase 1/2 (ERK1/2) and also increased the methylation of LPL promoter via down-regulating Tet methylcytosine dioxygenase 2 (TET2). Our results also revealed that CCDC80 significantly down-regulated TET2 expression through decreasing the phosphorylation of ERK1/2. In addition, we found that CCDC80 decreased binding of TET2 to forkhead box O3 (FOXO3a) but had no effect on FOXO3a expression. On the other hand, and that FOXO3a was partially involved in TET2-regulated LPL expression. CCDC80 down-regulated ERK1/2 phosphorylation and decreased expression of TET2 and its interaction with FOXO3a, leading to a reduction of LPL expression and acceleration of atherosclerosis.
Asunto(s)
Aterosclerosis/metabolismo , Proteínas de Unión al ADN/metabolismo , Glicoproteínas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Lipoproteína Lipasa/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Metilación de ADN , Dioxigenasas , Proteínas de la Matriz Extracelular , Proteína Forkhead Box O3/metabolismo , Lipoproteína Lipasa/genética , Masculino , Ratones Noqueados para ApoE , Músculo Liso Vascular/citología , Fosforilación , Triglicéridos/sangreRESUMEN
BACKGROUND: Lipoprotein lipase (LPL) plays an important role in triglyceride metabolism. It is translocated across endothelial cells to reach the luminal surface of capillaries by glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1), where it hydrolyzes triglycerides in lipoproteins. MicroRNA 377 (miR-377) is highly associated with lipid levels. However, how miR-377 regulates triglyceride metabolism and whether it is involved in the development of atherosclerosis remain largely unexplored. MethodsâandâResults: The clinical examination displayed that miR-377 expression was markedly lower in plasma from patients with hypertriglyceridemia compared with non-hypertriglyceridemic subjects. Bioinformatics analyses and a luciferase reporter assay showed that DNA methyltransferase 1 (DNMT1) was a target gene of miR-377. Moreover, miR-377 increased LPL binding to GPIHBP1 by directly targeting DNMT1 in human umbilical vein endothelial cells (HUVECs) and apolipoprotein E (ApoE)-knockout (KO) mice aorta endothelial cells (MAECs). In vivo, hematoxylin-eosin (H&E), Oil Red O and Masson's trichrome staining showed that ApoE-KO mice treated with miR-377 developed less atherosclerotic plaques, accompanied by reduced plasma triglyceride levels. CONCLUSIONS: It is concluded that miR-377 upregulates GPIHBP1 expression, increases the LPL binding to GPIHBP1, and reduces plasma triglyceride levels, likely through targeting DNMT1, inhibiting atherosclerosis in ApoE-KO mice.
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Aorta/metabolismo , Aterosclerosis/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , MicroARNs/metabolismo , Placa Aterosclerótica/metabolismo , Triglicéridos/metabolismo , Animales , Aorta/patología , Aterosclerosis/genética , Aterosclerosis/patología , ADN (Citosina-5-)-Metiltransferasa 1/genética , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Lipoproteína Lipasa/genética , Lipoproteína Lipasa/metabolismo , Ratones , Ratones Noqueados para ApoE , MicroARNs/genética , Placa Aterosclerótica/genética , Placa Aterosclerótica/patología , Receptores de Lipoproteína/biosíntesis , Receptores de Lipoproteína/genéticaRESUMEN
OBJECTIVE: The aim of this study was to determine whether the apolipoprotein A-1 (apoA-1) mimetic peptide ELK-2A2K2E regulates inflammatory cytokine expression through activating the adenosine triphosphate-binding cassette transporter A1 (ABCA1)-janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3)-tristetraprolin (TTP) signaling pathway in THP-1 macrophage-derived foam cells. METHODS AND RESULTS: The cells were treated with the apoA-1 mimetic peptide ELK-2A2K2E at different concentrations (0, 20, 40, and 80 µg/mL) or incubated with ELK-2A2K2E (40 µg/mL) for different times (0, 6, 12, and 24 hours). Our results showed that the levels of the cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1), were decreased at both concentration- and time-dependent manners. When the cells were exposed to lipopolysaccharides and actinomycin D, ELK-2A2K2E significantly decreased the mRNA stability of inflammatory cytokines at different time points (0, 30, 60, and 120 minutes) by increasing TTP expression as analyzed by real-time quantitative polymerase chain reaction. The effect of ELK-2A2K2E on TTP was obviously blocked by the inhibition of the JAK-STAT3 pathway. Furthermore, we found that ELK-2A2K2E activated the JAK-STAT3-TTP pathway through the upregulation of ABCA1 and then decreased inflammatory cytokine expression. CONCLUSIONS: ApoA-I mimetic peptide ELK-2A2K2E increases the degradation of TNF-α, IL-6, and MCP-1 mRNA and reduces the levels of inflammatory cytokines through activating the JAK2-STAT3-TTP signaling pathway that is dependent on the upregulation of ABCA1.
Asunto(s)
Transportador 1 de Casete de Unión a ATP/metabolismo , Antiinflamatorios/farmacología , Apolipoproteína A-I/farmacología , Citocinas/metabolismo , Células Espumosas/efectos de los fármacos , Mediadores de Inflamación/metabolismo , Janus Quinasa 2/metabolismo , Oligopéptidos/farmacología , Factor de Transcripción STAT3/metabolismo , Tristetraprolina/metabolismo , Citocinas/genética , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Células Espumosas/metabolismo , Humanos , Imitación Molecular , Transducción de Señal/efectos de los fármacos , Células THP-1 , Factores de TiempoRESUMEN
BACKGROUND AND AIMS: Recent studies have suggested that heat shock protein 70 (HSP70) may play critical roles in cardiovascular disease. However, the effects of HSP70 on the development of atherosclerosis in apoE-/- mice remain largely unknown. This study was to investigate the role and potential mechanism of HSP70 in atherosclerosis. METHODS: HSP70 was overexpressed in apoE-/- mice and THP-1-derived macrophages with lentiviral vectors. Oil Red O, hematoxylin-eosin, and Masson staining were performed to evaluate atherosclerotic plaque in apoE-/- mice fed the Western type diet. Moreover, immunostaining was employed to detect the expression of relative proteins in aortic sinus. Reporter gene and chromatin immunoprecipitation were performed to analyze the effect of Elk-1 on the promoter activity of ABCA1 and ABCG1; [3H] labeled cholesterol was used to assess the capacity of cholesterol efflux and reverse cholesterol transport (RCT). RESULTS: Our results showed that HSP70 increased lipid accumulation in arteries and promoted the formation of atherosclerotic lesion. The capacity of cholesterol efflux was reduced in peritoneal macrophages isolated from HSP70-overexpressed apoE-/- mice. The levels of ABCA1 and ABCG1 expression were also reduced in the peritoneal macrophages and the aorta from apoE-/- mice in response to HSP70. The c-Jun N-terminal kinase (JNK) and ETS transcription factor (Elk-1) played a critical role in HSP70-induced downregulation ABCA1 and ABCG1. Further, HSP70 reduced RCT from macrophages to plasma, liver, and feces in apoE-/- mice. CONCLUSIONS: HSP70 promotes the progression of atherosclerosis in apoE-/- mice by suppressing the expression of ABCA1 and ABCG1 through the JNK/Elk-1 pathway.
Asunto(s)
Transportador 1 de Casete de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/metabolismo , Aterosclerosis/patología , Proteínas HSP70 de Choque Térmico/metabolismo , Transportador 1 de Casete de Unión a ATP/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/genética , Animales , Aterosclerosis/etiología , Línea Celular , Colesterol/metabolismo , Dieta Occidental/efectos adversos , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Regulación hacia Abajo , Humanos , Sistema de Señalización de MAP Quinasas , Macrófagos , Masculino , Ratones , Ratones Noqueados para ApoE , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patología , Regiones Promotoras Genéticas , Seno Aórtico/metabolismo , Seno Aórtico/patología , Proteína Elk-1 con Dominio ets/metabolismoRESUMEN
BACKGROUND: It has previously been demonstrated that apolipoprotein A-1 (apoA-1) binding protein (AIBP) promotes apoA-1 binding to ATP-binding cassette transporter A1 (ABCA1) and prevents ABCA1 protein degradation so as to inhibit foam cell formation. Because apoA-1 inhibits inflammatory signaling pathways, whether AIBP has an inhibitory effect on inflammatory signaling pathways in THP-1-derived macrophages is investigated.MethodsâandâResults:Analysis of inflammation-related gene expression indicated that AIBP decreased lipopolysaccharide (LPS)-mediated macrophage inflammation. AIBP significantly prevented NF-κB nuclear translocation. Further, AIBP prevented the activation of mitogen-activated protein kinases (MAPKs), including p38 MAPK, extracellular-signal regulated kinase and c-Jun N-terminal kinase. AIBP decreased MyD88 expression at both mRNA and protein levels, but did not have any effect on TLR4 expression. Moreover, treatment with both AIBP and apoA-1 decreased the abundance of TLR4 in the lipid raft fraction. AIBP lacking 115-123 amino acids (∆115-123), however, did not have such effects as described for intact AIBP. In addition, knockdown of ABCA1 inhibited the effects of AIBP on inflammatory factor secretion. CONCLUSIONS: These results suggest that AIBP inhibits inflammatory signaling pathways through binding to apoA-1 and stabilizing ABCA1, and subsequent alteration of lipid rafts and TLR4 in the cell membrane.
Asunto(s)
Apolipoproteína A-I/metabolismo , Proteínas Portadoras/metabolismo , Células Espumosas/metabolismo , Sistema de Señalización de MAP Quinasas , Microdominios de Membrana/metabolismo , Transportador 1 de Casete de Unión a ATP/metabolismo , Proteínas de Unión al ADN , Células Espumosas/patología , Células HEK293 , Humanos , Inflamación/metabolismo , Inflamación/patología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Microdominios de Membrana/patología , Células THP-1 , Receptor Toll-Like 4/metabolismo , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
BACKGROUND AND AIMS: ApoA-1 binding protein (AIBP) is a secreted protein that interacts with apoA-I and accelerates cholesterol efflux from cells. We have recently reported that AIBP promotes apoA-1 binding to ABCA1 in the macrophage cell membrane, partially through 115-123 amino acids. However, the effects of AIBP on the development of atherosclerosis in vivo remain unknown. METHODS: ApoE-/- mice with established atherosclerotic plaques were infected with rAAV-AIBP or rAAV-AIBP(Δ115-123), respectively. RESULTS: AIBP-treated mice showed reduction of atherosclerotic lesion formation, increase in circulating HDL levels and enhancement of reverse cholesterol transport to the plasma, liver, and feces. AIBP increased ABCA1 protein levels in aorta and peritoneal macrophages. Furthermore, AIBP could diminish atherosclerotic plaque macrophage content and the expression of chemotaxis-related factors. In addition, AIBP prevented macrophage inflammation by inactivating NF-κB and promoted the expression of M2 markers like Mrc-1 and Arg-1. However, lack of 115-123 amino acids of AIBP(Δ115-123) had no such preventive effects on the progression of atherosclerosis. CONCLUSIONS: Our observations demonstrate that AIBP inhibits atherosclerosis progression and suggest that it may be an effective target for prevention of atherosclerosis.
Asunto(s)
Apolipoproteínas E/fisiología , Aterosclerosis/prevención & control , Colesterol/metabolismo , Proteínas de Unión al ADN/uso terapéutico , Inflamación/metabolismo , Animales , Apolipoproteínas E/genética , Aterosclerosis/genética , Transporte Biológico/efectos de los fármacos , Proteínas de Unión al ADN/farmacología , Proteínas de Unión al ADN/fisiología , Masculino , RatonesRESUMEN
AIMS: Atherosclerosis is the most common cause of cardiovascular disease, such as myocardial infarction and stroke. Previous study revealed that microRNA (miR)-134 promotes lipid accumulation and proinflammatory cytokine secretion through angiopoietin-like 4 (ANGPTL4)/lipid lipoprotein (LPL) signaling in THP-1 macrophages. METHODS: ApoE KO male mice on a C57BL/6 background were fed a high-fat/high-cholesterol Western diet, from 8 to 16 weeks of age. Mice were divided into four groups, and received a tail vein injection of miR-134 agomir, miR-134 antagomir, or one of the corresponding controls, respectively, once every 2 weeks after starting the Western diet. After 8 weeks we measured aortic atherosclerosis, LPL Activity, mRNA and protein levels of ANGPTL4 and LPL, LPL/ low-density lipoprotein receptor related protein 1 Complex Formation, proinflammatory cytokine secretion and lipid levels. RESULTS: Despite this finding, the influence of miR-134 on atherosclerosis in vivo remains to be determined. Using the well-characterized mouse atherosclerosis model of apolipoprotein E knockout, we found that systemic delivery of miR-134 agomir markedly enhanced the atherosclerotic lesion size, together with a significant increase in proinflammatory cytokine secretion and peritoneal macrophages lipid contents. Moreover, overexpression of miR-134 decreased ANGPTL4 expression but increased LPL expression and activity in both aortic tissues and peritoneal macrophages, which was accompanied by increased formation of LPL/low-density lipoprotein receptor-related protein 1 complexes in peritoneal macrophages. However, an opposite effect was observed in response to miR-134 antagomir. CONCLUSIONS: These findings suggest that miR-134 accelerates atherogenesis by promoting lipid accumulation and proinflammatory cytokine secretion via the ANGPTL4/LPL pathway. Therefore, targeting miR-134 may offer a promising strategy for the prevention and treatment of atherosclerotic cardiovascular disease.
Asunto(s)
Proteína 4 Similar a la Angiopoyetina/sangre , Proteína 4 Similar a la Angiopoyetina/genética , Aterosclerosis/genética , MicroARNs/sangre , MicroARNs/genética , Animales , Aterosclerosis/metabolismo , Colesterol/metabolismo , Citocinas/metabolismo , Células Espumosas/metabolismo , Inflamación , Lípidos/química , Lipoproteína Lipasa/metabolismo , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados para ApoERESUMEN
BACKGROUND: Lipoprotein lipase (LPL) expressed in macrophages plays an important role in promoting the development of atherosclerosis or atherogenesis. MicroRNA-182 (miR-182) is involved in the regulation of lipid metabolism and inflammation. However, it remains unclear how miR-182 regulates LPL and atherogenesis.MethodsâandâResults:Using bioinformatics analyses and a dual-luciferase reporter assay, we identified histone deacetylase 9 (HDAC9) as a target gene of miR-182. Moreover, miR-182 upregulated LPL expression by directly targetingHDAC9in THP-1 macrophages. Hematoxylin-eosin (H&E), Oil Red O and Masson's trichrome staining showed that apolipoprotein E (ApoE)-knockout (KO) mice treated with miR-182 exhibited more severe atherosclerotic plaques. Treatment with miR-182 increased CD68 and LPL expression in atherosclerotic lesions in ApoE-KO mice, as indicated by double immunofluorescence staining in the aortic sinus. Increased miR-182-induced increases in LPL expression in ApoE-KO mice was confirmed by real-time quantitative polymerase chain reaction and western blotting analyses. Treatment with miR-182 also increased plasma concentrations of proinflammatory cytokines and lipids in ApoE-KO mice. CONCLUSIONS: The results of the present study suggest that miR-182 upregulates LPL expression, promotes lipid accumulation in atherosclerotic lesions, and increases proinflammatory cytokine secretion, likely through targetingHDAC9, leading to an acceleration of atherogenesis in ApoE-KO mice.
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Aterosclerosis/inducido químicamente , Lipoproteína Lipasa/efectos de los fármacos , MicroARNs/farmacología , Proteínas Represoras/antagonistas & inhibidores , Animales , Biología Computacional , Citocinas/efectos de los fármacos , Células HEK293 , Histona Desacetilasas , Humanos , Inflamación/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Macrófagos , Ratones , Ratones Noqueados para ApoE , Células THP-1RESUMEN
It was reported that puerarin decreases the total cholesterol, low-density lipoprotein cholesterol (LDL-C), triglyceride (TG) and increases high-density lipoprotein cholesterol (HDL-C) level, but the underlying mechanism is unclear. This study was designed to determine whether puerarin decreased lipid accumulation via up-regulation of ABCA1-mediated cholesterol efflux in THP-1 macrophage-derived foam cells. Our results showed that puerarin significantly promoted the expression of ATP-binding cassette transporter A1 (ABCA1) mRNA and protein via the AMP-activated protein kinase (AMPK)-peroxisome proliferator-activated receptor gamma (PPARγ)-liver X receptor-alpha (LXR-α) pathway and decreased cellular lipid accumulation in human THP-1 macrophage-derived foam cells. The miR-7 directly targeted 3' untranslated region of STK11 (Serine/Threonine Kinase 11), which activated the AMPK pathway. Transfection with miR-7 mimic significantly reduced STK11 expression in puerarin-treated macrophages, decreased the phosphorylation of AMPK, down-regulated the expression of the PPARγ-LXR-α-ABCA1 expression. Additionally, treatment with miR-7 decreased cholesterol efflux and increased cholesterol levels in THP-1 macrophage-derived foam cells. Our study demonstrates that puerarin promotes ABCA1-mediated cholesterol efflux and decreases intracellular cholesterol levels through the pathway involving miR-7, STK11, and the AMPK-PPARγ-LXR-α-ABCA1 cascade.
Asunto(s)
Transportador 1 de Casete de Unión a ATP/metabolismo , Colesterol/metabolismo , Isoflavonas/farmacología , Macrófagos/metabolismo , Quinasas de la Proteína-Quinasa Activada por el AMP , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Receptores X del Hígado/genética , Receptores X del Hígado/metabolismo , Macrófagos/citología , Macrófagos/efectos de los fármacos , MicroARNs/genética , MicroARNs/metabolismo , PPAR gamma/genética , PPAR gamma/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Atherosclerotic lesions are characterized by the accumulation of abundant lipids and chronic inflammation. Previous researches have indicated that macrophage-derived lipoprotein lipase (LPL) promotes atherosclerosis progression by accelerating lipid accumulation and pro-inflammatory cytokine secretion. Although apelin-13 has been regarded as an atheroprotective factor, it remains unclear whether it can regulate the expression of LPL. The aim of this study was to explore the effects of apelin-13 on the expression of LPL and the underlying mechanism in THP-1 macrophage-derived foam cells. Apelin-13 significantly decreased cellular levels of total cholesterol, free cholesterol, and cholesterol ester at the concentrations of 10 and 100 nM. ELISA analysis confirmed that treatment with apelin-13 reduced pro-inflammatory cytokine secretion, such as interleukin-6 (IL-6), interleukin-1ß (IL-1ß) and tumor necrosis factor-alpha (TNF-α). It was also found that apelin-13 inhibited the expression of LPL as revealed by western blot and real-time PCR analyses. Bioinformatics analyses and dual-luciferase reporter assay indicated that miR-361-5p directly downregulated the expression of LPL by targeting the 3'UTR of LPL. In addition, apelin-13 + miR-361-5p mimic significantly downregulated the expression of LPL in cells. Finally, we demonstrated that apelin-13 downregulated the expression of LPL through activating the activity of PKCα. Taken together, our results showed that apelin-13 downregulated the expression of LPL via activating the APJ/PKCα/miR-361-5p signaling pathway in THP-1 macrophage-derived foam cells, leading to inhibition of lipid accumulation and pro-inflammatory cytokine secretion. Therefore, our studies provide important new insight into the inhibition of lipid accumulation and pro-inflammatory cytokine secretion by apelin-13, and highlight apelin-13 as a promising therapeutic target in atherosclerosis.
Asunto(s)
Células Espumosas/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Lipoproteína Lipasa/genética , Macrófagos/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Regiones no Traducidas 3'/genética , Receptores de Apelina/metabolismo , Línea Celular Tumoral , Citocinas/metabolismo , Células Espumosas/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Lipoproteína Lipasa/metabolismo , Macrófagos/metabolismo , MicroARNs/genética , Proteína Quinasa C-alfa/genética , Proteína Quinasa C-alfa/metabolismo , Interferencia de ARN , Transducción de Señal/genéticaRESUMEN
Atherosclerotic lesions are lipometabolic disorder characterized by chronic progressive inflammation in arterial walls. Previous studies have shown that macrophage-derived lipoprotein lipase (LPL) might be a key factor that promotes atherosclerosis by accelerating lipid accumulation and proinflammatory cytokine secretion. Increasing evidence indicates that microRNA-27 (miR-27) has beneficial effects on lipid metabolism and inflammatory response. However, it has not been fully understood whether miR-27 affects the expression of LPL and subsequent development of atherosclerosis in apolipoprotein E knockout (apoE KO) mice. To address these questions and its potential mechanisms, oxidized low-density lipoprotein (ox-LDL)-treated THP-1 macrophages were transfected with the miR-27 mimics/inhibitors and apoE KO mice fed high-fat diet were given a tail vein injection with miR-27 agomir/antagomir, followed by exploring the potential roles of miR-27. MiR-27 agomir significantly down-regulated LPL expression in aorta and peritoneal macrophages by western blot and real-time PCR analyses. We performed LPL activity assay in the culture media and found that miR-27 reduced LPL activity. ELISA showed that miR-27 reduced inflammatory response as analyzed in vitro and in vivo experiments. Our results showed that miR-27 had an inhibitory effect on the levels of lipid both in plasma and in peritoneal macrophages of apoE KO mice as examined by HPLC. Consistently, miR-27 suppressed the expression of scavenger receptors associated with lipid uptake in ox-LDL-treated THP-1 macrophages. In addition, transfection with LPL siRNA inhibited the miR-27 inhibitor-induced lipid accumulation and proinflammatory cytokines secretion in ox-LDL-treated THP-1 macrophages. Finally, systemic treatment revealed that miR-27 decreased aortic plaque size and lipid content in apoE KO mice. The present results provide evidence that a novel antiatherogenic role of miR-27 was closely related to reducing lipid accumulation and inflammatory response via downregulation of LPL gene expression, suggesting a potential strategy to the diagnosis and treatment of atherosclerosis.
Asunto(s)
Apolipoproteínas E/deficiencia , Aterosclerosis/metabolismo , Aterosclerosis/prevención & control , Inflamación/metabolismo , Lipoproteína Lipasa/farmacocinética , MicroARNs/metabolismo , Animales , Apolipoproteínas E/genética , Aterosclerosis/sangre , Aterosclerosis/genética , Línea Celular , Quimiocina CCL2/sangre , Cromatografía Líquida de Alta Presión , Ensayo de Inmunoadsorción Enzimática , Humanos , Inflamación/sangre , Inflamación/genética , Interleucina-1beta/sangre , Interleucina-6/sangre , Metabolismo de los Lípidos/genética , Metabolismo de los Lípidos/fisiología , Lipoproteína Lipasa/genética , Macrófagos/metabolismo , Masculino , Ratones , Ratones Noqueados , MicroARNs/genética , Células RAW 264.7 , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Depuradores/metabolismo , Factor de Necrosis Tumoral alfa/sangreRESUMEN
RATIONALE: Previous studies have shown that apolipoprotein-1 (apoA-1) binding protein (AIBP) is highly associated with the regulation of apoA-1 metabolism, suggesting its role in the treatment of atherosclerosis. However, how AIBP regulates foam cell formation remains largely unexplored. OBJECTIVE: To investigate the mechanisms underlying AIBP inhibition of foam cell formation from macrophages. METHODS AND RESULTS: THP-1-derived macrophages were incubated without or with apoA-1 and AIBP, followed by assessing the formation of foam cells and the potential mechanisms. Our results showed that AIBP and apoA-1 enhanced cholesterol efflux, altered the levels of cellular free cholesterol and cholesterol ester and prevented lipid accumulation so as to reduce the formation of foam cells. Meanwhile, lack of AIBP 115-123 amino acids resulted in the loss of AIBP binding to apoA-1. Moreover, our chemiluminescent analysis showed that AIBP promoted biotin-labeled apoA-1 binding to macrophages. Besides with AIBP, more apoA-1 bound to ABCA1, a key transporter responsible for cholesterol efflux to apoA-1, as indicated by our co-immunoprecipitation assay. Our results also showed that AIBP did not regulate ABCA1 mRNA expression, but stabilized its protein from CSN2-mediated degradation. CONCLUSIONS: AIBP promotes apoA-1 binding to ABCA1 on the cell membrane of macrophages and prevents ABCA1 protein from CSN2-mediated degradation so as to prevent foam cell formation. AIBP 115-123 amino acids is at least partially responsible for its binding to apoA-1.
Asunto(s)
Transportador 1 de Casete de Unión a ATP/metabolismo , Apolipoproteína A-I/metabolismo , Colesterol/metabolismo , Macrófagos/citología , Transportador 1 de Casete de Unión a ATP/genética , Animales , Apolipoproteína A-I/genética , Aterosclerosis/metabolismo , Transporte Biológico , Biotina/química , Complejo del Señalosoma COP9 , Línea Celular , Línea Celular Tumoral , Membrana Celular/metabolismo , Cromatografía Líquida de Alta Presión , Cricetinae , Células Espumosas/citología , Humanos , Macrófagos/metabolismo , Ratones , Unión Proteica , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Represoras/metabolismo , UbiquitinaciónRESUMEN
Macrophage-activating lipopeptide-2 (MALP-2) has been shown to promote the development of atherosclerosis. ATP-binding cassette transporter A1 (ABCA1), a transmembrane protein, plays a critical role in mediating cholesterol export from macrophages to apolipoprotein A-I (apoA-I). However, whether MALP-2 can regulate the expression of ABCA1 is still largely unknown. The aim of this study was to explore the effects of MALP-2 on ABCA1 expression in THP-1 macrophages and the underlying mechanisms. Our results showed that the treatment of cells with MALP-2 decreased ABCA1 level and suppressed cholesterol efflux in both concentration- and time-dependent manners. The contents of intracellular cholesterol were significantly increased in the presence of MALP-2. Moreover, MALP-2-mediated inhibition of ABCA1 expression was abolished by siRNA of either Toll-like receptor 2 (TLR2) or nuclear factor κB (NF-κB). A similar effect was produced by treatment with the NF-κB inhibitor pyrrolidine dithiocarbamate. In addition, MALP-2-induced activation of NF-κB markedly increased zinc finger protein 202 (ZNF202) level, and ZNF202 siRNA impaired the effects of MALP-2 on ABCA1 expression. Taken together, these results suggest that MALP-2 can decrease ABCA1 expression and subsequent cholesterol efflux through activation of the TLR2/NF-κB/ZNF202 signaling pathway in THP-1 macrophages.
Asunto(s)
Transportador 1 de Casete de Unión a ATP/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Lipopéptidos/farmacología , Macrófagos/metabolismo , FN-kappa B/metabolismo , Proteínas Represoras/metabolismo , Receptor Toll-Like 2/metabolismo , Transporte Biológico , Línea Celular , Colesterol/metabolismo , HumanosRESUMEN
This study was designed to evaluate whether CSE/H2S system, which is regulated by miR-216a, regulated ABCA1-mediated cholesterol efflux and cholesterol contents in THP-1 macrophages-derived foam cells. Our qPCR and western blotting results showed that CSE/H2S significantly up-regulated the expression of ATP-binding cassette transporter A1 (ABCA1) mRNA and protein via PI3K/AKT pathway in foam cells derived from human THP-1 macrophages. The miR-216a directly targeted 3' untranslated region of CSE. It significantly reduced CSE and ABCA1 expression, and also decreased the phosphorylation of PI3K and AKT. Additionally, cholesterol efflux decreased, and cholesterol levels increased in THP-1 macrophage-derived foam cells in response to treatment with miR-216a. Our study demonstrates that CSE/H2S system is regulated by miR-216a, and regulates ABCA1-mediated cholesterol efflux and cholesterol levels through the PI3K/AKT pathway.