RESUMO
Electronegative low-density lipoprotein (LDL(-)) has been found in the plasma of familial hypercholesterolemia and acute myocardial infarction and has been implicated in atherosclerosis and cardiovascular disease. However, less is known about the involvement of LDL(-) in atherosclerosis-related inflammation. This study aims at investigating the inducibility of LDL(-) by atherogenic diet in rabbits and at exploring the proinflammatory potential of the diet-induced LDL(-) in macrophages. Rabbits were fed with an atherogenic diet; LDL was isolated from plasma by NaBr density gradient ultracentrifugation and was then resolved into nLDL and LDL(-) by anion-exchange chromatography. Isolated nLDL and LDL(-) were directly used or incubated with 10 µM CuSO4 for 24 h to produce copper- (Cu-) ox-nLDL and Cu-ox-LDL(-). The effects of these LDLs on inflammation were evaluated in THP-1-derived macrophages. Macrophages were treated with nLDL, LDL(-), and extensively oxidized LDL (ox-LDL), then the levels of interleukin- (IL-) 1ß, IL-6, and tumor necrosis factor- (TNF-) α in a culture medium were determined by ELISA, and the levels of total and phosphorylated IκB, p65, p38, JNK, and ERK in cell lysates were determined by Western blotting. The LDL(-) induced significantly higher levels of IL-1ß, IL-6, and TNF-α in the medium. The levels of phosphorylated/total IκB, p65, p38, JNK, and ERK were also upregulated by LDL(-). In contrast, nLDL, Cu-ox-nLDL, and Cu-ox-LDL(-) exhibited much less effect. Knockdown of lectin-type oxidized LDL receptor- (LOX-) 1 resulted in significant reduction in LDL(-)-induced IL-1ß, IL-6, and TNF-α. In addition, these LDL(-) effects were also markedly attenuated by inhibition of NF-κB and ERK1/2. The data suggested that LDL(-) induced inflammation through LOX-1-, NF-κB-, and ERK1/2-dependent pathways. Taken together, our results show that rabbits fed with atherogenic diet produce a highly proinflammatory LDL(-) that is more potent in inducing inflammation than nLDL and extensively oxidize LDL in macrophages. The results thus provide a novel link between diet-induced hypercholesterolemia and inflammation.
Assuntos
Dieta Aterogênica/efeitos adversos , Interleucina-1beta/sangue , Lipoproteínas LDL/sangue , Macrófagos/metabolismo , Animais , Western Blotting , Linhagem Celular Tumoral , Humanos , Interleucina-6/sangue , Masculino , NF-kappa B/sangue , Coelhos , Células THP-1 , Fator de Necrose Tumoral alfa/sangueRESUMO
BACKGROUND: Homocysteine has been long considered a risk factor for atherosclerosis. However, cardiovascular events cannot be reduced through homocysteine lowering by B vitamin supplements. Although several association studies have reported an elevation of serum homocysteine levels in cardiovascular diseases, the relationship of homocysteine with ST-segment elevation myocardial infarction (STEMI) is not well established. METHODS: We prospectively enrolled STEMI patients who were consecutively admitted to an intensive care unit following coronary intervention in a single medical center in Taiwan. Control subjects were individuals who presented to the outpatient or emergency department with acute chest pain but subsequently revealed patent coronary arteries by coronary arteriography. The association between serum homocysteine levels and STEMI was investigated. A culture system using human coronary artery endothelial cells was also established to examine the toxic effects of homocysteine at the cellular level. RESULTS: Patients with chest pain were divided into two groups. The STEMI group included 56 patients who underwent a primary percutaneous coronary intervention. The control group included 17 subjects with patent coronary arteries. There was no difference in serum homocysteine levels (8.4 ± 2.2 vs. 7.6 ± 1.9 µmol/L, p = 0.142). When stratifying STEMI patients by the Killip classification into higher (Killip III-IV) and lower (Killip I-II) grades, CRP (3.3 ± 4.1 vs. 1.4 ± 2.3 mg/L, p = 0.032), peak creatine kinase (3796 ± 2163 vs. 2305 ± 1822 IU/L, p = 0.023), and SYNTAX scores (20.4 ± 11.1 vs. 14.8 ± 7.6, p = 0.033) were significantly higher in the higher grades, while serum homocysteine levels were similar. Homocysteine was not correlated with WBCs, CRP, or the SYNTAX score in STEMI patients. In a culture system, homocysteine at even a supraphysiological level of 100 µmol/L did not reduce the cell viability of human coronary artery endothelial cells. CONCLUSIONS: Homocysteine was not elevated in STEMI patients regardless of Killip severity, suggesting that homocysteine is a bystander instead of a causative factor of STEMI. Our study therefore supports the current notion that homocysteine-lowering strategies are not essential in preventing cardiovascular disease.
Assuntos
Homocisteína/sangue , Infarto do Miocárdio com Supradesnível do Segmento ST/sangue , Idoso , Biomarcadores/sangue , Estudos de Casos e Controles , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/patologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Feminino , Homocisteína/toxicidade , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Infarto do Miocárdio com Supradesnível do Segmento ST/diagnóstico , TaiwanRESUMO
L5-LDL, the most electronegative LDL associated with major cardiovascular risks, significantly rises in patients with ST-segment elevation myocardial infarction (STEMI). The inflammatory nature of atherosclerotic vascular diseases has prompted us to investigate whether L5-LDL induces the production of inflammatory cytokines, especially vascular ischemia-related interleukin (IL)-1ß, in the pathogenesis of STEMI. Clinical data showed that plasma levels of L5-LDL and IL-1ß were higher in the STEMI patients than in the controls (P < 0.05). In THP-1-derived human macrophages, L5-LDL significantly increased the levels of both IL-1ß and cleaved caspase-1, indicating the activation of NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasomes by L5-LDL. Knockdown of NLRP3 and its adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) resulted in decreased L5-LDL-induced IL-1ß. Furthermore, knock down of the lectin-type oxidized LDL receptor (LOX-1) in THP-1 cells attenuated L5-LDL-induced activation of NF-κB and caspase-1, leading to subsequent inhibition of IL-1ß in macrophages. Furthermore, blockade LOX-1 with neutralizing antibody also inhibited L5-LDL-induced IL-1ß in human peripheral blood mononuclear cell-derived macrophages. In conclusion, L5-LDL induces IL-1ß production in macrophages by activation of NF-κB and caspase-1 through the LOX-1-dependent pathway. This study represents the evidence linking L5-LDL and the inflammatory cytokine IL-1ß in STEMI, and identifies L5-LDL as a novel therapeutic target in acute myocardial infarction. NEW & NOTEWORTHY: This study represents the evidence linking L5-LDL and the inflammatory cytokine IL-1ß in ST-segment elevation myocardial infarction (STEMI). We elucidate the molecular mechanism underlying L5-LDL-induced production of IL-1ß in macrophages. The results showed that L5-LDL induced activation of caspase-1 and NF-κB through the lectin-type oxidized LDL receptor (LOX-1)-dependent pathway, leading to the production of IL-1ß.
Assuntos
Interleucina-1beta/imunologia , Lipoproteínas LDL/imunologia , Macrófagos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Infarto do Miocárdio com Supradesnível do Segmento ST/imunologia , Receptores Depuradores Classe E/imunologia , Western Blotting , Proteínas Adaptadoras de Sinalização CARD , Caspase 1/imunologia , Linhagem Celular , Proteínas do Citoesqueleto/genética , Técnicas de Silenciamento de Genes , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Humanos , Inflamassomos/imunologia , Interleucina-1beta/genética , Interleucina-6/genética , Interleucina-6/imunologia , Leucócitos Mononucleares , NF-kappa B/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Interferência de RNA , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Infarto do Miocárdio com Supradesnível do Segmento ST/genética , Receptores Depuradores Classe E/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
BACKGROUND: Granulocyte-colony stimulating factor (G-CSF) is a major regulator of the production and survival of neutrophils. Regulation of G-CSF expression is complex and occurs at both transcription and post-transcription levels. Two distinct types of cis-acting elements in the 3' untranslated region (3'UTR) of G-CSF mRNA have been identified as destabilizing elements; these consist of adenylate uridylate-rich elements (AUREs) and a stem-loop destabilizing element (SLDE). Regulation of the stability of mRNA by p38 mitogen-activated protein kinase (MAPK) has been indicated to be linked to AUREs in the 3'UTR. However, whether p38 MAPK is involved in the regulation of the stability of G-CSF mRNA has not been elucidated. This study investigated the effect of SB203580, an inhibitor of p38 MAPK, on the lipopolysaccharide-induced G-CSF expression in macrophages at the post-transcription level. RESULTS: Our study showed surprising results that SB203580 augmented the lipopolysaccharide-induced increase in the G-CSF mRNA levels in RAW264.7 mouse macrophages, mouse bone marrow-derived macrophages and in THP-1 human macrophages. This effect was also seen in p38α MAPK knockdown RAW264.7 cells, showing that it was not due to inhibition of p38 MAPK activity. In the presence of actinomycin D, the decay of G-CSF mRNA was slower in SB203580-treated cells than in control cells, showing that SB203580 increased the stability of G-CSF mRNA. Reporter genes containing luciferase with or without the 3'UTR of G-CSF were constructed and transfected into RAW264.7 cells and the results showed that the presence of the 3'UTR reduced the luciferase mRNA levels and luciferase activity. Furthermore, SB203580 increased the luciferase mRNA levels and activity in RAW264.7 cells transfected with the luciferase reporter containing the 3'UTR, but not in cells transfected with the luciferase reporter without the 3'UTR. Mutations of the highly conserved SLDE in the 3'UTR abolished these effects, showing that the SLDE was essential for the SB203580-induced increase in the stability of mRNA. CONCLUSIONS: SB203580 increases G-CSF expression in macrophages by increasing the stability of G-CSF mRNA via its 3'UTR, and the effect was not due to its inhibition of p38 MAPK activity. The results of this study also highlight a potential target for boosting endogenous production of G-CSF during neutropenia.
Assuntos
Regiões 3' não Traduzidas/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/biossíntese , Imidazóis/farmacologia , Piridinas/farmacologia , Dobramento de RNA/efeitos dos fármacos , Estabilidade de RNA/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Linhagem Celular , Fator Estimulador de Colônias de Granulócitos/genética , Humanos , Camundongos , Dobramento de RNA/genética , Estabilidade de RNA/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
Chronic inflammation, coupled with alcohol, betel quid, and cigarette consumption, is associated with oral squamous cell carcinoma (OSCC). Interleukin-1 beta (IL-1ß) is a critical mediator of chronic inflammation and implicated in many cancers. In this study, we showed that increased pro-IL-1ß expression was associated with the severity of oral malignant transformation in a mouse OSCC model induced by 4-Nitroquinolin-1-oxide (4-NQO) and arecoline, two carcinogens related to tobacco and betel quid, respectively. Using microarray and quantitative PCR assay, we showed that pro-IL-1ß was upregulated in human OSCC tumors associated with tobacco and betel quid consumption. In a human OSCC cell line TW2.6, we demonstrated nicotine-derived nitrosamine ketone (NNK) and arecoline stimulated IL-1ß secretion in an inflammasome-dependent manner. IL-1ß treatment significantly increased the proliferation and dysregulated the Akt signaling pathways of dysplastic oral keratinocytes (DOKs). Using cytokine antibodies and inflammation cytometric bead arrays, we found that DOK and OSCC cells secreted high levels of IL-6, IL-8, and growth-regulated oncogene-α following IL-1ß stimulation. The conditioned medium of IL-1ß-treated OSCC cells exerted significant proangiogenic effects. Crucially, IL-1ß increased the invasiveness of OSCC cells through the epithelial-mesenchymal transition (EMT), characterized by downregulation of E-cadherin, upregulation of Snail, Slug, and Vimentin, and alterations in morphology. These findings provide novel insights into the mechanism underlying OSCC tumorigenesis. Our study suggested that IL-1ß can be induced by tobacco and betel quid-related carcinogens, and participates in the early and late stages of oral carcinogenesis by increasing the proliferation of dysplasia oral cells, stimulating oncogenic cytokines, and promoting aggressiveness of OSCC.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Transformação Celular Neoplásica/metabolismo , Regulação Neoplásica da Expressão Gênica , Interleucina-1beta/metabolismo , Neoplasias Bucais/metabolismo , Animais , Arecolina/farmacologia , Transformação Celular Neoplásica/efeitos dos fármacos , Células Cultivadas , Humanos , Queratinócitos/citologia , CamundongosRESUMO
BACKGROUND/PURPOSE: The aim of this study is to comprehensively analyze the potential factors affecting the failure rates of three types of mini-implants used for orthodontic anchorage. METHODS: Data were collected on 727 mini-implants (miniplates, predrilled titanium miniscrews, and self-drilling stainless steel miniscrews) in 220 patients. The factors related to mini-implant failure were investigated using a Chi-square test for univariate analysis and a generalized estimating equation model for multivariate analysis. RESULTS: The failure rate for miniplates was significantly lower than for miniscrews. All types of mini-implants, especially the self-drilling stainless steel miniscrews, showed decreased stability if the previous implantation had failed. The stability of predrilled titanium miniscrews and self-drilling stainless steel miniscrews were comparable at the first implantation. However, the failure rate of stainless steel miniscrews increased at the second implantation. The univariate analysis showed that the following variables had a significant influence on the failure rates of mini-implants: age of patient, type of mini-implant, site of implantation, and characteristics of the soft tissue around the mini-implants. The generalized estimating equation analysis revealed that mini-implants with miniscrews used in patients younger than 35 years, subjected to orthodontic loading after 30 days and implanted on the alveolar bone ridge, have a significantly higher risk of failure. CONCLUSION: This study revealed that once the dental surgeon becomes familiar with the procedure, the stability of orthodontic mini-implants depends on the type of mini-implant, age of the patient, implantation site, and the healing time of the mini-implant. Miniplates are a more feasible anchorage system when miniscrews fail repeatedly.
Assuntos
Processo Alveolar/cirurgia , Implantes Dentários/normas , Análise do Estresse Dentário , Falha de Equipamento/estatística & dados numéricos , Procedimentos de Ancoragem Ortodôntica/normas , Adulto , Distribuição de Qui-Quadrado , Feminino , Humanos , Masculino , Análise Multivariada , Estudos Retrospectivos , TaiwanRESUMO
Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is an RNA-dependent RNA polymerase (RdRp) that acts as a key player in the HCV replication complex. Understanding the interplay between the viral and cellular components of the HCV replication complex could provide new insight for prevention of the progression of HCV-associated hepatocellular carcinoma (HCC). In this study, the NS5B protein was used as the bait in a pulldown assay to screen for NS5B-interacting proteins that are present in Huh7 hepatoma cell lysates. After mass spectrophotometric analysis, fatty acid synthase (FASN) was found to interact with NS5B. Coimmunoprecipitation and double staining assays further confirmed the direct binding between NS5B and FASN. The domain of NS5B that interacts with FASN was also determined. Moreover, FASN was associated with detergent-resistant lipid rafts and colocalized with NS5B in active HCV replication complexes. In addition, overexpression of FASN enhanced HCV expression in Huh7/Rep-Feo cells, while transfection of FASN small interfering RNA (siRNA) or treatment with FASN-specific inhibitors decreased HCV replication and viral production. Notably, FASN directly increased HCV NS5B RdRp activity in vitro. These results together indicate that FASN interacts with NS5B and modulates HCV replication through a direct increase of NS5B RdRp activity. FASN may thereby serve as a target for the treatment of HCV infection and the prevention of HCV-associated HCC progression.
Assuntos
Ácido Graxo Sintases/metabolismo , Hepacivirus/enzimologia , Hepacivirus/fisiologia , Hepatite C/enzimologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Linhagem Celular Tumoral , Ácido Graxo Sintases/genética , Hepacivirus/genética , Hepatite C/genética , Hepatite C/virologia , Humanos , Ligação Proteica , Proteínas não Estruturais Virais/genéticaRESUMO
BACKGROUND: Oxidized LDL (oxLDL) is involved in the development of atherosclerotic heart disease through a mechanism that is not fully understood. In this study, we examined the role of malondialdehyde (MDA), an important oxidative stress epitope of oxLDL, in mediating coronary endothelial cytotoxicity. RESULTS: Human coronary artery endothelial cells (HCAECs) were treated with oxLDL in the presence or absence of antibody against MDA (anti-MDA) or apoB100 (anti-apoB100). In HCAECs treated with oxLDL (100 µg/ml) alone, DNA synthesis, cell viability, and expression of prosurvival fibroblast growth factor 2 (FGF2) were significantly reduced (P < 0.01 vs phosphate buffered saline-treated cells). These inhibitory effects of oxLDL were significantly attenuated in HCAECs cotreated with anti-MDA (0.15 µg/ml; P < 0.05 vs oxLDL-treated cells), but not in those cotreated with anti-apoB100. When we tested the effects of a panel of signal transduction modifiers on the signal transduction pathways of MDA in oxLDL-treated HCAECs, we found that MDA-induced cytotoxicity was mediated partly through the Akt pathway. Using a reporter gene assay, we identified an oxLDL-response element in the FGF2 promoter that was responsible for the transcriptional repression of FGF2 by oxLDL. The results of bisulfite genomic DNA sequencing showed that in HCAECs treated with oxLDL, the GC-rich promoter of FGF2 was heavily methylated at cytosine residues, whereas cotreatment with anti-MDA markedly reduced oxLDL-induced FGF2 promoter methylation. CONCLUSION: OxLDL disrupts the growth and survival of HCAECs through an MDA-dependent pathway involving methylation of the FGF2 promoter and repression of FGF2 transcription. This novel epigenetic mechanism of oxLDL may underlie its atherogenicity in patients with atherosclerotic cardiovascular disease.
Assuntos
Aterosclerose/metabolismo , Fator 2 de Crescimento de Fibroblastos/genética , Lipoproteínas LDL/metabolismo , Malondialdeído/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Anticorpos/administração & dosagem , Aterosclerose/etiologia , Aterosclerose/patologia , Sobrevivência Celular/efeitos dos fármacos , Vasos Coronários/citologia , Vasos Coronários/metabolismo , DNA/biossíntese , Metilação de DNA/genética , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Humanos , Lipoproteínas LDL/toxicidade , Malondialdeído/antagonistas & inibidores , Malondialdeído/imunologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genéticaRESUMO
The strong associations between oral squamous cell carcinoma (OSCC) and dietary habits such as alcohol consumption (A), betel quid chewing (B) and cigarette smoking (C) and its predominance in men have been well documented; however, systemic analysis of OSCC is limited. Our study applied high-throughput screening methods to identify causative epigenetic targets in a cohort of men with ABC-associated OSCC. We identified BEX1 and LDOC1 as two epigenetically silenced X-linked tumour suppressors and demonstrated a functional link between the transcription of BEX1 and LDOC1 and promoter hypermethylation. Methylation of the BEX1 and LDOC1 promoters was associated significantly (p < 0.0001) with OSCC and were detected in 75% (42/56) and 89% (50/56) of the samples, respectively. We observed concordant increases in the methylation of both genes in 71% (40/56) of the tumours, and potent in vitro and in vivo growth inhibitory effects in OSCC cells ectopically expressing BEX1 and/or LDOC1. Restored expression of BEX1 and LDOC1 suppressed the nuclear factor-κB (NF-κB) signalling pathway, which is the most frequently hyperactivated signalling pathway in OSCC. This suppression might result from decreased p50 and p65 expression. These findings suggest that silencing of BEX1 and LDOC1 by promoter hypermethylation might represent a critical event in the molecular pathogenesis of OSCC and account for the oncogenic effects of ABC exposure and the male predominance of OSCC occurrence. Microarray data are available in the Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/)
Assuntos
Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Bucais/genética , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Proteínas Supressoras de Tumor/genética , Animais , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular , Estudos de Coortes , Metilação de DNA , Regulação para Baixo , Epigênese Genética , Inativação Gênica , Genes Ligados ao Cromossomo X , Ensaios de Triagem em Larga Escala , Humanos , Masculino , Camundongos , Camundongos SCID , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , Distribuição Aleatória , Fatores Sexuais , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismoRESUMO
OBJECTIVE: ATP-binding cassette, sub-family B (MDR/TAP), member 1 (ABCB1) is a drug transporter protein expressed on the epithelial cells of the intestine and the endothelial cells of the blood-brain barrier. Intestinal ABCB1 actively transports drugs from the cell membrane and prevents them from entering the blood stream whereas the blood-brain barrier ABCB1 prevents drugs from entering the central nervous system. In this study, we tested whether genetic polymorphisms within the ABCB1 gene are associated with the severity of depression and the effectiveness of the antidepressant, escitalopram (S-CIT), in treating major depressive disorder (MDD). METHODS: Twenty single nucleotide polymorphisms in the ABCB1 gene were selected and genotyped in 100 MDD patients who had undergone S-CIT treatment continuously for 8 weeks. The serum concentrations of S-CIT and its metabolites (S-desmethylcitalopram and S-didesmethylcitalopram) were then measured at weeks 2, 4, and 8. RESULTS: The ABCB1 genotypes of rs1922242 (P=0.0028) and rs1202184 (P=0.0021) showed significant association with the severity of depressive symptoms as assessed by the Hamilton Rating Scale for Depression adjusted with Hamilton Rating Scale for Anxiety. The haplotype block, rs1882478-rs2235048-rs2235047-rs1045642-rs6949448 (from intron 27 to intron 26), of ABCB1 was found strongly associated with the remission rate (global P=0.003, d.f.=69) in which haplotype T-T-T-C-C was associated with a slower remission rate on S-CIT treatment (P=0.001). The haplotypes may not be indicators of the severity of depression or anxiety. CONCLUSION: Our findings suggest that single nucleotide polymorphisms in the ABCB1 gene may be indicators of the severity of depression and of the likely S-CIT treatment remission response in MDD.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Antidepressivos de Segunda Geração/uso terapêutico , Citalopram/uso terapêutico , Transtorno Depressivo Maior/tratamento farmacológico , Transtorno Depressivo Maior/genética , Polimorfismo Genético , Subfamília B de Transportador de Cassetes de Ligação de ATP , Antidepressivos de Segunda Geração/sangue , Antidepressivos de Segunda Geração/farmacologia , Citalopram/sangue , Citalopram/farmacologia , Genótipo , Haplótipos , HumanosRESUMO
Granulocyte-colony stimulating factor (G-CSF) is a cytokine which involves in anti-inflammation and inflammation as well. Rapamycin is an inhibitor of mTOR which also plays a role in innate immunity. This study investigated the effect of rapamycin on the lipoteichoic acid (LTA)-induced expression of G-CSF in macrophages and its underlying mechanism. Our data show that LTA induced G-CSF expression in RAW264.7 and bone marrow-derived macrophages and that this effect was inhibited by rapamycin. Analysis of the G-CSF 5' flanking sequence revealed that the -283 to +35 fragment, which contains CSF and octamer elements, was required for maximal promoter activity in response to LTA stimulation. Western blot analyses of proteins that bind to the CSF and octamer element show that LTA increased protein levels of NF-κB, C/EBPß and Oct-2, and that rapamycin inhibited the LTA-induced increase in Oct-2 protein levels, but not the others. Knockdown of Oct-2 by RNA interference resulted in a decrease in LTA-induced G-CSF mRNA levels. Moreover, forced expression of Oct-2 by transfection with the pCG-Oct-2 plasmid overcame the inhibitory effect of rapamycin on the LTA-induced increase in G-CSF mRNA levels and promoter activity. This study demonstrates that rapamycin reduces G-CSF expression in LTA-treated macrophages by inhibiting Oct-2 expression.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/biossíntese , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Sirolimo/farmacologia , Ácidos Teicoicos/antagonistas & inibidores , Ácidos Teicoicos/farmacologia , Região 5'-Flanqueadora/genética , Animais , Células da Medula Óssea/citologia , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Fator Estimulador de Colônias de Granulócitos/genética , Fator Estimulador de Colônias de Granulócitos/metabolismo , Macrófagos/citologia , Camundongos , NF-kappa B/metabolismo , Fator 2 de Transcrição de Octâmero/metabolismo , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Electronegative LDL (LDL(-)) and free fatty acids (FFAs) are circulating risk factors for cardiovascular diseases (CVDs) and have been associated with inflammation. Interleukin-1 beta (IL-1ß) represents a key cytokine in the development of CVD; however, the initial trigger of IL-1ß in CVD remains to be explored. In this study, we investigated the combined effects of LDL(-) from the plasma of ST-segment elevation myocardial infarction (STEMI) patients or diet-induced hypercholesterolemic rabbits and bovine serum albumin bound palmitic acid (PA-BSA) on IL-1ß production in macrophages. Macrophages derived from THP-1 cells or human peripheral blood mononuclear cells were independently treated with LDL(-), PA-BSA or cotreated with LDL(-) and PA-BSA. The results showed that nLDL and/or PA-BSA had no effect on IL-1ß, and LDL(-) slightly increased IL-1ß; however, cotreatment with LDL(-) and PA-BSA resulted in abundant secretion of IL-1ß in macrophages. Rabbit LDL(-) induced the elevation of cellular pro-IL-1ß and p-Iκ-Bα, but PA-BSA had no effect on pro-IL-1ß or p-Iκ-Bα. In potassium-free buffer, LDL(-)-induced IL-1ß reached a level similar to that induced by cotreatment with LDL(-) and PA-BSA. Moreover, LDL(-) and PA-BSA-induced IL-1ß was inhibited in lectin-type oxidized LDL receptor-1 (LOX-1) knockdown cells and by blockers of voltage-gated potassium (Kv) channels. LDL(-) from diet-induced hypercholesterolemic rabbit had a similar effect as STEMI LDL(-) on IL-1ß in macrophages. These results show that PA-BSA cooperates with LDL(-) to trigger IL-1ß production in macrophages via a mechanism involving the LOX-1 and Kv channel pathways, which may play crucial roles in the regulation of inflammation in CVD.
Assuntos
Interleucina-1beta/metabolismo , Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , Ácido Palmítico/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Receptores Depuradores Classe E/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Hipercolesterolemia/metabolismo , Lipoproteínas LDL/farmacologia , Macrófagos/imunologia , Masculino , Ácido Palmítico/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , Coelhos , Infarto do Miocárdio com Supradesnível do Segmento ST/metabolismo , Receptores Depuradores Classe E/genética , Transdução de Sinais , Células THP-1RESUMO
Maternal hypercholesterolemia induces early onset of cardiovascular diseases in offspring; however, its underlying mechanism remains poorly understood. We hypothesized that maternal hypercholesterolemia increases offspring susceptibility to atherosclerosis in adulthood through developmental modifications of macrophages. Female apolipoprotein E (ApoE)-deficient mice were fed a Western-type diet (WD) or a control diet (CD) prior to and throughout gestation and lactation. The offspring were all fed a WD after weaning. Sixteen-week-old female offspring of WD-fed dams showed a significant increase in atherosclerotic lesions of the aorta and aortic root compared with those of CD-fed dams. This effect was associated with increased macrophage accumulation within lesions, macrophage inflammation and an increase in circulating Ly6Chigh monocyte and F4/80 macrophage counts. We further evidenced that in utero WD exposure promoted macrophage polarization toward the M1 phenotype by elevating M1 markers (Cd86, Inos/Nos2) without affecting M2 markers (Cd206, Arg1). Proinflammatory cytokine synthesis was also enhanced in response to LPS. Finally, maternal WD intake strongly inhibited the macrophage expression of Pparg and Lxra, which was associated with aberrant DNA methylation of Lxra promoter. Our findings demonstrate that maternal hypercholesterolemia exacerbates atherosclerosis, in part by altering the epigenetic state of the macrophage genome of the offspring, imprinting gene expression, and changing macrophage polarization, which ultimately contributes to plaque macrophage burden.
Assuntos
Fenômenos Fisiológicos da Nutrição Animal , Aterosclerose/metabolismo , Hipercolesterolemia/metabolismo , Macrófagos/metabolismo , Fenômenos Fisiológicos da Nutrição Materna , Efeitos Tardios da Exposição Pré-Natal , Animais , Aorta/metabolismo , Apolipoproteínas E/metabolismo , Aterosclerose/genética , Aterosclerose/patologia , Dieta Ocidental , Modelos Animais de Doenças , Feminino , Expressão Gênica , Humanos , Hipercolesterolemia/genética , Hipercolesterolemia/patologia , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Fenótipo , GravidezRESUMO
UNLABELLED: Liver cirrhosis is characterized by progressive accumulation of extracellular matrix following chronic liver injuries. In the extracellular space, the constant turnover of liver matrix is regulated by the matrix metalloproteinase (MMP) class of enzyme. To assess whether genetic variations in MMP would result in diversity of liver cirrhosis, a case-control study of 320 patients with hepatocellular carcinoma, with or without cirrhosis, was conducted. Ten single-nucleotide polymorphism markers from four potential fibrosis-associated genes were selected for genotyping. Among these genes, a nonsynonymous single-nucleotide polymorphism which generates the variation of Gly-137 and Asp-137 in the MMP-7 gene was found to be strongly associated with the development of liver cirrhosis. In contrast to MMP-7(Gly-137) that predominantly secretes out into the cell culture medium, the cirrhosis-associated MMP-7(Asp-137) variant is preferentially localized on the extracellular membranes where it exerts its proteolytic activity on pericellular substrates. Functional analysis demonstrated an increased ability of the MMP-7(Asp-137) variant to associate with the cell surface CD151 molecule. In wound-healing and Boyden chamber assays, cell motility was specifically enhanced with the expression of MMP-7(Asp-137) as compared to the cells expressing MMP-7(Gly-137). These results demonstrate that the MMP-7(Asp-137) variant confers a gain-of-function phenotype for MMP-7. CONCLUSION: We have identified a novel genetic association of MMP-7(Asp-137) variant with liver cirrhosis in patients with hepatocellular carcinoma. Whether the MMP-7 variant can be a new marker for liver cirrhosis will be further studied.
Assuntos
Carcinoma Hepatocelular/metabolismo , Predisposição Genética para Doença/genética , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Neoplasias Hepáticas/metabolismo , Metaloproteinase 7 da Matriz/genética , Metaloproteinase 7 da Matriz/metabolismo , Adulto , Idoso , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Estudos de Casos e Controles , Membrana Celular/metabolismo , Feminino , Genótipo , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genética , Fatores de Risco , Tetraspanina 24RESUMO
BACKGROUND: We compared the haemodynamic and metabolic effects of acetyl-L-carnitine (one of the carnitine derivatives) and of oxfenicine (a carnitine palmitoyltransferase-1 inhibitor) in streptozotocin-induced diabetes in male Wistar rats. MATERIALS AND METHODS: Diabetes was induced by a single tail vein injection of 55mgkg(-1) streptozotocin. The diabetic animals daily treated with either acetyl-L-carnitine (150mgkg(-1) in drinking water) or oxfenicine (150mgkg(-1) by oral gavage) for 8weeks,were compared with the untreated age-matched diabetic controls. Arterial wave reflection was derived using the impulse response function of the filtered aortic input impedance spectra. Thiobarbituric acid reactive substances (TBARS) measurement was used to estimate malondialdehyde (MDA) content. RESULTS: Oxfenicine, but not acetyl-L-carnitine, increased total peripheral resistance in diabetes, which paralleled its elevation in plasma levels of free fatty acids. By contrast, acetyl-L-carnitine, but not oxfenicine, resulted in a significant increase in wave transit time and a decrease in wave reflection factor, suggesting that acetyl-L-carnitine may attenuate the diabetes-induced deterioration in systolic loading condition for the left ventricle. This was in parallel with its lowering of MDA/TBARS content in plasma and aortic walls in diabetes. Acetyl-L-carnitine therapy also prevented the diabetes-related cardiac hypertrophy, as evidenced by the reduction in ratio of the left ventricular weight to body weight. CONCLUSION: Acetyl-L-carnitine, but not oxfenicine, attenuates aortic stiffening and cardiac hypertrophy, possibly through its decrease of lipid oxidation-derived MDA/TBARS in the rats with insulin deficiency.
Assuntos
Acetilcarnitina/uso terapêutico , Aorta/efeitos dos fármacos , Carnitina O-Palmitoiltransferase/antagonistas & inibidores , Diabetes Mellitus Experimental/tratamento farmacológico , Inibidores Enzimáticos/uso terapêutico , Complexo Vitamínico B/uso terapêutico , Análise de Variância , Animais , Carnitina O-Palmitoiltransferase/uso terapêutico , Diabetes Mellitus Experimental/fisiopatologia , Masculino , Ratos , Ratos WistarRESUMO
Homocysteine (Hcy) contributes to atherogenesis and angiostasis by altering the phenotype of arterial endothelial cells (ECs). The present study was aimed at elucidating potential mechanisms by which Hcy can slow EC proliferation and induce EC apoptosis, thereby disrupting endothelial integrity. Given the strong mitogenic and antiapoptotic properties of fibroblast growth factor (FGF)2, we examined whether Hcy can modulate its expression. In cultured human coronary and bovine aortic ECs, Hcy exerted time- and concentration-dependent (0 to 500 micromol/L) reduction of the mRNA and protein levels of FGF2, whereas vascular endothelial growth factor expression was not affected until Hcy reached a proapoptotic 500 micromol/L. By testing a panel of signal transduction inhibitors, we found that the Hcy-induced downregulation of FGF2 was specifically attenuated by pertussis toxin, an inhibitor of Gi protein signaling. Hcy induced cell cycle arrest at the G(1)/S transition and increased TUNEL-positive apoptotic cells in a graded manner. These effects were effectively counteracted by exogenous FGF2. Reporter gene assays showed that Hcy downregulated FGF2 by transcriptional repression of the gene promoter encompassed in a CpG dinucleotide-rich island. This region was heavily methylated at the cytosine residues by Hcy despite decreased methylation potential (S-adenosylmethionine to S-adenosylhomocysteine ratio). Normal levels of FGF2 transcription were restored to ECs simultaneously exposed to Hcy and 5-aza-deoxycytidine. We conclude that homocysteine disrupts the growth and survival of ECs through a G protein-mediated pathway associated with altered promoter DNA methylation and the transcriptional repression of FGF2.
Assuntos
Artérias/citologia , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/citologia , Fator 2 de Crescimento de Fibroblastos/genética , Proteínas de Ligação ao GTP/metabolismo , Homocistina/farmacologia , Animais , Bovinos , Metilação de DNA , Regulação para Baixo/efeitos dos fármacos , Transcrição GênicaRESUMO
In response to environmental stimuli, monocytes undergo polarization into classically activated (M1) or alternatively activated (M2) states. M1 and M2 macrophages exert opposing pro- and anti-inflammatory properties, respectively. Electronegative low-density lipoprotein (LDL) (LDL(-)) is a naturally occurring mildly oxidized LDL found in the plasma of patients with hypercholesterolemia, diabetes, and acute myocardial infarction, and has been shown to involve in the pathogenesis of atherosclerosis. In this study, we examined the effects of LDL(-) on macrophage polarization and the involvement of lectin-like oxidized LDL receptor-1 (LOX-1) in this process. THP-1 macrophages were treated with native LDL (nLDL) or LDL(-), and then the expression of M1/M2-related surface markers and cytokines were evaluated. The results show that treatment with LDL(-) resulted in profound increase in proinflammatory cytokines, IL-1ß, IL-6, and TNF-α, and M1-surface marker CD86; however, M2-related cytokines, IL-10 and TGF-ß, and M2-surface marker CD206 were not changed by LDL(-). Untreated or nLDL-treated cells were used as control. LDL(-)-induced M1 polarization and secretion of proinflammatory cytokines were diminished in LOX-1 knockdown cells. Taken together, the results show that LDL(-) promotes differentiation of human monocytes to M1 macrophages through a LOX-1-dependent pathway, and explore the contribution of LDL(-) and LOX-1 to the development of chronic inflammation in atherosclerosis.
Assuntos
Polaridade Celular/fisiologia , Lipoproteínas LDL/farmacologia , Macrófagos/metabolismo , Receptores Depuradores Classe E/metabolismo , Transdução de Sinais/fisiologia , Animais , Polaridade Celular/efeitos dos fármacos , Dieta Hiperlipídica/efeitos adversos , Humanos , Macrófagos/efeitos dos fármacos , Masculino , Coelhos , Transdução de Sinais/efeitos dos fármacos , Células THP-1RESUMO
BACKGROUND: Macrophages engulf oxidized-LDL (oxLDL) leading to accumulation of cellular cholesterol and formation of foam cells, which is a hallmark of atherosclerosis. Moreover, recent studies showed that accumulation of free cholesterol in macrophages leading to activation of NLRP3 inflammasome and production of interleukin-1ß (IL-1ß) has been linked to atherosclerosis-associated inflammation. However, it is not clear if cholesterol accumulation is associated with hepatic inflammation and fibrosis in the liver. In this study, we investigated the association of free cholesterol and oxLDL accumulation in portal vein with the inflammation, atherosclerosis, and fibrosis in human nonalcoholic fatty liver disease (NAFLD). METHODS: Serial sections derived from surgical specimens of NAFLD were stained with filipin and antibodies against IL-1ß, CD68, α-smooth muscle actin (α-SMA), oxLDL and lectin-like oxLDL receptor-1 (LOX-1). RESULTS: We show that free cholesterol was colocalized with oxLDL in the wall of portal vein, and which was associated with lumen narrowing, plaque formation, endothelium deformation, and portal venous inflammation. The inflammation was evidenced by the colocalization of Kupffer cells and IL-1ß and the expression of LOX-1. Notably, ruptured plaque was closely associated with portal venous inflammation. Moreover, free cholesterol and oxLDL accumulation in periportal and sinusoidal fibrosis, which was associated with regional stellate cell activation and chicken-wire fibrosis. CONCLUSION: These findings reveal a direct association between cholesterol accumulation, portal venous inflammation and fibrosis in NAFLD.
RESUMO
Although resistin was first suggested as a possible link between obesity and diabetes, we have demonstrated previously that expression of resistin is induced by LPS (lipopolysaccharide). In the present study, we showed that LPS increased levels of resistin mRNA and promoter activity in murine RAW264.7 macrophages. Investigation of cis-regulatory elements in the mouse resistin promoter required for LPS-mediated induction showed that an Octamer (ATTTGCAT) element, located at -914 to -907, was required for maximal promoter activity in response to LPS stimulation. Co-transfection of RAW264.7 cells with a resistin promoter-luciferase construct and an Oct-1 or Oct-2 expression plasmid (pCG-Oct-1 or pCG-Oct-2) showed that Oct-2, but not Oct-1, activated the resistin promoter upon LPS treatment. Binding of Oct-2 to the Octamer element was demonstrated by supershift DNA-affinity precipitation and chromatin immunoprecipitation assays. Reverse transcription-PCR and Western blot results showed that levels of Oct-2 mRNA and protein were both up-regulated by LPS in RAW264.7 cells. The LPS-induced increase in Oct-2 protein was inhibited by LY294002 (a phosphoinositide 3-kinase inhibitor) post-transcriptionally, and the inhibition also resulted in a lower response of both resistin mRNA and promoter activity to LPS treatment. Moreover, specific knockdown of Oct-2 by RNA interference impaired the LPS-induced increase in resistin mRNA and promoter activity. Together, these results indicate that Oct-2 is involved in the LPS-mediated induction of resistin gene expression in macrophages and suggest that activation of Oct-2 is a part of LPS signalling pathways in macrophages.
Assuntos
Expressão Gênica/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Fator 2 de Transcrição de Octâmero/metabolismo , Resistina/genética , Região 5'-Flanqueadora , Animais , Sequência de Bases , Linhagem Celular , Cromonas/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Dados de Sequência Molecular , Morfolinas/farmacologia , Mutagênese/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Fator 2 de Transcrição de Octâmero/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Interferência de RNA , RNA Mensageiro/genética , Resistina/metabolismo , Elementos de Resposta/genética , Transativadores/genética , TransfecçãoRESUMO
Modifications of both carriers and host barriers have been investigated for efficient inhalation gene delivery to lung. Here we used a biocompatible, non-ionic poly(ethyleneoxide)-poly(propyleneoxide)-poly(ethyleneoxide) (PEO-PPO-PEO) polymeric micelles (PM) as a carrier and combined it with ethanol to enhance membrane penetration of delivered DNA. The inhalation delivery with six 100 microg doses of pCMV-Lac Z with PM co-formulated with 10%-40% ethanol to nude mice in 2 days at 8 h interval was performed. The beta-galatosidase (beta-Gal) activity was assessed using chlorophenol red-beta-d galactopyranoside (CPRG) and X-gal staining for quantitative and qualitative analysis in tissues. The results showed that beta-Gal activity was significantly increased by 38% in lung around bronchioles when inhalation with PM and 10% ethanol was given. The 10% ethanol also increased the intracellular apparent permeability by 42% in stomach and by 141% in intestine at 48 h after the first dosage of delivery. Also delivery of DNA encoding a functional human cystic fibrosis transmembrane protein (CFTR) using the same inhalation delivery method co-formulated with 10% ethanol, an increased expression of CFTR in lung was detected by immunostaining. We concluded that 10% ethanol co-formulated with the PM system could enhance inhaled gene delivery to airway and gastrointestinal (GI) tract.