RESUMO
BACKGROUND: Empagliflozin is a sodium-glucose cotransporter 2 (SGLT2) inhibitor that has demonstrated cardiovascular and renal protection in patients with type 2 diabetes (T2D). We hypothesized that empaglifozin (EMPA) could modulate ectopic fat stores and myocardial energetics in high-fat-high-sucrose (HFHS) diet mice and in type 2 diabetics (T2D). METHODS: C57BL/6 HFHS mice (n = 24) and T2D subjects (n = 56) were randomly assigned to 12 weeks of treatment with EMPA (30 mg/kg in mice, 10 mg/day in humans) or with placebo. A 4.7 T or 3 T MRI with 1H-MRS evaluation-myocardial fat (primary endpoint) and liver fat content (LFC)-were performed at baseline and at 12 weeks. In humans, standard cardiac MRI was coupled with myocardial energetics (PCr/ATP) measured with 31P-MRS. Subcutaneous (SAT) abdominal, visceral (VAT), epicardial and pancreatic fat were also evaluated. The primary efficacy endpoint was the change in epicardial fat volume between EMPA and placebo from baseline to 12 weeks. Secondary endpoints were the differences in PCr/ATP ratio, myocardial, liver and pancreatic fat content, SAT and VAT between groups at 12 weeks. RESULTS: In mice fed HFHS, EMPA significantly improved glucose tolerance and increased blood ketone bodies (KB) and ß-hydroxybutyrate levels (p < 0.05) compared to placebo. Mice fed HFHS had increased myocardial and liver fat content compared to standard diet mice. EMPA significantly attenuated liver fat content by 55%, (p < 0.001) but had no effect on myocardial fat. In the human study, all the 56 patients had normal LV function with mean LVEF = 63.4 ± 7.9%. Compared to placebo, T2D patients treated with EMPA significantly lost weight (- 2.6 kg [- 1.2; - 3.7]) and improved their HbA1c by 0.88 ± 0.74%. Hematocrit and EPO levels were significantly increased in the EMPA group compared to placebo (p < 0.0001, p = 0.041). EMPA significantly increased glycosuria and plasma KB levels compared to placebo (p < 0.0001, p = 0.012, respectively), and significantly reduced liver fat content (- 27 ± 23 vs. - 2 ± 24%, p = 0.0005) and visceral fat (- 7.8% [- 15.3; - 5.6] vs. - 0.1% [- 1.1;6.5], p = 0.043), but had no effect on myocardial or epicardial fat. At 12 weeks, no significant change was observed in the myocardial PCr/ATP (p = 0.57 between groups). CONCLUSIONS: EMPA effectively reduced liver fat in mice and humans without changing epicardial, myocardial fat or myocardial energetics, rebutting the thrifty substrate hypothesis for cardiovascular protection of SGLT2 inhibitors. Trial registration NCT, NCT03118336. Registered 18 April 2017, https://clinicaltrials.gov/ct2/show/NCT03118336.
Assuntos
Tecido Adiposo/efeitos dos fármacos , Compostos Benzidrílicos/uso terapêutico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Metabolismo Energético/efeitos dos fármacos , Glucosídeos/uso terapêutico , Fígado/efeitos dos fármacos , Miocárdio/metabolismo , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Animais , Compostos Benzidrílicos/efeitos adversos , Biomarcadores/sangue , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Modelos Animais de Doenças , Método Duplo-Cego , França , Glucosídeos/efeitos adversos , Hemoglobinas Glicadas/metabolismo , Humanos , Fígado/metabolismo , Fígado/patologia , Camundongos Endogâmicos C57BL , Miocárdio/patologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Espectroscopia de Prótons por Ressonância Magnética , Inibidores do Transportador 2 de Sódio-Glicose/efeitos adversos , Fatores de Tempo , Resultado do Tratamento , Redução de Peso/efeitos dos fármacosRESUMO
BACKGROUND/OBJECTIVES: Lymphocytes have a critical role in visceral adipose tissue (AT) inflammation. The CD28 costimulatory molecule is required for lymphocyte activation and for the development of a functional regulatory T cells (Tregs) compartment; however, its role during obesity is unknown. METHODS: During diet-induced obesity, we investigated the effects of selective interference with CD28 signaling using knockout mice (Cd28KO) and a CTLA4-Ig fusion protein inhibiting CD28-B7 interactions. RESULTS: Cd28 deficiency decreased pathogenic T cells and Treg content within AT without changing the macrophages number. Cd28KO epididymal but not subcutaneous fat was characterized by enlarged adipocytes, reduced levels of inflammatory cytokines and increased Glut4, adiponectin and lipogenic enzyme mRNA levels. This was associated with reduced inflammation, fat accumulation and enhanced glucose metabolism in liver. Weight gain and fasting glucose tolerance were not affected. CTLA4-Ig injections reduced the number of T cells in epididymal AT (epiAT) but not the inflammatory cytokines levels and failed to improve liver fat accumulation. CONCLUSIONS: Deletion of CD28 creates a new pro/anti-inflammatory balance in epiAT and liver and exerts a protective effect against hepatic steatosis.
Assuntos
Tecido Adiposo/patologia , Antígenos CD28/genética , Fígado Gorduroso/patologia , Deleção de Genes , Inflamação/patologia , Fígado/patologia , Obesidade/patologia , Animais , Modelos Animais de Doenças , Inflamação/metabolismo , Resistência à Insulina , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metabolismo dos Lipídeos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Membro 7 da Superfamília de Receptores de Fatores de Necrose TumoralRESUMO
BACKGROUND: The role of plasminogen activator inhibitor type-1 (PAI-1) in abdominal sepsis remains elusive. OBJECTIVES: To study the influence of inhibition and over-expression of PAI-1 upon survival in cecal ligation and puncture (CLP) sepsis. METHODS: (i) Mice underwent moderate CLP and received 10 mg kg(-1) of either monoclonal anti-PAI-1 (MA-MP6H6) or control (MA-Control) antibody intravenously at 0, 18 or 30 h post-CLP. The 30-h treatment group was additionally stratified into mice predicted to survive (P-SUR) or die (P-DIE) based on IL 6 measured at 24 h post-CLP. (ii) PAI-1 expression was induced with pLIVE.PAI-1 plasmid administered 72 h pre-CLP. Blood was sampled for 5 days and survival was monitored for 28 days. RESULTS: MA-MP6H6 effectively neutralized active PAI-1 and fully restored fibrinolysis while PAI-1 over-expression was liver-specific and correlated with PAI-1 increase in the blood. Without stratification, MA-MP6H6 co-/post-treatment conferred no survival benefit. Prospective stratification (IL-6 cut-off: 14 ng mL(-1) ) suggested increased mortality by MA-MP6H6 treatment in P-SUR that reached 30% difference (vs. MA-Control; P < 0.05) after a retrospective cut-off readjustment to 3.3 ng mL(-1) for better P-SUR homogeneity. Subsequent prospective anti-PAI-1 treatment in P-SUR mice with 3.3 ng mL(-1) cut-off demonstrated a negative but statistically insignificant effect: mortality was higher by 17% after MA-MP6H6 vs. MA-Control. Over-expression of PAI 1 did not alter post-CLP survival. Neither PAI-1 inhibition nor over-expression meaningfully modified inflammatory response and/or organ function. CONCLUSIONS: Restoration of fibrinolysis in early abdominal sepsis was not beneficial and it may prove detrimental in subjects with the lowest risk of death, while preemptive PAI-1 up-regulation at the current magnitude was not protective.
Assuntos
Anticorpos Monoclonais/farmacologia , Ceco/cirurgia , Terapia Genética , Fígado/efeitos dos fármacos , Peritonite/terapia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Sepse/terapia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/toxicidade , Biomarcadores/sangue , Ceco/microbiologia , Modelos Animais de Doenças , Esquema de Medicação , Feminino , Fibrinólise/efeitos dos fármacos , Fibrinólise/genética , Mediadores da Inflamação/sangue , Injeções Intravenosas , Interleucina-6/sangue , Ligadura , Fígado/metabolismo , Fígado/microbiologia , Camundongos , Peritonite/sangue , Peritonite/genética , Peritonite/microbiologia , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/imunologia , Punções , Sepse/sangue , Sepse/genética , Sepse/microbiologia , Fatores de Tempo , Regulação para CimaRESUMO
We reported, in previous experiments, that AA is a global regulator of cAMP pools. In this study, we demonstrate that K873, an analog of AA we synthesized and presenting antiproliferative properties, has also an impact on cAMP production. However, K873 has no antioxidant activity, at the contrary of AA. It definitively demonstrates that action of AA on the cAMP production is not linked to antioxidant activity. These data suggest that AA, and derivatives of this molecule, could be promising drug acting on biological processes that are under the control of cAMP dependent pathway.
Assuntos
Ácido Ascórbico/análogos & derivados , Ácido Ascórbico/química , AMP Cíclico/metabolismo , Organofosfatos/farmacologia , Animais , Antioxidantes/metabolismo , Ácido Ascórbico/farmacologia , Linhagem Celular , Proliferação de Células , Relação Dose-Resposta a Droga , Camundongos , Proteínas da Mielina/metabolismo , Técnicas de Cultura de Órgãos/métodos , Ratos , Células de Schwann/citologia , Nervo Isquiático/metabolismo , beta Caroteno/metabolismoRESUMO
AIMS/HYPOTHESIS: Obesity is associated with adipose tissue inflammation. The CD40 molecule, TNF receptor superfamily member 5 (CD40)/CD40 ligand (CD40L) pathway plays a role in the onset and maintenance of the inflammatory reaction, but has not been studied in human adipose tissue. Our aim was to examine CD40 expression by human adipocytes and its participation in adipose tissue inflammation. METHODS: CD40 expression was investigated in human whole adipose tissue and during adipocyte differentiation by real-time PCR, Western blot and immunohistochemistry. The CD40/CD40L pathway was studied using recombinant CD40L (rCD40L) in adipocyte culture and neutralising antibodies in lymphocyte/adipocyte co-culture. RESULTS: CD40 mRNA levels in subcutaneous adipose tissue were higher in the adipocyte than in the stromal-vascular fraction. CD40 expression was upregulated during adipocyte differentiation. Addition of rCD40L to adipocytes induced mitogen activated protein kinase (MAPK) activation, stimulated inflammatory adipocytokine production, and decreased insulin-induced glucose transport in parallel with a downregulation of IRS1 and GLUT4 (also known as SCL2A4). rCD40L decreased the expression of lipogenic genes and increased lipolysis. CD40 mRNA levels were significantly higher in subcutaneous adipose tissue than in visceral adipose tissue of obese patients and were positively correlated with BMI, and with IL6 and leptin mRNA levels. Lymphocyte/adipocyte co-culture led to an upregulation of proinflammatory adipocytokines and a downregulation of leptin and adiponectin. Physical separation of the two cell types attenuated these effects, suggesting the involvement of a cell-cell contact. Blocking the CD40/CD40L interaction with neutralising antibodies reduced IL-6 secretion from adipocytes. CONCLUSIONS/INTERPRETATION: Adipocyte CD40 may contribute to obesity-related inflammation and insulin resistance. T lymphocytes regulate adipocytokine production through both the release of soluble factor(s) and heterotypic contact with adipocytes involving CD40.
Assuntos
Adipócitos/metabolismo , Antígenos CD40/genética , Antígenos CD40/metabolismo , Linfócitos/metabolismo , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adiponectina/metabolismo , Adrenomedulina/metabolismo , Animais , Western Blotting , Ligante de CD40/farmacologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Técnicas de Cocultura , Expressão Gênica/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Linfócitos/efeitos dos fármacos , Camundongos , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Tumor necrosis factor (TNF) is a major cytokine involved in inflammatory reaction and a mortality predictor in patients with coronary artery disease (CAD). Plasma levels of soluble TNF (sTNF) depend on the rate of its synthesis but also on its shedding from cell surface, a mechanism mainly regulated by the TNF alpha converting enzyme (TACE or ADAM17). We investigated the relationship between ADAM17 and TNF polymorphisms, circulating levels of shed ADAM17 substrates (sTNF, sTNFR1 and sTNFR2), and cardiovascular risk in a prospective cohort of CAD patients. Five tag single-nucleotide polymorphisms (SNPs) of the ADAM17 gene as well as four previously described TNF SNPs were genotyped in the Atherogene Study composed of 1,400 CAD patients among which 136 died from a cardiovascular (CV) cause. sTNF, sTNFR1, and sTNFR2 concentrations were all significantly elevated in patients with future CV death, independently of other clinical/biological variables. While none of the studied TNF SNPs was associated with sTNF, sTNFR1, nor sTNFR2 levels, the ADAM17 -154A allele was found associated with a 14% increase of sTNF levels as compared to the -154C allele (p = 0.0066). Moreover, individuals carrying the 747Leu allele displayed a borderline increased risk of future cardiovascular death [odds ratio, 2.06 (1.05-4.04), p = 0.03]. These results suggest a role of ADAM17 in the regulation of sTNF plasma levels and identifies ADAM17 gene as a candidate for CAD. Tumor necrosis factor (TNF) is a major cytokine involved in inflammatory reaction and a mortality predictor in patients with coronary artery disease (CAD). We have studied the association of ADAM17 and TNF polymorphisms with circulating levels of shed ADAM17 substrates (sTNF, sTNFR1 and sTNFR2) and with cardiovascular risk in a large population of individuals with CAD (Atherogene Study, n = 1,400). Two newly identified polymorphisms, obtained by a systematic sequencing of the ADAM17 gene, C-154A and Ser747leu, slightly influence respectively sTNF plasma levels and the risk of cardiovascular death.
Assuntos
Proteínas ADAM/genética , Doença da Artéria Coronariana/genética , Polimorfismo de Nucleotídeo Único , Fator de Necrose Tumoral alfa/genética , Proteína ADAM17 , Idoso , Alelos , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/mortalidade , Feminino , Frequência do Gene , Predisposição Genética para Doença/genética , Genótipo , Haplótipos , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Receptores Tipo I de Fatores de Necrose Tumoral/sangue , Receptores Tipo II do Fator de Necrose Tumoral/sangue , Taxa de Sobrevida , Fator de Necrose Tumoral alfa/sangueRESUMO
TNFalpha (TNF) critically regulates inflammation-driven atherosclerosis. Because the transmembrane (tmTNF) and soluble (sTNF) forms of TNF possess distinct immuno-modulatory properties, we hypothesized that they might differentially regulate atherosclerosis progression. Three groups of male ApoE(-/-) mice were studied: one expressing wild-type TNF (WT-TNF); one expressing exclusively a mutated non-cleavable form of TNF (KI-TNF); and one deficient in TNF (KO-TNF). Mice aged 5 weeks were fed the high-fat diet for 5 (T5) and 15 weeks (T15) or a standard chow diet for 15 weeks. At T5, in mice fed the high-fat diet, no significant differences in lesion area were observed among the three groups, either in valves or in aortas. At T15, lesion areas in valves were significantly lower in KO-TNF mice compared with those in WT-TNF mice, whereas in KI-TNF mice, they were intermediate between KO- and WT-TNF mice but not significantly different from these two groups. In aortas, lesions in KI-TNF were comparable to those of KO-TNF, both being significantly lower than those in WT-TNF. Theses differences were not linked to circulating lipids, or to macrophage, actin, and collagen contents of lesions. At T15, in mice fed the chow diet, lesion areas in valves and the aortic arch were not significantly different between the three groups. Levels of IL-6, IFNgamma, IL-10, and Foxp3 mRNAs in spleens and production of IL-6, IL-10, MCP-1, RANTES, and TNFR-2 by peritoneal macrophages at T15 of the high-fat diet showed a decrease in pro-inflammatory status, more marked in KO-TNF than in KI-TNF mice. Apoptosis was reduced only in KO-TNF mice. In conclusion, these data show that TNF effects on atherosclerosis development are detectable at stages succeeding fatty streaks and that wild-type TNF is superior to tmTNF alone in promoting atherosclerosis. TNF-dependent progression of atherosclerosis is probably linked to the differential production of pro-inflammatory mediators whether tmTNF is preponderant or essentially cleaved.
Assuntos
Apolipoproteínas E/deficiência , Aterosclerose/patologia , Fator de Necrose Tumoral alfa/genética , Animais , Doenças da Aorta/genética , Doenças da Aorta/metabolismo , Doenças da Aorta/patologia , Valva Aórtica/patologia , Apoptose , Aterosclerose/genética , Aterosclerose/metabolismo , Colesterol/sangue , Dieta Aterogênica , Progressão da Doença , Expressão Gênica , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , RNA Mensageiro/genética , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
BACKGROUND: Plasma plasminogen activator inhibitor-1 (PAI-1) level rises during sepsis and confers a worse prognosis. PAI-1 participation to sepsis has been poorly documented and was mainly associated with fibrin deposits. Beside fibrin deposits, increased tissue PAI-1 expression may contribute to the poor outcome of endotoxemia through other mechanisms. OBJECTIVE AND METHODS: During lipopolysaccharide (LPS) challenge, the role of PAI-1 in the early phase of inflammation was examined in the lungs of transgenic mice that either overexpress or lack the PAI-1 gene (PAI-1Tg or PAI-1(-/-)). RESULTS: Analysis of leukocytes revealed that neutrophil and macrophage infiltrations did not differ for PAI-1Tg and wild-type (WT) mice. Remarkably, CD25+ lymphocyte infiltration was totally blunted in PAI-1Tg lungs and inversely correlated with fibrin depositions. In parallel, mRNA levels of the regulatory T cell (Treg) markers FoxP3, CTLA-4, and GITR were significantly lower in PAI-1Tg than in WT lungs after LPS challenge. These data are supported by opposite results in PAI-1(-/-) lungs. The systemic compartments (spleen and peripheral blood) showed no decrease in CD25+, CD4+ CD25+ lymphocytes, and Treg markers in PAI-1Tg mice after LPS injection compared with WT mice. In addition, plasma and lung concentrations of interleukin-6 (IL-6) and macrophage inflammatory protein-1alpha (MIP-1alpha) were significantly higher in PAI-1Tg mice than WT mice. CONCLUSION: Our results suggest that chronic tissue PAI-1 overexpression influences the early phase of the inflammatory response during endotoxemia through the control of T lymphocyte traffic.
Assuntos
Quimiotaxia de Leucócito , Endotoxemia/metabolismo , Imunidade Inata , Inflamação/etiologia , Subunidade alfa de Receptor de Interleucina-2/análise , Pulmão/metabolismo , Serpinas/metabolismo , Linfócitos T/imunologia , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Antígeno CTLA-4 , Quimiocina CCL3/metabolismo , Modelos Animais de Doenças , Endotoxemia/induzido quimicamente , Endotoxemia/complicações , Endotoxemia/imunologia , Endotoxemia/patologia , Fibrina/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Proteína Relacionada a TNFR Induzida por Glucocorticoide , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Interferon gama/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos , Pulmão/imunologia , Pulmão/patologia , Macrófagos/imunologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Neutrófilos/imunologia , Fibrose Pulmonar/etiologia , Fibrose Pulmonar/imunologia , Fibrose Pulmonar/metabolismo , RNA Mensageiro/metabolismo , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/metabolismo , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/metabolismo , Serpina E2 , Serpinas/deficiência , Serpinas/genética , Linfócitos T/metabolismo , Linfócitos T/patologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Fatores de Tempo , Regulação para CimaRESUMO
AIMS/HYPOTHESIS: Inflammation is associated with obesity and has been implicated in the development of diabetes and atherosclerosis. During gram-negative bacterial infection, lipopolysaccharide causes an inflammatory reaction via toll-like receptor 4 (TLR4), which has an essential function in the induction of innate and adaptative immunity. Our aim was to determine what role TLR4 plays in the development of metabolic phenotypes during high-fat feeding. MATERIALS AND METHODS: We evaluated metabolic consequences of a high-fat diet in TLR4 mutant mice (C3H/HeJ) and their respective controls. RESULTS: TLR4 inactivation reduced food intake without significant modification of body weight, but with higher epididymal adipose tissue mass and adipocyte hypertrophy. It also attenuated the inflammatory response and increased glucose transport and the expression levels of adiponectin and lipogenic markers in white adipose tissue. In addition, TLR4 inactivation blunted insulin resistance induced by lipopolysaccharide in differentiated adipocytes. Increased feeding efficiency in TLR4 mutant mice was associated with lower mass and lower expression of uncoupling protein 1 gene in brown adipose tissue. Finally, TLR4 inactivation slowed the development of hepatic steatosis, reducing the liver triacylglycerol content and also expression levels of lipogenic and fibrosis markers. CONCLUSIONS/INTERPRETATION: TLR4 influences white adipose tissue inflammation and insulin sensitivity, as well as liver fat storage, and is important in the regulation of metabolic phenotype during a fat-enriched diet.
Assuntos
Tecido Adiposo/fisiologia , Gorduras na Dieta/farmacologia , Resistência à Insulina/genética , Mutação , Receptor 4 Toll-Like/genética , Tecido Adiposo/fisiopatologia , Animais , Glicemia/metabolismo , Glicólise , Inflamação , Lipídeos/fisiologia , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Triglicerídeos/sangueRESUMO
Inhibitors of HMG-CoA reductase (statins) lower the level of circulating LDL-C by blocking the activity of HMG-CoA reductase. Their efficiency to prevent cardiovascular events was demonstrated in several clinical trials for primary and secondary prevention. However, subgroups analysis of trials together with experimental studies have increasingly documented that the beneficial effects of statins extend beyond the sole reduction in LDL-C. These effects include improvements of vasoreactivity, haemostasis and plaque stability, reduction of pro-inflammatory events such as a decrease in monocyte adhesion and infiltration, oxidation level and pericellular proteolysis. Possible repair of ischemic tissues through enhancement of mobilization of endothelial progenitor cells are also described, although more investigation are needed to clearly identify the role and safety of statins in angiogenesis. These pleiotropic effects are generally explained by the fact that statins inhibit the intracellular production of metabolites located downstream of mevalonate in the cholesterol pathway, such as isoprenoids (farnesyl pyrophosphate and geranylgeranylpyrophosphate). These hydrophobic metabolites allow the membrane anchorage of small G proteins (Ras and Rho) as well as the Gy subunit of heterotrimeric G proteins, a post-translational step that is critical in the regulation of G protein signaling activity. These drugs are therefore a valuable tool not only for the clinician but also for the biologist, allowing to investigate the regulation of gene expression that is controlled by the intracellular activity of membrane-anchored prenylated signaling proteins.
Assuntos
Doença da Artéria Coronariana/prevenção & controle , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Proteína C-Reativa/análise , LDL-Colesterol/sangue , LDL-Colesterol/efeitos dos fármacos , Humanos , Inflamação/sangue , Neovascularização Patológica/tratamento farmacológico , Sistema Vasomotor/efeitos dos fármacosRESUMO
Migration and proliferation of arterial smooth muscle cells (SMCs) play a prominent role in the development of atherosclerotic plaques and restenosis lesions. Most of the growth-regulatory molecules potentially involved in these pathological conditions also demonstrate chemotactic properties. Extracellular purine and pyrimidine nucleotides have been shown to induce cell cycle progression and to elicit growth of cultured vascular SMCs. Moreover, the P2Y(2) ATP/UTP receptor was overexpressed in intimal thickening, suggesting a role of these nucleotides in vascular remodeling. Using the Transwell system migration assay, we demonstrate that extracellular ATP, UTP, and UDP exhibit a concentration-dependent chemotactic effect on cultured rat aortic SMCs. UTP, the most powerful nucleotide inducer of migration, elicited significant responses from 10 nmol/L. In parallel, UTP increased osteopontin expression dose-dependently. The blockade of osteopontin or its integrin receptors alpha(v)beta(3)/beta(5) by specific antibodies or antagonists inhibited UTP-induced migration. Moreover, the blockade of ERK-1/ERK-2 MAP kinase or rho protein pathways led to the inhibition of both UTP-induced osteopontin increase and migration, demonstrating the central role of osteopontin in this process. Taken together, these results suggest that extracellular nucleotides, and particularly UTP, can induce arterial SMC migration via the action of osteopontin.
Assuntos
Movimento Celular/fisiologia , Espaço Extracelular/metabolismo , Músculo Liso Vascular/metabolismo , Nucleotídeos/farmacologia , Sialoglicoproteínas/metabolismo , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Aorta , Cálcio/metabolismo , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/fisiologia , Cultura em Câmaras de Difusão , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Oligopeptídeos/farmacologia , Osteopontina , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Ratos Wistar , Difosfato de Uridina/farmacologia , Uridina Trifosfato/farmacologia , Quinases Associadas a rho , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
The fibrinolytic inhibitor plasminogen activator inhibitor type 1 (PAI-1) plays a role in the development of atherothrombosis and is produced by macrophages that infiltrate the atherosclerotic vessel wall. Because statins are effective in reducing atherosclerosis, we investigated if they modulate the synthesis of PAI-1 in human monocytes/macrophages. To this end, we studied the effect of atorvastatin in different models of monocyte/macrophage differentiation, such as differentiated human promyelocytic cell line HL-60 and human peripheral blood monocyte-derived macrophages. HL-60 cells were differentiated along monocyte lineage by phorbol myristate acetate (PMA) or a mixture of transforming growth factor-beta type 1 (TGF-beta1)/1alpha,25-dihydroxyvitamin D3 (D3). In these conditions, PAI-1 synthesis was strongly induced and atorvastatin upregulated this synthesis, especially during TGF-beta1/D3-induced differentiation. Recombinant human tumor necrosis factor-alpha (TNF-alpha) strongly upregulated PAI-1 synthesis in PMA- or TGF-beta1/D3-differentiated cells, and the potentiating effect of atorvastatin was of the same order as in the absence of TNF-alpha. Mevalonate reversed the enhancing effect of atorvastatin. In mature human monocyte-derived macrophages, atorvastatin, alone or in combination with TNF-alpha, TGF-beta1, or PMA, did not exert any significant effect on PAI-1 synthesis. Basal production of urokinase (uPA), which was below detection limits in HL-60 cells and very low in human monocyte-derived macrophages, was not altered by atorvastatin. These results show that atorvastatin upregulates PAI-1 synthesis during the early stages of monocyte/macrophage differentiation, but has no effect on PAI-1 and uPA synthesis in mature human monocyte-derived macrophages. Atorvastatin did not significantly interact with the upregulating action of TNF-alpha on PAI-1 synthesis during differentiation.
Assuntos
Ácidos Heptanoicos/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Macrófagos/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Inibidor 1 de Ativador de Plasminogênio/biossíntese , Pirróis/farmacologia , Atorvastatina , Células HL-60 , Humanos , Macrófagos/metabolismo , Monócitos/metabolismo , Acetato de Tetradecanoilforbol/metabolismo , Fator de Crescimento Transformador beta/biossíntese , Fator de Necrose Tumoral alfa/biossínteseRESUMO
We investigated intracellular mechanisms involved in the up-regulation of plasminogen activator inhibitor I (PAI-1) synthesis by human recombinant tumor necrosis factor-alpha (TNF) during monocyte differentiation of HL-60 cells triggered by the transforming growth factor-beta1/vitamin D(3) (TGF/D3) mixture. TGF/D3-treated cells expressed surface monocytic markers and produced noticeable amounts of PAI-1 but stopped to proliferate. A reduced p70 S6 kinase (p70(S6K)) phosphorylation was also observed and, in this situation, TNF dramatically enhanced PAI-1 synthesis. Similarly, TNF significantly up-regulated PAI-1 synthesis when p70(S6K) phosphorylation was inhibited by rapamycin. This phenomenon was not due to a general decrease in protein synthesis but involved the activation of gene transcription rather than PAI-1 mRNA stabilization. The level of the transcriptional regulator factor E2F1, a repressor of PAI-1 gene expression, was shown to be down-modulated in TGF/D3- as well as in rapamycin-treated cells. Furthermore, the apoptotic effect of TNF in HL-60 cells appeared to be prevented by the addition of either TGF/D3 or rapamycin. In conclusion, these results indicate that inhibition of p70(S6K) phosphorylation during TGF/D3-induced monocyte differentiation of HL-60 cells is a determinant factor that allows TNF to exert its up-regulating effect on PAI-1 synthesis while protecting cells from apoptosis.
Assuntos
Proteínas de Ciclo Celular , Diferenciação Celular/efeitos dos fármacos , Colecalciferol/farmacologia , Proteínas de Ligação a DNA , Monócitos/efeitos dos fármacos , Inibidor 1 de Ativador de Plasminogênio/biossíntese , Proteínas Quinases S6 Ribossômicas/antagonistas & inibidores , Fator de Crescimento Transformador beta/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Citometria de Fluxo , Células HL-60 , Humanos , Monócitos/citologia , Monócitos/metabolismo , Inibidor 1 de Ativador de Plasminogênio/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional , Regulação para CimaRESUMO
Inhibitors of HMG-CoA reductase, namely statins, improve endothelial function independently of their cholesterol-lowering effects. Plasminogen activator inhibitor type-1 (PAI-1) plays a critical role in vascular pathophysiology both at the intra- and extravascular levels. We therefore investigated the effects of atorvastatin (ATOR) and fluvastatin (FLU) on PAI-1 and also tissue-type plasminogen activator (t-PA) synthesis in 20% fetal calf serum-cultured human umbilical vein endothelial cells (HUVEC) stimulated or not by recombinant human pro-inflammatory cytokines, i.e. tumor necrosis factor alpha (TNFalpha) and interleukin 1alpha (IL-1alpha). In non-stimulated HUVEC, ATOR and FLU significantly diminished (-50% at 2.0 micromol/l) the constitutive production of PAI-1 (mRNA level and protein secretion). This effect was prevented by addition of mevalonate (100 micromol/l). In HUVEC cultivated in 20% fetal calf serum, the t-PA antigen accumulation was not significantly altered, whereas in low serum concentration (1%) a significant stimulatory effect of ATOR (+30%) and FLU (+76%) was observed. In TNFalpha-stimulated cells, ATOR and FLU had a modest down-modulating effect (-17 and -20%, respectively) on TNFalpha-induced increase in PAI-1 synthesis. No effect of statins was observed in IL-1alpha-stimulated HUVEC, suggesting that statins do not interfere with the up-regulation of PAI-1 synthesis by pro-inflammatory cytokines. However, ATOR and FLU inhibited the TNFalpha-induced decrease in t-PA release. In conclusion, these results show that statins favorably modulate the expression of fibrinolytic factors produced by human endothelial cells.
Assuntos
Anticolesterolemiantes/farmacologia , Endotélio Vascular/metabolismo , Ácidos Graxos Monoinsaturados/farmacologia , Ácidos Heptanoicos/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Indóis/farmacologia , Inibidor 1 de Ativador de Plasminogênio/biossíntese , Pirróis/farmacologia , Atorvastatina , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Fluvastatina , Humanos , Interleucina-1/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativador de Plasminogênio Tecidual/biossíntese , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Tumor necrosis factor-alpha (TNFalpha) critically regulates several cellular functions during monocyte/macrophage differentiation. We therefore investigated during the phorbol ester (phorbol 12-myristate 13-acetate (PMA))-induced monocyte/macrophage differentiation of the human HL-60 leukemia cells, if TNFalpha contributed to plasminogen activator inhibitor type-1 (PAI-1) synthesis that is initiated by a protein kinase Cbeta-extracellular signal-regulated kinase 2-dependent pathway (Lopez, S., Peiretti, F., Morange, P., Laouar, A., Fossat, C., Bonardo, B., Huberman, E., Juhan-Vague, I., and Nalbone, G. (1999) Thromb. Haemostasis 81, 415-422). Following PMA treatment, the level of TNFalpha mRNA strongly increased and appeared earlier than PAI-1 mRNA. An anti-TNFalpha antibody significantly inhibited the PMA-induced PAI-1 mRNA and protein levels. The recombinant human TNFalpha, which is inactive on native HL-60 cells in terms of PAI-1 synthesis, optimally potentiates it once HL-60 cells are committed into the differentiation process. The use of 1) the HL-525 cell line, a clone issued from HL-60 cells rendered resistant to PMA-induced differentiation, and 2) the transforming growth factorbeta-1/vitamin D3 differentiative mixture confirmed the relationships between the induction of differentiation and the potency of TNFalpha to up-regulate PAI-1 synthesis. In conclusion, we showed that during the induction of monocyte/macrophage differentiation, TNFalpha and PAI-1 gene expressions are activated and that synthesized TNFalpha up-regulates and prolongs, in an autocrine manner, the synthesis of PAI-1.
Assuntos
Comunicação Autócrina , Leucemia/metabolismo , Leucemia/patologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Diferenciação Celular , Células HL-60 , Humanos , Monócitos/metabolismo , Monócitos/patologia , Regulação para CimaRESUMO
HL-60 cells treated by PMA develop the monocyte adherent phenotype and synthesize plasminogen activator inhibitor type-1 (PAI-1). We focused our study on the identification of the PMA-activated protein kinase C (PKC) isoform and its downstream transduction pathway activating PAI-1 synthesis. Acquisition of the monocytic phenotype was evidenced by cell adherence (90-95%) and a sharp increase of CD 36 and receptor for urokinase plasminogen activator (uPAR) surface expression. Ro 31-8220, a specific inhibitor of PKC, prevented PMA-induced PAI-1 synthesis (mRNA and protein levels) and cell adhesion. To identify the PKC isoform, we took advantage of the HL-525 cell line, an HL-60 cell variant deficient in PKCbeta gene expression. This defect prevents PMA to induce the differentiation process. HL-525 stimulated by PMA did not synthesize PAI-1 nor become adherent. However, in HL-525 cells either pretreated by retinoic acid that reinduces PKCbeta gene expression or transfected with PKCbeta cDNA, PMA significantly activated PAI-1 synthesis and adhesion of cells. Immunoblotting of active Mitogen Activated Protein Kinase (MAPK) p42/p44 in HL-60 cells showed a preferential and sustained activation of the p42 isoform by PMA over the p44 isoform. Ro 31-8220 significantly attenuated this activation. PD 098059 and U0126, both highly specific MEK inhibitors, efficiently prevented PMA-induced PAI-1 synthesis (mRNA and protein levels) and cell adhesion whereas SB203580, a specific inhibitor of stress-activated MAPK p38, did not. Results obtained from HL-60 and HL-525 cells indicate that the PMA-activated transduction pathway of uPAR expression involves a PKC isoform other than PKCbeta. In conclusion, we propose that the pathway PKCbeta-MEK-MAPK p42 is a potential linear route for PAI-1 synthesis leading to morphological changes and adherence linked to PMA-induced differentiation in HL-60 cells.
Assuntos
Carcinógenos/farmacologia , Isoenzimas/metabolismo , MAP Quinase Quinase Quinase 1 , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Inibidor 1 de Ativador de Plasminogênio/biossíntese , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Células HL-60 , Humanos , Isoenzimas/genética , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase C beta , Transdução de Sinais/efeitos dos fármacosRESUMO
Plasminogen activator inhibitor 1 (PAI-1) is likely to play a role in vascular disease, primarily in subjects with android obesity. It has been demonstrated that PAI-1 is overexpressed in adipose tissue from obese subjects and that visceral adipose tissue produced more PAI-1 than subcutaneous fat. In the present study, the effect of insulin and glucocorticoids, which are key mediators of adipose tissue metabolism, was examined in relation to PAI-1 synthesis by human adipose tissue explants (HAT), collagenase isolated human adipocytes (IHA), cultured human stromal cells (cSC), and differentiated adipocytes from the murine clonal cell line 3T3-F442A. A significant increase in PAI-1 antigen release (1.5-fold) from HAT was detectable after 16 h of treatment with insulin concentrations of at least 10(-8) mol/l. This was associated with a PAI-1 mRNA increase. Concomitant addition of insulin (10(-8) mol/l) to forskolin (5 x 10(-5) mol/l) reversed the decrease in PAI-1 antigen caused by forskolin alone. No effect on PAI-1 antigen was observed when insulin was incubated with IHA or cSC. 3T3 F442A cells were sensitive to insulin with a four- and twofold increase in PAI-1 antigen and mRNA levels, respectively, after 16 h of stimulation with 10(-8) mol/l. Dexamethasone (DXM) significantly enhanced PAI-1 antigen and mRNA expression by HAT (1.5- and 2.5-fold increase, respectively) at concentrations of at least 10(-8) mol/l. A higher stimulation was observed with IHA (sevenfold increase) and with the differentiated 3T3 F442 cell line. Cortisol was found to be less potent than DXM. No effect was observed when glucocorticoids were incubated with cSC. Coincubation of HAT with insulin (10(-7) mol/l) and DXM (10(-7) mol/l) led to an additive effect on PAI-1 synthesis. These results support the hypothesis that PAI-1 expression in human adipose tissue is controlled by insulin and glucocorticoids and may help to explain the increase in plasma PAI-1 levels observed in patients with android obesity.
Assuntos
Tecido Adiposo/metabolismo , Glucocorticoides/farmacologia , Insulina/farmacologia , Inibidor 1 de Ativador de Plasminogênio/biossíntese , Células 3T3 , Tecido Adiposo/citologia , Tecido Adiposo/efeitos dos fármacos , Adulto , Animais , Técnicas de Cultura , Dexametasona/farmacologia , Combinação de Medicamentos , Feminino , Humanos , Camundongos , Pessoa de Meia-Idade , Inibidor 1 de Ativador de Plasminogênio/genética , RNA Mensageiro/metabolismoRESUMO
We investigated in human umbilical vein endothelial cells (HUVECs) the interaction between the signaling pathways triggered by calcium mobilization and those affected by human recombinant tumor necrosis factor-alpha (TNF) on the expression of type-1 plasminogen activator inhibitor (PAI-1). Calcium ionophore A23187 alone exerted a modest increase (50%) on PAI-1 synthesis. TNF alone increased PAI-1 accumulation in the culture medium in a time- and dose-dependent fashion, but this increase was abolished when A23187 was added simultaneously with TNF. The downregulating effect of A23187 was not the result of impaired protein secretion, proteolysis, cytotoxicity, or an apoptotic process. A23187 did not decrease the TNF-enhanced PAI-1 mRNA level but did provoke a significant shift in the distribution pattern of PAI-1 transcripts by increasing the 2.3-kb relative to the 3.2-kb form. Comparable inhibitory effects on PAI-1 protein synthesis were observed when A23187 was added 7 hours after the onset of TNF stimulation, strongly suggesting a posttranscriptional inhibitory action of calcium signaling on TNF-stimulated PAI-1 synthesis. However, treatment with actinomycin D showed that PAI-1 mRNA stability was not altered by the various treatments. Chelation of extracellular calcium by EGTA did not prevent the A23187-induced inhibition of TNF-stimulated PAI-1 protein synthesis, emphasizing the role of internal calcium stores in the inhibition of PAI-1 synthesis. Sucrose gradient fractionation of cell lysates revealed that regardless of which treatment was used, both PAI-1 mRNA transcripts exhibited similar sedimentation profiles in the actively translating polysomal pool, suggesting that the A23187-induced shift had no functional consequence on translation. However, in TNF-stimulated cells, A23187 induced a higher proportion of PAI-1 mRNAs that sedimented in fractions corresponding to less dense polysomes, a phenomenon that usually reflects a slower initiation rate during mRNA translation. A23187 also abolished the increase in PAI-1 synthesis induced by recombinant human interleukin 1 beta, and thapsigargin exerted effects comparable to those of A23187 on PAI-1 synthesis in TNF-stimulated cells. It is proposed that in HUVECs, the A23187-induced release of calcium from endoplasmic stores suppresses at the translational level the increase in PAI-1 synthesis triggered by proinflammatory cytokines.
Assuntos
Cálcio/metabolismo , Endotélio Vascular/citologia , Membranas Intracelulares/química , Inibidor 1 de Ativador de Plasminogênio/biossíntese , Fator de Necrose Tumoral alfa/farmacologia , Apoptose/efeitos dos fármacos , Calcimicina/farmacologia , Células Cultivadas , Meios de Cultivo Condicionados , Estabilidade de Medicamentos , Endotélio Vascular/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Inibidor 1 de Ativador de Plasminogênio/sangue , Inibidor 1 de Ativador de Plasminogênio/genética , Biossíntese de Proteínas , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Cordão Umbilical/irrigação sanguínea , Veias/químicaRESUMO
Plasminogen activator inhibitor type 1 (PAI-1) contributes to the pathogenesis of atherothrombosis. Its plasma level is strongly correlated with parameters that define the insulin resistance syndrome, in particular with BMI and visceral accumulation of body fat, suggesting that PAI-1 may be an adipose tissue-derived circulating peptide. The present study was designed to investigate PAI-1 expression by human adipose tissue and its different cellular fractions. Special interest has been paid to the amount of PAI-1 antigen produced by omental versus subcutaneous fat. PAI-1 protein detected by immunolocalization was present at the stromal and adipocyte levels. PAI-1 mRNA was detected in stromal vascular cells freshly isolated and under culture conditions. It was also detected in whole adipose tissue and adipocyte fraction under culture conditions. The mRNA signal from the adipocyte fraction was detected as early as 2 h of incubation. The increase in PAI-1 mRNA was followed by an increase in PAI-1 antigen in the conditioned medium that was suppressed by treatment with cycloheximide. Transforming growth factor-beta1 significantly increased PAI-1 antigen production by the adipocyte fraction, whereas tumor necrosis factor-alpha did not have any effect. Interestingly, after 5 h of incubation, omental tissue explants produced significantly more PAI-1 antigen than did subcutaneous tissue from the same individual, whereas similar production of leptin by the two territories was observed. These results strongly suggest that human adipose tissue, in particular visceral tissue, can be an important contributor to the elevated plasma PAI-1 levels observed in central obesity.
Assuntos
Tecido Adiposo/metabolismo , Inibidor 1 de Ativador de Plasminogênio/biossíntese , Adipócitos/metabolismo , Adulto , Idoso , Antígenos/análise , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/imunologia , RNA Mensageiro/metabolismo , Vísceras/químicaRESUMO
The main function of plasminogen activator inhibitor type 1 (PAI-1) is to decrease fibrinolysis, which leads to fibrin accumulation. An elevated plasma PAI-1 concentration has been identified as a risk factor for the development of myocardial infarction, and an association between 1 polymorphism of the PAI-1 promoter and plasma PAI-1 levels has been described. Our aim was to identify new polymorphisms in the PAI-1 gene and to further examine the relationship between PAI-1 genotypes and circulating PAI-1 levels. We report the presence of 4 new polymorphisms that were identified by non-isotopic single-strand conformational polymorphism analysis followed by sequencing. These polymorphisms were investigated in relation to PAI-1 levels in a sample of 256 healthy men, aged 50-59 years, from France and Northern Ireland. Two G/A substitutions were detected at positions -844 and +9785. The former is in strong positive linkage disequilibrium with the previously described 4G/5G polymorphism at position -675. Two polymorphisms in the 3' untranslated region were identified. One corresponds to a T/G substitution at position +11,053 and is in negative linkage disequilibrium with the G/A substitution (+9785). The other is a 9-nucleotide insertion/deletion located between nucleotides +11,320 and +11,345 in a threefold-repeated sequence. This polymorphism is in strong positive linkage disequilibrium with the G/A substitution (+9785). The overall heterozygosity provided by the 5 PAI-1 polymorphisms (including the 4 new variants and the 4G/5G polymorphism) was .77. No significant association was found between PAI activity and genotypes; furthermore, the well known associations between PAI activity and body mass index, serum triglycerides, or insulin were homogeneous according to PAI-1 genotypes.