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1.
PLoS One ; 14(5): e0216405, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31071151

RESUMO

Statins exert pleiotropic and beneficial anti-inflammatory and antioxidant effects. We have previously reported that macrophages treated with statins increased the expression of heme oxygenase-1 (HO-1), an inducible anti-inflammatory and cytoprotective stress protein, responsible for the degradation of heme. In the present study, we investigated the effects of atorvastatin on inflammation in mice and analyzed its mechanism of action in vivo. Air pouches were established in 8 week-old female C57BL/6J mice. Atorvastatin (5 mg/kg, i.p.) and/or tin protoporphyrin IX (SnPPIX), a heme oxygenase inhibitor (12 mg/kg, i.p.), were administered for 10 days. Zymosan, a cell wall component of Saccharomyces cerevisiae, was injected in the air pouch to trigger inflammation. Cell number and levels of inflammatory markers were determined in exudates collected from the pouch 24 hours post zymosan injection by flow cytometry, ELISA and quantitative PCR. Analysis of the mice treated with atorvastatin alone displayed increased expression of HO-1, arginase-1, C-type lectin domain containing 7A, and mannose receptor C-type 1 in the cells of the exudate of the air pouch. Flow cytometry analysis revealed an increase in monocyte/macrophage cells expressing HO-1 and in leukocytes expressing MRC-1 in response to atorvastatin. Mice treated with atorvastatin showed a significant reduction in cell influx in response to zymosan, and in the expression of proinflammatory cytokines and chemokines such as interleukin-1α, monocyte chemoattractant protein-1 and prostaglandin E2. Co-treatment of mice with atorvastatin and tin protoporphyrin IX (SnPPIX), an inhibitor of heme oxygenase, reversed the inhibitory effect of statin on cell influx and proinflammatory markers, suggesting a protective role of HO-1. Flow cytometry analysis of air pouch cell contents revealed prevalence of neutrophils and to a lesser extent of monocytes/macrophages with no significant effect of atorvastatin treatment on the modification of their relative proportion. These findings identify HO-1 as a target for the therapeutic actions of atorvastatin and highlight its potential role as an in vivo anti-inflammatory agent.


Assuntos
Anti-Inflamatórios/farmacologia , Atorvastatina/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Heme Oxigenase-1/biossíntese , Proteínas de Membrana/biossíntese , Zimosan/toxicidade , Animais , Movimento Celular/efeitos dos fármacos , Feminino , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/epidemiologia , Inflamação/patologia , Macrófagos/enzimologia , Macrófagos/patologia , Metaloporfirinas/farmacologia , Camundongos , Monócitos/enzimologia , Monócitos/patologia , Neutrófilos/enzimologia , Neutrófilos/patologia , Protoporfirinas/farmacologia
2.
Med Sci Monit ; 25: 2112-2121, 2019 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-30898992

RESUMO

BACKGROUND Our research was designed to investigate the relationship of spleen tyrosine kinase (Syk) and inflammatory factors with coronary heart disease (CHD) and the risk factors of CHD. MATERIAL AND METHODS In our study, 226 patients were enrolled, from October 2017 to March 2018. Clinical and biochemical data were collected. We collected samples of peripheral blood monocytes (PBMs) from the enrolled patients. The patients were divided in 4 groups: patients without coronary artery disease (control group), patients with stable angina pectoris (SAP group), patients with non-ST-segment elevation acute coronary syndrome (NSTE-ACS group), and patients with ST-segment elevation acute myocardial infarction group (STEMI group). We detect the protein levels of Syk and inflammatory factors expression by western blot. RESULTS Our results found the protein levels of Syk and inflammatory factors expression in the NSTE-ACS and STEMI groups were higher than those in the SAP and control groups. The protein levels of Syk and inflammatory factors expression in the SAP group were higher than those in the control group. Moreover, there were many risk factors significantly associated with Syk. Besides that, these risk factors were also independent risk factors of CHD. CONCLUSIONS Our results found that the level of Syk was associated with the severity of CHD. From our study, we found that higher levels of Syk and inflammatory factors protein were associated with worse results of the CHD. For the first time, Syk was reported to be a promising therapeutic factor for CHD patients.


Assuntos
Quinase Syk/biossíntese , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Humanos , Inflamação/sangue , Inflamação/enzimologia , Masculino , Pessoa de Meia-Idade , Monócitos/enzimologia , Intervenção Coronária Percutânea/métodos , Fatores de Risco , Quinase Syk/sangue , Transcriptoma
3.
Transfus Clin Biol ; 26(2): 128-129, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30898432

RESUMO

Patients with sickle cell disease (SCD) suffer from intravascular hemolysis associated with vascular injury and dysfunction. Painful vaso-occlusive crisis (VOC) involving increased attachment of sickle erythrocytes and activated leukocytes to damaged vascular endothelium is a hallmark of SCD. Patrolling monocytes, which normally scavenge damaged cells and debris from the vasculature, express higher levels of anti-inflammatory heme oxygenase 1 (HO-1), a heme degrading enzyme with anti-cytotoxic and anti-inflammatory properties. Recent data show that patients with SCD have a novel subset of patrolling monocytes expressing very high levels of HO-1 (HO-1hi) which are decreased in numbers in patients who had a recent VOC episode. This patrolling monocyte subset was responsible for protection of endothelium against sickle RBC stasis in an experimental model. This raises the possibility that patrolling monocytes may also offer protection against vascular endothelium damage in hyperhemolytic conditions in SCD.


Assuntos
Anemia Falciforme/sangue , Heme Oxigenase-1/sangue , Monócitos/enzimologia , Reação Transfusional/sangue , Anemia Falciforme/terapia , Animais , Modelos Animais de Doenças , Endotélio Vascular/patologia , Hemólise , Humanos , Camundongos , Fagocitose , Reação Transfusional/etiologia , Doenças Vasculares/etiologia
4.
Vascul Pharmacol ; 112: 79-90, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30213580

RESUMO

Upon myocardial infarction (MI) immune system becomes activated by extensive necrosis of cardiomyocytes releasing intracellular molecules called damage-associated molecular patterns. Overactive and prolonged immune responses are likely to be responsible for heart failure development and progression in patients surviving the ischemic episode. Heme oxygenase-1 (HO-1) plays a crucial role in heme degradation and in this way releases carbon monoxide, free iron, and biliverdin. This stress-inducible enzyme is induced by various oxidative and inflammatory signals. Consequently, biological actions of HO-1 are not limited to degradation of a toxic heme released from hemoproteins, but also provide an adaptive cellular response against chronic inflammation and oxidative injury. Indeed, the immunomodulatory and anti-inflammatory properties of HO-1 were demonstrated in several experimental studies, as well as in human cases of genetic HO-1 deficiency. HO-1 was shown to suppress the production, myocardial infiltration and inflammatory properties of monocytes and macrophages what resulted in limitation of post-MI cardiac damage. This review specifically addresses the role of HO-1, heme and its degradation products in macrophage biology and post-ischemic cardiac repair. A more complete understanding of these mechanisms is essential to develop new therapeutic approaches.


Assuntos
Fármacos Cardiovasculares/farmacologia , Terapia Genética/métodos , Heme Oxigenase-1/metabolismo , Macrófagos/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Miocárdio/enzimologia , Animais , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Insuficiência Cardíaca/enzimologia , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/imunologia , Insuficiência Cardíaca/patologia , Heme Oxigenase-1/genética , Humanos , Mediadores da Inflamação/metabolismo , Macrófagos/enzimologia , Macrófagos/imunologia , Terapia de Alvo Molecular , Monócitos/enzimologia , Monócitos/imunologia , Miocárdio/imunologia , Miocárdio/patologia , Necrose , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
5.
Bioorg Med Chem Lett ; 29(1): 1-7, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30466896

RESUMO

Myeloperoxidase (MPO), an abundant hemoprotein present in neutrophils and monocytes, plays a significant role in immune surveillance and host defense mechanisms. However, increased MPO activity has been linked to a number of pathologies with compelling evidence in initiation and progression of inflammatory events. As a result, search for active compounds that can efficiently inhibit MPO activity and subsequently decrease inflammatory events has been focus of the current research. This perspective provides an overview of the development of MPO inhibitors, their mechanism of action and the review of molecules that were in clinical trials as promising MPO inhibitors.


Assuntos
Inibidores Enzimáticos/farmacologia , Peroxidase/antagonistas & inibidores , Animais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Estrutura Molecular , Monócitos/enzimologia , Neutrófilos/enzimologia , Peroxidase/metabolismo , Relação Estrutura-Atividade
6.
Exp Parasitol ; 197: 93-102, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30562480

RESUMO

The intraerythrocytic malaria parasite digests haemoglobin to provide amino acids for metabolism and releases toxic haem that is sequestered into haemozoin, a non-toxic, insoluble, crystalline pigment. Following erythrocyte rupture, haemozoin is released into circulation and phagocytosed by monocytes. Phagocytosed haemozoin and antimalarial drugs have both been reported to modulate monocyte functions. This study determined the effects of therapeutic concentrations of seven antimalarial drugs; amodiaquine, artemisinin, chloroquine, doxycycline, primaquine, pyrimethamine and quinine, on the phagocytosis of ß-haematin (synthetic haemozoin) by two monocytic cell lines, J774A.1 and U937, and human peripheral blood mononuclear cells. A novel spectrophotometric method based on the absorbance (O.D 400 nm) of alkali/SDS treated monocytes containing ß-haematin was developed to complement counting phagocytosis with microscopy. The method has potential use for the large scale screening of monocyte phagocytic activity. Artemisinin, quinine, primaquine and pyrimethamine activated ß-haematin phagocytosis by 12% or more, whereas amodiaquine, chloroquine and doxycyline inhibited ß-haematin phagocytosis. In contrast, antimalarial drugs had minimal inhibitory effects on the phagocytosis of latex beads with only quinine resulting in more than 20% inhibition. Antimalarial drugs appear to alter monocyte phagocytic activity which has implications for the treatment, pathogenicity and adjunct therapies for malaria.


Assuntos
Antimaláricos/farmacologia , Hemeproteínas/metabolismo , Monócitos/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Amodiaquina/farmacologia , Animais , Artemisininas/farmacologia , Contagem de Células , Linhagem Celular , Cloroquina/farmacologia , Doxiciclina/farmacologia , Microanálise por Sonda Eletrônica , Heme/análise , Hemeproteínas/biossíntese , Hemeproteínas/química , Hemeproteínas/ultraestrutura , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Camundongos , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Monócitos/enzimologia , Monócitos/metabolismo , Peroxidase/isolamento & purificação , Peroxidase/metabolismo , Primaquina/farmacologia , Pirimetamina/farmacologia , Quinina/farmacologia , Espectrofotometria , Temperatura , Células U937
7.
Gut ; 68(10): 1872-1883, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-30580251

RESUMO

OBJECTIVE: Acute-on-chronic liver failure (ACLF) is associated with dysfunctional circulating monocytes whereby patients become highly susceptible to bacterial infections. Here, we identify the pathways underlying monocyte dysfunction in ACLF and we investigate whether metabolic rewiring reinstates their phagocytic and inflammatory capacity. DESIGN: Following phenotypic characterisation, we performed RNA sequencing on CD14+CD16- monocytes from patients with ACLF and decompensated alcoholic cirrhosis. Additionally, an in vitro model mimicking ACLF patient-derived features was implemented to investigate the efficacy of metabolic regulators on monocyte function. RESULTS: Monocytes from patients with ACLF featured elevated frequencies of interleukin (IL)-10-producing cells, reduced human leucocyte antigen DR isotype (HLA-DR) expression and impaired phagocytic and oxidative burst capacity. Transcriptional profiling of isolated CD14+CD16- monocytes in ACLF revealed upregulation of an array of immunosuppressive parameters and compromised antibacterial and antigen presentation machinery. In contrast, monocytes in decompensated cirrhosis showed intact capacity to respond to inflammatory triggers. Culturing healthy monocytes in ACLF plasma mimicked the immunosuppressive characteristics observed in patients, inducing a blunted phagocytic response and metabolic program associated with a tolerant state. Metabolic rewiring of the cells using a pharmacological inhibitor of glutamine synthetase, partially restored the phagocytic and inflammatory capacity of in vitro generated- as well as ACLF patient-derived monocytes. Highlighting its biological relevance, the glutamine synthetase/glutaminase ratio of ACLF patient-derived monocytes positively correlated with disease severity scores. CONCLUSION: In ACLF, monocytes feature a distinct transcriptional profile, polarised towards an immunotolerant state and altered metabolism. We demonstrated that metabolic rewiring of ACLF monocytes partially revives their function, opening up new options for therapeutic targeting in these patients.


Assuntos
Insuficiência Hepática Crônica Agudizada/tratamento farmacológico , Infecções Bacterianas/tratamento farmacológico , Glutamato-Amônia Ligase/antagonistas & inibidores , Imunossupressores/uso terapêutico , Monócitos/enzimologia , Insuficiência Hepática Crônica Agudizada/imunologia , Insuficiência Hepática Crônica Agudizada/metabolismo , Adulto , Infecções Bacterianas/metabolismo , Infecções Bacterianas/patologia , Células Cultivadas , Citocinas/metabolismo , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Fagocitose , Estudos Retrospectivos
8.
Arterioscler Thromb Vasc Biol ; 38(11): 2590-2600, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30354246

RESUMO

Objective- Inhibition of HMGCR (3-hydroxy-3-methylglutaryl-coenzyme A reductase) is atheroprotective primarily by decreasing plasma LDL (low-density lipoprotein)-cholesterol. However, it is unknown whether inhibition of HMGCR in myeloid cells contributes to this atheroprotection. We sought to determine the role of myeloid HMGCR in the development of atherosclerosis. Approach and Results- We generated mice with genetically reduced Hmgcr in myeloid cells ( Hmgcr m- /m-) using LysM (Cre) and compared various functions of their macrophages to those of Hmgcr fl/fl control mice. We further compared the extent of atherosclerosis in Hmgcr m-/ m- and Hmgcr fl/fl mice in the absence of Ldlr (LDL receptor). Hmgcr m-/ m- macrophages and granulocytes had significantly lower Hmgcr mRNA expression and cholesterol biosynthesis than Hmgcr fl/fl cells. In vitro, Hmgcr m-/ m- monocytes/macrophages had reduced ability to migrate, proliferate, and survive compared with Hmgcr fl/fl monocytes/macrophages. However, there was no difference in ability to adhere, phagocytose, store lipids, or polarize to M1 macrophages between the 2 types of macrophages. The amounts of plasma membrane-associated small GTPase proteins, such as RhoA (RAS homolog family member A), were increased in Hmgcr m-/ m- macrophages. In the setting of Ldlr deficiency, Hmgcr m-/ m- mice developed significantly smaller atherosclerotic lesions than Hmgcr fl/fl mice. However, there were no differences between the 2 types of mice either in plasma lipoprotein profiles or in the numbers of proliferating or apoptotic cells in the lesions in vivo. The in vivo migration of Hmgcr m-/ m- macrophages to the lesions was reduced compared with Hmgcr fl/fl macrophages. Conclusions- Genetic reduction of HMGCR in myeloid cells may exert atheroprotective effects primarily by decreasing the migratory activity of monocytes/macrophages to the lesions.


Assuntos
Aorta/enzimologia , Doenças da Aorta/enzimologia , Aterosclerose/enzimologia , Movimento Celular , Hidroximetilglutaril-CoA Redutases/metabolismo , Macrófagos Peritoneais/enzimologia , Monócitos/enzimologia , Transferência Adotiva , Animais , Aorta/patologia , Doenças da Aorta/genética , Doenças da Aorta/patologia , Doenças da Aorta/prevenção & controle , Aterosclerose/genética , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Modelos Animais de Doenças , Feminino , Predisposição Genética para Doença , Hidroximetilglutaril-CoA Redutases/genética , Lipídeos/sangue , Macrófagos Peritoneais/patologia , Macrófagos Peritoneais/transplante , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/patologia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Fenótipo , Receptores de LDL/deficiência , Receptores de LDL/genética , Transdução de Sinais
9.
Sci Rep ; 8(1): 15446, 2018 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-30337601

RESUMO

Missense mutations in glucocerebrosidase (GBA1) that impair the activity of the encoded lysosomal lipid metabolism enzyme (GCase) are linked to an increased risk of Parkinson's disease. However, reduced GCase activity is also found in brain tissue from Parkinson's disease patients without GBA1 mutations, implicating GCase dysfunction in the more common idiopathic form of Parkinson's disease. GCase is very highly expressed in monocytes, and thus we measured GCase activity in blood samples from recently diagnosed Parkinson's disease patients. Flow cytometry and immunoblotting assays were used to measure levels of GCase activity and protein in monocytes and lymphocytes from patients with Parkinson's disease (n = 48) and matched controls (n = 44). Gene sequencing was performed to screen participants for GBA1 missense mutations. In the Parkinson's disease patients, GCase activity was significantly reduced in monocytes, but not lymphocytes, compared to controls, even when GBA1 mutation carriers were excluded. Monocyte GCase activity correlated with plasma ceramide levels in the Parkinson's disease patients. Our results add to evidence for GCase dysfunction in idiopathic Parkinson's disease and warrant further work to determine if monocyte GCase activity associates with Parkinson's disease progression.


Assuntos
Ceramidas/sangue , Glucosilceramidase/deficiência , Monócitos/enzimologia , Doença de Parkinson/enzimologia , Idoso , Progressão da Doença , Feminino , Citometria de Fluxo , Genótipo , Glucosilceramidase/análise , Glucosilceramidase/genética , Humanos , Linfócitos/enzimologia , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Doença de Parkinson/sangue , Interferência de RNA , RNA Interferente Pequeno/genética
10.
Artigo em Inglês | MEDLINE | ID: mdl-30285552

RESUMO

The enzyme thymidine phosphorylase (TP) is important for activation of capecitabine and 5-fluorouracil. Assessment of TP phenotype might be suitable for identification of patients at risk of fluoropyrimidine-induced toxicity. In this paper, we describe the development and validation an assay for TP activity in peripheral blood mononuclear cells (PBMCs). The assay was based on ex vivo conversion of the TP substrate thymidine to thymine. The amount of thymine formed was determined by high-performance liquid chromatography - ultraviolet detection (HPLC-UV) with 5-bromouracil as internal standard. Lymphocytes and monocytes were purified from isolated PBMCs to examine cell-specific TP activity. TP activity in PBMCs demonstrated Michaelis-Menten kinetics. The lower limit of quantification was 2.3 µg PBMC protein and assay linearity was demonstrated up to 22.7 µg PBMC protein. Within-day and between-day precisions were ≤9.2% and ≤6.0%, respectively. Adequate stability TP activity was demonstrated after long-term storage of PBMC dry pellets and lysates at -80 °C. In monocytes, TP activity was approximately 3 times higher than in lymphocytes. Clinical applicability was demonstrated in samples that were collected from five cancer patients. A simple, precise and sensitive HPLC-UV assay for quantification of TP activity in PBMCs was developed that can be applied for clinical research.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Leucócitos Mononucleares/enzimologia , Timidina Fosforilase/sangue , Antimetabólitos Antineoplásicos/uso terapêutico , Capecitabina/uso terapêutico , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Limite de Detecção , Linfócitos/enzimologia , Monócitos/enzimologia , Neoplasias/sangue , Sensibilidade e Especificidade , Timidina/metabolismo , Timidina Fosforilase/metabolismo , Timina/metabolismo
11.
Sci Rep ; 8(1): 14496, 2018 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-30262819

RESUMO

Despite the high success rate, antiretroviral therapy does not cure the disease completely due to presence of latent viral reservoirs. Although several studies have addressed this issue earlier, the role of serum starvation/deprivation in HIV-1 latency has not been studied. So, we investigated the role of serum starvation in regulating HIV-1 latency. The impact of serum starvation on HIV-1 latency was assessed in latently infected monocytes U1 and T-cells J1.1. Serum starvation breaks HIV-1 latency in U1 cells. Under similar conditions, J1.1 cells failed to show reactivation of virus. We investigated the involvement of cell death pathway and autophagy during the serum starvation in viral reactivation. Inhibition of these pathways did not affect viral reactivation. Furthermore, other crucial factors like NF-κB, SP1 and AKT did not play any role in regulating viral latency. Here, we report that serum deprivation up-regulates ERK/JNK pathway. This leads to phosphorylation of c-Jun which plays an important role in viral reactivation. Treatment of cells with U0126, an ERK kinase inhibitor, potently inhibited viral replication. In summary, we show that serum starvation leads to reactivation of HIV-1 in latently infected monocytes through the ERK/JNK pathway.


Assuntos
Infecções por HIV/enzimologia , HIV-1/fisiologia , Sistema de Sinalização das MAP Quinases , Monócitos , Ativação Viral/fisiologia , Latência Viral/fisiologia , Autofagia , Linhagem Celular , Infecções por HIV/patologia , Humanos , Monócitos/enzimologia , Monócitos/patologia , Monócitos/virologia , Ativação Viral/efeitos dos fármacos , Latência Viral/efeitos dos fármacos
12.
Acta Biochim Pol ; 65(3): 409-414, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29959857

RESUMO

Cyclic guanosine monophosphate (cGMP) is synthesized by two types of enzymes: particulate (membrane-bound) guanylyl cyclases (pGCs) and soluble (cytosolic) guanylyl cyclases (sGCs). sGCs are primarily activated by binding of nitric oxide to their prosthetic heme group while pGCs are activated by binding of peptide ligands to their extracellular domains. One of them, pGC type A (GC-A) is activated by atrial and brain natriuretic peptides (ANP and BNP, respectively). Human monocytes isolated from peripheral blood mononuclear cells have been found to display sGC expression without concomitant expression of GC-A. However, GC-A activity appears in monocytes under certain conditions but a molecular mechanism of GC-A expression is still poorly understood. In this report we show that phorbol ester (PMA) induces transcription of a gene encoding GC-A in human monocytic THP-1 cells. Moreover, we find that PMA-treated THP-1 cells raise cGMP content following treatment with ANP. Studies using pharmacological inhibitors of protein kinases suggest involvement of protein kinase C (PKC), mitogen extracellular kinases (MEK1/2), and extracellular signal-regulated kinases (ERK1/2) in PMA-induced expression of the GC-A encoding gene in THP-1 cells. Finally, we show that PMA stimulates binding of Sp1 transcription factor to GC-rich DNA sequences and mithramycin A (a selective Sp1 inhibitor) inhibits expression of the GC-A mRNA in PMA-treated THP-1 cells. Taken together, our findings suggest that the PMA-stimulated PKC and MEK/ERK signaling pathways induce Sp1-mediated transcription of the GC-A encoding gene in human monocytic THP-1 cells.


Assuntos
Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Guanilato Ciclase/genética , Monócitos/enzimologia , Fator de Transcrição Sp1/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , DNA/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Humanos , Plicamicina/análogos & derivados , Plicamicina/farmacologia , Ligação Proteica/efeitos dos fármacos , Proteínas Quinases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição Sp1/antagonistas & inibidores , Células THP-1 , Transcrição Genética/efeitos dos fármacos
13.
Circ Res ; 123(6): 700-715, 2018 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-29970365

RESUMO

RATIONALE: Giant cell arteritis (GCA)-a primary vasculitis of medium and large arteries-is associated with vessel wall damage, elastic membrane fragmentation, and vascular remodeling. Proteinases are believed to contribute to pathogenesis by degrading extracellular matrix and causing tissue injury. OBJECTIVE: The MMP (matrix metalloproteinase)-9-a type IV collagenase-is produced in the vasculitic lesions of GCA. It is unknown which pathogenic processes are MMP-9 dependent. METHODS AND RESULTS: The tissue transcriptome of GCA-affected temporal arteries contained high amounts of MMP-9 transcripts, and immunostaining for pro-MMP-9 localized the enzyme to wall-infiltrating macrophages. MMP-2 and MMP-9 transcripts were also abundant in monocytes and monocyte-derived macrophages from patients with GCA. Patient-derived monocytes outperformed healthy monocytes in passing through engineered basement membranes. GCA CD (cluster of differentiation) 4+ T cells required MMP-9-producing monocytes to penetrate through matrix built from type IV collagen. In vivo functions of MMP-9 were tested in a human artery-SCID (severe combined immunodeficiency) chimera model by blocking enzyme activity with a highly specific monoclonal antibody or by injecting rMMP-9 (recombinant MMP-9). Inhibiting MMP-9 activity profoundly suppressed vascular injury, decreased the density of inflammatory infiltrates ( P<0.001), reduced intramural neoangiogenesis ( P<0.001), and prevented intimal layer hyperplasia ( P<0.001). rMMP-9 amplified all domains of vasculitic activity, promoted assembly of T-cell infiltrates ( P<0.05), intensified formation of new microvessels ( P<0.001), and worsened intimal thickening ( P<0.001). Systemic delivery of N-acetyl-proline-glycine-proline-a matrikine produced by MMP-9-mediated gelatinolysis-had limited vasculitogenic effects. CONCLUSIONS: In large vessel vasculitis, MMP-9 controls the access of monocytes and T cells to the vascular wall. T cells depend on MMP-9-producing monocytes to pass through collagen IV-containing basement membrane. Invasion of vasculitogenic T cells and monocytes, formation of neoangiogenic networks, and neointimal growth all require the enzymatic activity of MMP-9, identifying this protease as a potential therapeutic target to restore the immunoprivilege of the arterial wall in large vessel vasculitis.


Assuntos
Artéria Axilar/enzimologia , Linfócitos T CD4-Positivos/enzimologia , Movimento Celular , Arterite de Células Gigantes/enzimologia , Metaloproteinase 9 da Matriz/metabolismo , Monócitos/enzimologia , Artérias Temporais/enzimologia , Remodelação Vascular , Idoso , Idoso de 80 Anos ou mais , Animais , Anti-Inflamatórios/farmacologia , Anticorpos Monoclonais/farmacologia , Artéria Axilar/efeitos dos fármacos , Artéria Axilar/imunologia , Artéria Axilar/patologia , Membrana Basal/enzimologia , Membrana Basal/patologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Estudos de Casos e Controles , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Arterite de Células Gigantes/imunologia , Arterite de Células Gigantes/patologia , Arterite de Células Gigantes/prevenção & controle , Humanos , Masculino , Metaloproteinase 9 da Matriz/genética , Inibidores de Metaloproteinases de Matriz/farmacologia , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Neointima , Neovascularização Patológica , Transdução de Sinais , Artérias Temporais/efeitos dos fármacos , Artérias Temporais/imunologia , Artérias Temporais/patologia , Remodelação Vascular/efeitos dos fármacos
14.
Mol Cell ; 70(5): 961-970.e5, 2018 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-29883611

RESUMO

HIV-1 expresses several accessory proteins to counteract host anti-viral restriction factors to facilitate viral replication and disease progression. One such protein, Vpr, has been implicated in affecting multiple cellular processes, but its mechanism remains elusive. Here we report that Vpr targets TET2 for polyubiquitylation by the VprBP-DDB1-CUL4-ROC1 E3 ligase and subsequent degradation. Genetic inactivation or Vpr-mediated degradation of TET2 enhances HIV-1 replication and substantially sustains expression of the pro-inflammatory cytokine interleukin-6 (IL-6). This process correlates with reduced recruitment of histone deacetylase 1 and 2 to the IL-6 promoter, thus enhancing its histone H3 acetylation level during resolution phase. Blocking IL-6 signaling reduced the ability of Vpr to enhance HIV-1 replication. We conclude that HIV-1 Vpr degrades TET2 to sustain IL-6 expression to enhance viral replication and disease progression. These results suggest that disrupting the Vpr-TET2-IL6 axis may prove clinically beneficial to reduce both viral replication and inflammation during HIV-1 infection.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , HIV-1/metabolismo , Mediadores da Inflamação/metabolismo , Interleucina-6/metabolismo , Monócitos/virologia , Proteínas Proto-Oncogênicas/metabolismo , Replicação Viral , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismo , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Ligação a DNA/genética , Células HEK293 , HIV-1/genética , HIV-1/crescimento & desenvolvimento , HIV-1/patogenicidade , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Interleucina-6/genética , Células Jurkat , Monócitos/enzimologia , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases , Proteólise , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais , Células THP-1 , Ubiquitina-Proteína Ligases , Ubiquitinação , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/genética
15.
Atherosclerosis ; 275: 11-21, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29852400

RESUMO

BACKGROUND AND AIMS: Lipoprotein(a) (Lp(a)) is a causal risk factor for cardiovascular disorders including coronary heart disease and calcific aortic valve stenosis. Apolipoprotein(a) (apo(a)), the unique glycoprotein component of Lp(a), contains sequences homologous to plasminogen. Plasminogen activation is markedly accelerated in the presence of cell surface receptors and can be inhibited in this context by apo(a). METHODS: We evaluated the role of potential receptors in regulating plasminogen activation and the ability of apo(a) to mediate inhibition of plasminogen activation on vascular and monocytic/macrophage cells through knockdown (siRNA or blocking antibodies) or overexpression of various candidate receptors. Binding assays were conducted to determine apo(a) and plasminogen receptor interactions. RESULTS: The urokinase-type plasminogen activator receptor (uPAR) modulates plasminogen activation as well as plasminogen and apo(a) binding on human umbilical vein endothelial cells (HUVECs), human acute monocytic leukemia (THP-1) cells, and THP-1 macrophages as determined through uPAR knockdown and overexpression. Apo(a) variants lacking either the kringle V or the strong lysine binding site in kringle IV type 10 are not able to bind to uPAR to the same extent as wild-type apo(a). Plasminogen activation is also modulated, albeit to a lower extent, through the Mac-1 (αMß2) integrin on HUVECs and THP-1 monocytes. Integrin αVß3 can regulate plasminogen activation on THP-1 monocytes and to a lesser extent on HUVECs. CONCLUSIONS: These results indicate cell type-specific roles for uPAR, αMß2, and αVß3 in promoting plasminogen activation and mediate the inhibitory effects of apo(a) in this process.


Assuntos
Apoproteína(a)/metabolismo , Células Endoteliais da Veia Umbilical Humana/enzimologia , Integrina alfaVbeta3/metabolismo , Antígeno de Macrófago 1/metabolismo , Macrófagos/enzimologia , Monócitos/enzimologia , Plasminogênio/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Ativação Enzimática , Humanos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Transdução de Sinais , Células THP-1
16.
Clin Gastroenterol Hepatol ; 16(9): 1488-1494.e5, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29723689

RESUMO

BACKGROUND & AIMS: Idiosyncratic drug-induced liver injury (iDILI) is one of the most challenging diagnoses in hepatology. It is frequently impossible to identify the agent that has caused iDILI in patients who take multiple medicines. We developed an in vitro method to identify drugs that cause liver injury in patients, based on drug toxicity to monocyte-derived hepatocyte-like (MH) cells from patient blood samples. We then collected data on patients who were re-exposed to drugs found to be toxic in the MH test to validate test performance. METHODS: We performed a prospective study of patients referred to the University Hospital in Munich, Germany, with acute liver injury believed to be caused by medications (300 patients were enrolled in the study and we present data from 40 patients with iDILI and re-exposure to implicated drugs). We collected data from patients on medical history, laboratory test and imaging results, findings from biopsy analyses, and medications taken. Blood samples were collected from all patients and MH cells were isolated and cultured for 10 days. MH cells were then incubated with drugs to which each patient had been exposed, and toxicity was measured based on release of lactate dehydrogenase. Agents found to be toxic to MH cells were considered as candidates for the cause of liver injury. Patients were followed up for up to 6 months after liver injury and data on drug re-exposures and subsequent liver damage within the following 3 to 24 months were associated with findings from MH tests. RESULTS: Our test identified 10 drugs that were toxic to MH cells from 13 patients (amoxicillin/clavulanate to cells from 2 patients; diclofenac to cells from 2 patients; methylprednisolone to cells from 2 patients; and atorvastatin, metamizole, pembrolizumab, piperacillin/tazobactam, moxifloxacin, duloxetine, or sertraline each to cells from 1 patient). Thirteen patients had a recurrence of liver injury after inadvertent re-exposure to a single drug, and the MH test correctly identified 12 of the 13 drugs that caused these liver re-injury events. All 86 drugs that were not toxic to MH cells in our assay were safely resumed by patients and were not associated with liver re-injury in 27 patients. Therefore, the MH test identifies drugs that cause liver injury with 92.3% sensitivity and 100% specificity (1 false-negative and 12 true-positive results). CONCLUSIONS: We developed a test to identify drugs that cause liver injury in patients based on their toxicity to MH cells isolated from patients with DILI. We validated results from the assay and found it to identify drugs that cause DILI with 92.3% sensitivity and 100% specificity. The MH cell test could be a tool to identify causes of iDILI, even in patients taking multiple medications. ClinicalTrials.gov no: NCT 02353455.


Assuntos
Sobrevivência Celular/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/diagnóstico , Técnicas Citológicas/métodos , Testes Diagnósticos de Rotina/métodos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/diagnóstico , Monócitos/efeitos dos fármacos , Adolescente , Adulto , Idoso , Células Cultivadas , Feminino , Alemanha , Humanos , L-Lactato Desidrogenase/análise , Masculino , Pessoa de Meia-Idade , Monócitos/enzimologia , Sensibilidade e Especificidade , Adulto Jovem
17.
Toxicology ; 406-407: 9-20, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29777723

RESUMO

Inflammation is an important factor in the development of many diseases of the central nervous system, including Alzheimer's disease and other types of dementia. ‪Given that acetylcholinesterase inhibitors are also currently believed to have anti-inflammatory properties, the purpose of this study was to investigate the effect of acetylcholinesterase inhibitors (rivastigmine, donepezil) on cyclooxygenase activity and expression using the proinflammatory action of fluoride (F-) on cultured macrophages obtained from THP-1 monocytes. COX-1 and COX-2 activity was determined through measurement of the products of prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) in cell culture supernatants. Expression of COX-1 and COX-2 proteins was examined immunocytochemically, and mRNA expression was determined by qRT PCR. ‪‪ Our study confirmed the inhibitory effects of donepezil and rivastigmine on the production of PGE2, TXB2, COX-1 and COX-2 mRNA and protein expression in macrophages. We also demonstrated that the pro-inflammatory effect of fluoride may be reduced by the use of both drugs. The additive effect of these drugs cannot be ruled out, and effects other than those observed in the use of one drug should also be taken into account.


Assuntos
Inibidores da Colinesterase/farmacologia , Ciclo-Oxigenase 1/biossíntese , Ciclo-Oxigenase 2/biossíntese , Donepezila/farmacologia , Fluoretos/toxicidade , Rivastigmina/farmacologia , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 2/genética , Regulação Enzimológica da Expressão Gênica , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Monócitos/efeitos dos fármacos , Monócitos/enzimologia , Células THP-1
18.
Food Chem Toxicol ; 116(Pt B): 238-248, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29630947

RESUMO

Chalcones present in edible plants possess anti-cancer and anti-inflammatory properties, with the Michael acceptor moiety reported to be responsible for their biological activities. In this study, two novel dihydrotriazine-chalcone compounds previously identified to exert anti-proliferative effects through dual-targeting of dihydrofolate reductase (DHFR) and thioredoxin reductase (TrxR), were evaluated for their anti-invasive and anti-inflammatory abilities. At non-lethal concentrations, the compounds suppressed in vitro migration of MDA-MB-231 breast carcinoma cells, which was correlated with a dose-dependent downregulation of phorbol 12-myristate 13-acetate (PMA)-induced matrix metalloproteinase-9 (MMP-9) expression and secretion. At similar concentrations, these chalcone-based compounds suppressed expression of inflammatory mediators inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lipopolysaccharides (LPS)-stimulated murine macrophage-like RAW 264.7 cells, as well as tumor necrosis factor alpha (TNF-α) in LPS-stimulated human monocytes isolated from healthy donors. Mechanistically, inhibition of cancer cell invasion and inflammation by the compounds were mediated through suppression of the nuclear factor-kappaB (NF-κB) signaling pathway, which corroborated with the reported mechanism of action of chalcones. Their abilities to target multiple biological mediators relevant to multi-step carcinogenesis and with bioactivities stronger than those of the parent chalcone scaffold have warranted dihydrotriazine-chalcone compounds as promising candidates for use in pharmacological intervention of aggressive cancers.


Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Chalcona/farmacologia , Inflamação/prevenção & controle , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Triazinas/farmacologia , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/metabolismo , Regulação para Baixo , Ensaio de Imunoadsorção Enzimática , Humanos , Inflamação/induzido quimicamente , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/toxicidade , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Monócitos/efeitos dos fármacos , Monócitos/enzimologia , Monócitos/metabolismo , Invasividade Neoplásica/prevenção & controle , Óxido Nítrico Sintase Tipo II/metabolismo , Células RAW 264.7 , Acetato de Tetradecanoilforbol/farmacologia
19.
Pharmazie ; 73(4): 202-206, 2018 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-29609686

RESUMO

OBJECTIVES: To investigate whether there are aberrant acetylation modifications in global histone and monocyte chemoattractant protein-1 (MCP-1) promoter in monocytes from patients with coronary artery disease (CAD) and demonstrate the potential mechanisms. METHODS: CD14+ monocytes were isolated from 13 patients with CAD and 18 confirmed non-CAD controls using magnetic beads. Global histone H3/H4 acetylation and H3K4/H3K27 tri-methylation levels were measured with enzyme-linked immunosorbent assay. Quantitative real time-PCR was performed to detect the mRNA expression levels of MCP-1 and enzymes involved in histone modification processes. Histone modification levels in MCP-1 promoter were assessed by ChIP-qPCR assay. RESULTS: Our results showed a markedly lower global histone H3 acetylation level in monocytes from CAD patients. Global H3K27 tri-methylation level was significantly increased in monocytes from CAD patients. Furthermore, the mRNA expression levels of epigenetic modification enzymes HDAC3, SIRT1, P300, JMJD3 and SUV39H1 were decreased significantly in monocytes from CAD patients, while HDAC7 mRNA expression level was markedly increased. MCP-1 mRNA expression level was increased histone H3/H4 acetylation levels in MCP-1 promoter were markedly increased in monocytes of CAD patients. CONCLUSION: Aberrant histone modifications, including acetylation and tri-methylation, were found both in global histone and specific MCP-1 gene locos in monocytes from patients with CAD. Aberrant epigenetic modification enzymes expressions may be the regulatory mechanism responsible for aberrant histone modifications.


Assuntos
Quimiocina CCL2/genética , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/metabolismo , Histonas/metabolismo , Receptores de Lipopolissacarídeos/genética , Receptores de Lipopolissacarídeos/metabolismo , Monócitos/metabolismo , Acetilação , Feminino , Histonas/química , Histonas/genética , Humanos , Masculino , Pessoa de Meia-Idade , Monócitos/enzimologia , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional
20.
Blood ; 131(14): 1600-1610, 2018 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-29437594

RESUMO

Patients with sickle cell disease (SCD) suffer from intravascular hemolysis associated with vascular injury and dysfunction in mouse models, and painful vaso-occlusive crisis (VOC) involving increased attachment of sickle erythrocytes and activated leukocytes to damaged vascular endothelium. Patrolling monocytes, which normally scavenge damaged cells and debris from the vasculature, express higher levels of anti-inflammatory heme oxygenase 1 (HO-1), a heme degrading enzyme. Here, we show that HO-1-expressing patrolling monocytes protect SCD vasculature from ongoing hemolytic insult and vaso-occlusion. We found that a mean 37% of patrolling monocytes from SCD patients express very high levels of HO-1 (HO-1hi) vs 6% in healthy controls and demonstrated that HO-1hi expression was dependent on uptake of heme-exposed endothelium. SCD patients with a recent VOC episode had lower numbers of HO-1hi patrolling monocytes. Heme-mediated vaso-occlusion by mouse SCD red blood cells was exacerbated in mice lacking patrolling monocytes, and reversed following transfer of patrolling monocytes. Altogether, these data indicate that SCD patrolling monocytes remove hemolysis-damaged endothelial cells, resulting in HO-1 upregulation and dampening of VOC, and that perturbation in patrolling monocyte numbers resulting in lower numbers of HO-1hi patrolling monocyte may predispose SCD patients to VOC. These data suggest that HO-1hi patrolling monocytes are key players in VOC pathophysiology and have potential as therapeutic targets for VOC.


Assuntos
Anemia Falciforme/enzimologia , Heme Oxigenase-1/metabolismo , Hemólise , Monócitos/enzimologia , Doenças Vasculares/prevenção & controle , Adolescente , Adulto , Anemia Falciforme/complicações , Anemia Falciforme/patologia , Criança , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Monócitos/patologia , Doenças Vasculares/enzimologia , Doenças Vasculares/genética , Doenças Vasculares/patologia
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