RESUMO
Peripheral arterial disease (PAD) is a chronic condition caused by atherosclerosis and is a severe complication of type 2 diabetes (T2D). We hypothesised that chronic condition of arterial disease engenders inflammation and endothelial damage in response to circulating cytokines released in the blood stream of PAD patients. We explored the levels of circulating cytokines in PAD patients with and without diabetes by multiplex cytokine array compared with non-PAD controls. Serum from PAD patients with or without diabetes showed high levels of VEGF, IFN-gamma, TNF-alpha, MCP-1, and EGF. VEGF levels correlated with TNF-alpha and IFN-gamma, significantly. Endothelial cells (ECs) were exposed to the different altered cytokines to evaluate changes in cell growth, migration and tubule-like formation, displaying impairment on proliferation, migration and tubule formation. Our findings demonstrate that a set of cytokines is significantly increased in the serum of PAD patients. These cytokines act to induce endothelial dysfunction synergistically. VEGF strongly correlated with TNF-alpha and IFN-gamma, opening new therapeutic perspectives.
Assuntos
Citocinas/sangue , Endotélio Vascular/fisiopatologia , Doença Arterial Periférica/sangue , Doença Arterial Periférica/fisiopatologia , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Hipóxia Celular , Movimento Celular , Proliferação de Células , Quimiocina CCL2/sangue , Citocinas/farmacologia , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/fisiopatologia , Endotélio Vascular/citologia , Fator de Crescimento Epidérmico/sangue , Feminino , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Interferon gama/sangue , Masculino , Pessoa de Meia-Idade , Doença Arterial Periférica/etiologia , Fator de Necrose Tumoral alfa/sangue , Fator A de Crescimento do Endotélio Vascular/sangueRESUMO
We show that human osteosarcoma cells (Saos-2) have downregulation of alpha3beta1-integrin compared to normal bone cells; this was further described in human osteosarcomas and in a primary murine sarcoma. The alpha3 gene was silenced in Saos-2 cells causing a low expression of alpha3beta1-integrin and reduction in collagen attachment with increasing migratory capacity. Chromatin immunoprecipitation assay performed on alpha3 promoter established that Myc and Yin Yang protein (YY1) cooperate in tandem to downregulate the alpha3 gene. This silencing mechanism involves the binding of Myc and YY1 to DNA and formation of complexes among Myc/Max, YY1, CREB-binding protein and deacetylation activity. The promoter containing deletions of E-boxes or YY1 cassettes failed to downregulate the transcription of a reporter gene as well as the inhibition of deacetylation activity. Overexpression of both Myc and YY1 was necessary to determine the alpha3-integrin promoter downregulation in normal osteoblasts. This downregulation of alpha3beta1-integrin can contribute to the acquisition of a more aggressive phenotype. YY1 regulated negatively the Myc activity through a direct interaction with the Myc/Max and deacetylase complexes. This represents a novel silencing mechanism with broad implications in the transcription machinery of tumours.
Assuntos
Inativação Gênica , Integrina alfa3/genética , Integrina alfa3beta1/metabolismo , Osteossarcoma/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fator de Transcrição YY1/metabolismo , Animais , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Western Blotting , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Imunoprecipitação da Cromatina , Colágeno/metabolismo , Regulação para Baixo , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Imunofluorescência , Humanos , Laminina/metabolismo , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteossarcoma/genética , Osteossarcoma/patologia , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , Transfecção , Células Tumorais Cultivadas , Fator de Transcrição YY1/antagonistas & inibidores , Fator de Transcrição YY1/genéticaRESUMO
Autophagy is an evolutionarily conserved process that mediates prominent homeostatic functions, both at the cellular and organismal level. Indeed, baseline autophagy not only ensures the disposal of cytoplasmic entities that may become cytotoxic upon accumulation, but also contributes to the maintenance of metabolic fitness in physiological conditions. Likewise, autophagy plays a fundamental role in the cellular and organismal adaptation to homeostatic perturbations of metabolic, physical, or chemical nature. Thus, the molecular machinery for autophagy is functionally regulated by a broad panel of sensors that detect indicators of metabolic homeostasis. Moreover, increases in autophagic flux have a direct impact on core metabolic circuitries including (but not limited to) glycolysis and mitochondrial respiration. Here, we detail a simple methodological approach to monitor these two processes in cultured cancer cells that mount a proficient autophagic response to stress.
Assuntos
Autofagia , Glicólise , Mitocôndrias/metabolismo , Técnicas de Cultura de Células/métodos , Células HCT116 , Humanos , Neoplasias/metabolismo , Consumo de OxigênioRESUMO
Macroautophagy is a specific variant of autophagy that involves a dedicated double-membraned organelle commonly known as autophagosome. Various methods have been developed to quantify the size of the autophagosomal compartment, which is an indirect indicator of macroautophagic responses, based on the peculiar ability of microtubule-associated protein 1 light chain 3 beta (MAP1LC3B; best known as LC3) to accumulate in forming autophagosomes upon maturation. One particularly convenient method to monitor the accumulation of mature LC3 within autophagosomes relies on a green fluorescent protein (GFP)-tagged variant of this protein and fluorescence microscopy. In physiological conditions, cells transfected temporarily or stably with a GFP-LC3-encoding construct exhibit a diffuse green fluorescence over the cytoplasm and nucleus. Conversely, in response to macroautophagy-promoting stimuli, the GFP-LC3 signal becomes punctate and often (but not always) predominantly cytoplasmic. The accumulation of GFP-LC3 in cytoplasmic dots, however, also ensues the blockage of any of the steps that ensure the degradation of mature autophagosomes, calling for the implementation of strategies that accurately discriminate between an increase in autophagic flux and an arrest in autophagic degradation. Various cell lines have been engineered to stably express GFP-LC3, which-combined with the appropriate controls of flux, high-throughput imaging stations, and automated image analysis-offer a relatively straightforward tool to screen large chemical or biological libraries for inducers or inhibitors of autophagy. Here, we describe a simple and robust method for the high-throughput quantification of GFP-LC3+ dots by automated fluorescence microscopy.
Assuntos
Autofagossomos/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Microscopia de Fluorescência/métodos , Proteínas Associadas aos Microtúbulos/análise , Automação , Linhagem Celular Tumoral , Citoplasma/metabolismo , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Humanos , Processamento de Imagem Assistida por Computador , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Neoplasias/metabolismo , Proteínas Recombinantes/análise , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Estradiol and progesterone receptors were assayed in tumors from 79 patients with primary colorectal and 56 patients with stomach adenocarcinomas. Eighteen of 79 colorectal cancers contained estradiol receptor, while 34 specimens were positive for progesterone receptor. In stomach cancer, the positive samples were 8 for estradiol and 14 for progesterone receptors. In both types of tumors, the Kd was in the range of 10(-10) M for estradiol and 10(-9) M for progesterone receptor, respectively. In colorectal adenocarcinomas, the presence of progesterone receptor seems to be partially correlated to the presence of estradiol receptor while, in stomach tumors, this correlation is lost. The positivity of at least one receptor in colorectal cancers is higher in the female sex. The contrary occurs for stomach cancer. Sucrose gradient centrifugation showed that cytoplasmic estradiol receptor of stomach cancer sedimented at 8S or 4 to 5S at low ionic strength. The isoelectric point of stomach cancer estradiol receptor is 6.5.
Assuntos
Adenocarcinoma/análise , Estradiol/análise , Neoplasias Gastrointestinais/análise , Receptores de Estrogênio/análise , Receptores de Progesterona/análise , Neoplasias do Colo/análise , Feminino , Humanos , Cinética , Masculino , Menopausa , Receptores de Estradiol , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Neoplasias Retais/análise , Neoplasias Gástricas/análiseRESUMO
HMG-CoA reductase inhibitors, such as Lovastatin and Simvastatin, cause cell cycle arrest by interfering with the mitogenic activity of mitogens present in culture media. Cells are induced to pause in G1 and can readily resume growth upon removal of the enzymatic block. Estrogens, acting via their nuclear receptor, are mitogens for different normal and transformed cell types, where they foster cell cycle progression and cell division. In estrogen-responsive MCF-7 human breast cancer cells, but not in non responsive cells, 17 beta-estradiol (E2) induces cells arrested with Lovastatin or Simvastatin to proliferate in the presence of inhibitor, without restoring HMG-CoA reductase activity or affecting the protein prenylation pattern. Mitogenic stimulation of G1-arrested MCF-7 cells with E2 includes primary transcriptional activation of c-fos, accompanied by transient binding in vivo of the estrogen receptor and/or other factors to the ERE and the estrogen-responsive DNA region of this proto-oncogene, as detected by dimethylsulphate genomic footprinting analysis. Mitogenic stimulation of growth-arrested MCF-7 cells by E2 occurs, under these conditions, without evident activation of ERK-1 and -2 kinases, and thus independently from the mitogen-responsive signal transduction pathways that converge on these enzymes.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Ciclo Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Estradiol/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases , Lovastatina/análogos & derivados , Lovastatina/farmacologia , Proteínas Quinases Ativadas por Mitógeno , Proteínas Proto-Oncogênicas c-fos/metabolismo , Sequência de Bases , Neoplasias da Mama , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Colesterol/biossíntese , Ativação Enzimática , Feminino , Fase G1/efeitos dos fármacos , Genes fos/efeitos dos fármacos , Humanos , Proteína Quinase 1 Ativada por Mitógeno , Proteína Quinase 3 Ativada por Mitógeno , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Proto-Oncogene Mas , Receptores de Estradiol/fisiologia , Sinvastatina , Ativação Transcricional/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
MCF-7 human breast cancer cells express functional estrogen receptor and grow in response to estrogen stimulation. G(1)-synchronized MCF-7 cells, made quiescent by exposure to the HMG-CoA reductase inhibitor Simvastatin in estrogen-free medium, readily resume cell cycle progression upon stimulation with 17beta-estradiol (E(2)), even under conditions where polypeptide growth factor-triggered signal transduction pathways are inhibited by the continuous presence of Simvastatin in the culture medium. Under these conditions, cyclin D(1) gene transcription is transiently induced within the first 1-9 h of stimulation, as shown by the accumulation of cyclin D(1) mRNA and protein (p36(D(1))) in the cell and by enhanced expression of stably transfected D(1) promoter-luciferase hybrid genes. Estrogen-induced p36(D(1)) associates readily with p32(cdk2) and p34(cdk4), but not with p31(cdk5), which is however abundantly expressed in these cells. Only p36(D(1))-p34(cdk4) complexes are activated by E(2), as detected in cell extracts by immunoprecipitation with anti-D(1) antibodies followed by assessment of phosphotransferase activity toward the retinoblastoma (Rb) gene product and by analysis of p105(Rb) phosphorylation in vivo. An estrogen-responsive regulatory region has been mapped within the first 944 bp upstream of the transcriptional startsite of the human D(1) gene. Sequence analysis of this DNA region reveals that the cis-acting elements responsive to estrogen are likely to be different in this case from the canonical EREs.
Assuntos
Neoplasias da Mama/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Estradiol/farmacologia , Fase G1/efeitos dos fármacos , Proteínas Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas , Proteína do Retinoblastoma/metabolismo , Transcrição Gênica/efeitos dos fármacos , Neoplasias da Mama/patologia , Ciclina D1 , Quinase 4 Dependente de Ciclina , Ciclinas/genética , DNA/biossíntese , Dactinomicina/farmacologia , Feminino , Expressão Gênica/efeitos dos fármacos , Genes Reporter , Humanos , Lovastatina/análogos & derivados , Lovastatina/farmacologia , Luciferases/genética , Luciferases/metabolismo , Proteínas Oncogênicas/genética , Fosforilação , RNA Mensageiro/metabolismo , Sinvastatina , Transfecção , Células Tumorais CultivadasRESUMO
New approaches to atherosclerosis-related diseases include novel uses of proven treatments and development of innovative agents. Several commonly used cardiovascular drugs such as dihydropyridine calcium antagonists, ACE inhibitors containing the sulphydryl group, or highly lipophilic beta-blockers have some anti-atherosclerotic activities. Moreover, new clinical trials suggesting that additional reduction of low-density lipoprotein cholesterol levels with statin therapy results in additional benefit in coronary heart disease prevention. Notably, new cholesterol transport or bile acid transport inhibitors have been found to produce significant reductions in intestinal cholesterol absorption and experimental atherosclerosis. Inhibitors of acyl coenzyme A:cholesterol acyltransferase, which can reduce cholesterol storage in macrophages and in arterial lesions, have also been developed. Finally, newer therapeutical strategies against atherogenesis may include the use of antioxidants and cholestyramine during pregnancy or the development of metalloproteinase inhibitors.
Assuntos
Arteriosclerose/tratamento farmacológico , Fármacos Cardiovasculares/uso terapêutico , Fármacos Cardiovasculares/classificação , Feminino , Humanos , Gravidez , Complicações na Gravidez/tratamento farmacológicoRESUMO
Ischemic preconditioning (IP) has been proposed as an endogenous form of protection against ischemia reperfusion injury. IP, however, does not prevent post-ischemic dysfunction in the aging heart but may be partially corrected by exercise training and food restriction. We investigated the role of exercise training combined with food restriction on restoring IP in the aging heart. Effects of IP against ischemia-reperfusion injury in isolated hearts from adult (A, 6 months old), sedentary 'ad libitum' fed (SL), trained ad libitum fed (TL), sedentary food-restricted (SR), trained- and food-restricted senescent rats (TR) (24 months old) were investigated. Norepinephrine release in coronary effluent was determined by high performance liquid cromatography. IP significantly improved final recovery of percent developed pressure in hearts from A (p<0.01) but not in those from SL (p=NS) vs unconditioned controls. Developed pressure recovery was partial in hearts from TL and SR (64.3 and 67.3%, respectively; p<0.05 vs controls) but it was total in those from TR (82.3%, p=NS vs A; p<0.05 vs hearts from TL and SR). Similarly, IP determined a similar increase of norepinephrine release in A (p<0.001) and in TR (p<0.001, p=NS vs adult). IP was abolished by depletion of myocardial norepinephrine stores by reserpine in all groups. Thus, IP reduces post-ischemic dysfunction in A but not in SL. Moreover, IP was preserved partially in TR and SR and totally in TR. Complete IP maybe due to full restoration of norepinephrine release in response to IP stimulus.
Assuntos
Envelhecimento/fisiologia , Restrição Calórica , Precondicionamento Isquêmico Miocárdico , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Condicionamento Físico Animal/fisiologia , Animais , Peso Corporal/fisiologia , Ventrículos do Coração/patologia , Masculino , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Norepinefrina/metabolismo , Tamanho do Órgão/fisiologia , Ratos , Ratos WistarRESUMO
Estrogen hormones induce transient transcriptional activation of c-fos during the early phases of mitogenic stimulation of target cells. This is mediated by a functional estrogen response element (ERE) that in the human c-fos gene is localized 1kb up-stream of the transcription start site. This is the first known example of transient transcriptional activation induced by a steroid hormone acting via its nuclear receptor. Starting with the hypothesis that the product of c-fos (Fos) interferes with estrogen receptor (ER) activity on this gene promoter, generating in this way a feedback inhibition mechanism responsible for the rapid transcriptional down-regulation detected in vivo, we tested the effects of Fos overexpression on ER-mediated activation of the c-fos promoter in transfected HeLa cells. Transient transfection of an ER expression vector is followed by hormone-dependent trans-activation of reporter genes comprising the c-fos ERE linked to its own promoter. Coexpression of Fos in the cell induces a significant reduction in the activity of ER on the reporter genes. Fos antagonism is effective on both transcription activation functions of the receptor molecule and is independent of the nature of the target promoter. Furthermore, under the same experimental conditions, the estrogen-receptor complex antagonizes activation of an AP-1-responsive test gene by Fos. ER mutants deprived of the DNA-binding domain are efficient inhibitors of Fos activity, indicating that reciprocal antagonism is likely to be mediated by the formation of inactive complexes between the two factors. These results reveal the existence of a functional interference between the ER and Fos for regulation of c-fos protooncogene transcription. It is the first case in which the product of an estrogen-induced growth-related gene is shown to exert a negative feedback control on ER regulation of its own promoter.
Assuntos
Genes fos , Proteínas Proto-Oncogênicas c-fos/farmacologia , Receptores de Estrogênio/metabolismo , Ativação Transcricional/efeitos dos fármacos , Animais , Sequência de Bases , Antagonismo de Drogas , Estradiol/farmacologia , Retroalimentação , Regulação da Expressão Gênica , Células HeLa/efeitos dos fármacos , Humanos , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Sequências Reguladoras de Ácido Nucleico , TransfecçãoRESUMO
Understanding the molecular and morphological basis of estrogen responsiveness in the various tissues and organs that make up an adult organism and its onset during ontogenesis requires identification of the genetic controls that determine timed expression of the estrogen receptor (ER) gene in multiple cell types. With this goal in mind, we describe here the results of the functional analysis of the mouse (m) ER gene promoter, carried out in vivo in transgenic mice. The mER gene promoter was cloned and spliced to the coding sequence of the bacterial lacZ gene (fused to the nuclear localization signal of SV40 large T: nls-beta-GAL) and then stably reintegrated into the genome of mice. Analysis of beta-GAL mRNA and protein expression in multiple organs of both female and male transgenic animals was then performed. Results show that the transgenic mER promoter, much like the endogenous one, is active in several organs and tissues of adult female and male mice. The first 0.4 kilobases of 5'-flanking DNA (up to -364) are sufficient to direct widespread expression of the transgene in mouse organs. This indicates that genetic elements functional in various cell types are included in this segment. Furthermore, the first exon and intron of the mER gene are necessary to achieve sexually dimorphic expression of the transgene in neurons located at specific sites within the central nervous system. These mER promoter transgenic mice will be useful in mapping estrogen- responsive cell types under different physiological and pathological conditions in vivo, in defining ontogenesis of estrogen action in the mouse, and in studying the mechanisms that regulate ER gene transcription.
Assuntos
Regiões Promotoras Genéticas , Receptores de Estrogênio/genética , Animais , Encéfalo/fisiologia , Clonagem Molecular , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Idade Gestacional , Masculino , Camundongos , Camundongos Transgênicos/embriologia , RNA Mensageiro/genética , Mapeamento por Restrição , Transgenes , beta-Galactosidase/genéticaRESUMO
Autophagy is a cellular recycling program that retards ageing by efficiently eliminating damaged and potentially harmful organelles and intracellular protein aggregates. Here, we show that the abundance of phosphatidylethanolamine (PE) positively regulates autophagy. Reduction of intracellular PE levels by knocking out either of the two yeast phosphatidylserine decarboxylases (PSD) accelerated chronological ageing-associated production of reactive oxygen species and death. Conversely, the artificial increase of intracellular PE levels, by provision of its precursor ethanolamine or by overexpression of the PE-generating enzyme Psd1, significantly increased autophagic flux, both in yeast and in mammalian cell culture. Importantly administration of ethanolamine was sufficient to extend the lifespan of yeast (Saccharomyces cerevisiae), mammalian cells (U2OS, H4) and flies (Drosophila melanogaster). We thus postulate that the availability of PE may constitute a bottleneck for functional autophagy and that organismal life or healthspan could be positively influenced by the consumption of ethanolamine-rich food.
Assuntos
Autofagia/fisiologia , Longevidade/fisiologia , Fosfatidiletanolaminas/fisiologia , Animais , Linhagem Celular Tumoral , Drosophila melanogaster , Humanos , LevedurasRESUMO
Several natural compounds found in health-related food items can inhibit acetyltransferases as they induce autophagy. Here we show that this applies to anacardic acid, curcumin, garcinol and spermidine, all of which reduce the acetylation level of cultured human cells as they induce signs of increased autophagic flux (such as the formation of green fluorescent protein-microtubule-associated protein 1A/1B-light chain 3 (GFP-LC3) puncta and the depletion of sequestosome-1, p62/SQSTM1) coupled to the inhibition of the mammalian target of rapamycin complex 1 (mTORC1). We performed a screen to identify the acetyltransferases whose depletion would activate autophagy and simultaneously inhibit mTORC1. The knockdown of only two acetyltransferases (among 43 candidates) had such effects: EP300 (E1A-binding protein p300), which is a lysine acetyltranferase, and NAA20 (N(α)-acetyltransferase 20, also known as NAT5), which catalyzes the N-terminal acetylation of methionine residues. Subsequent studies validated the capacity of a pharmacological EP300 inhibitor, C646, to induce autophagy in both normal and enucleated cells (cytoplasts), underscoring the capacity of EP300 to repress autophagy by cytoplasmic (non-nuclear) effects. Notably, anacardic acid, curcumin, garcinol and spermidine all inhibited the acetyltransferase activity of recombinant EP300 protein in vitro. Altogether, these results support the idea that EP300 acts as an endogenous repressor of autophagy and that potent autophagy inducers including spermidine de facto act as EP300 inhibitors.
Assuntos
Proteína p300 Associada a E1A/antagonistas & inibidores , Espermidina/farmacologia , Autofagia/efeitos dos fármacos , Autofagia/fisiologia , Linhagem Celular Tumoral , Proteína p300 Associada a E1A/metabolismo , HumanosRESUMO
BACKGROUND: Essential hypertension is associated with enhanced LDL oxidation and impaired endothelium-dependent vasodilation. The antioxidant status is linked to the nitric oxide (NO) pathway. Sulfhydryl angiotensin-converting enzyme (ACE) inhibitors inhibit oxidative stress and atherogenesis in experimental models; therefore we tested whether this beneficial antioxidant activity could be also clinically relevant in patients with essential hypertension. METHODS: Plasma LDL oxidizability was investigated initially in untreated normocholesterolemic patients with moderate essential hypertension without clinically evident target organ damage (n = 96) and in control normotensive subjects (n = 46). Patients were then randomly assigned into two age- and sex-matched groups to receive the new sulfhydryl ACE inhibitor zofenopril (15 to 30 mg/d; n = 48) or enalapril (20 mg/d, n = 48). LDL oxidizability was evaluated (generation of malondialdehyde, MDA) and systemic oxidative stress was evaluated by isoprostanes (8-isoPGF2alpha). Asymmetrical dimethyl-L-arginine (ADMA), a competitive inhibitor of endothelial NO synthase, and plasma nitrite and nitrates (NOx) were also measured. RESULTS: LDL from hypertensive subjects had enhanced susceptibility to oxidation in vitro compared with that in control subjects (P <.05). Similarly, isoprostanes were significantly increased (P <.01) in hypertensive subjects versus control subjects. After 12-week treatment, MDA levels were significantly reduced by zofenopril (P <.05) but not enalapril treatment (P = not significant). Isoprostanes were normalized after zofenopril treatment (P <.03), whereas enalapril was ineffective. After treatment with both ACE inhibitors, plasma NOx concentrations were significantly reduced (P <.05). Similarly, hypertension increased ADMA concentration compared with the normotensive state, whereas ACE inihibition elicited a significant decrease. However, the reduction of ADMA concentration was significantly higher in patients receiving sulfhydryl ACE inhibition (P <.05 vs enalapril). CONCLUSIONS: The sulfhydryl ACE inhibitor zofenopril reduces oxidative stress and improves the NO pathway in patients with essential hypertension. If confirmed in a large multicenter clinical trial, our data suggest a possible vasculoprotective effect of the compound in retarding vascular dysfunction and atherogenesis that often develops rapidly in hypertensive patients.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Arginina/análogos & derivados , Captopril/análogos & derivados , Captopril/farmacologia , Enalapril/farmacologia , Hipertensão/tratamento farmacológico , Óxido Nítrico/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Arginina/metabolismo , Captopril/uso terapêutico , Enalapril/uso terapêutico , Feminino , Humanos , Hipertensão/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Lipoproteínas LDL/metabolismo , Masculino , Pessoa de Meia-Idade , Nitratos/sangue , Nitritos/sangueRESUMO
Estrogens induce transcriptional activation of c-fos and c-myc proto-oncogenes during mitogenic stimulation of human, chicken, mouse and rat cells in vivo and in vitro. In this paper we show that 17 beta-estradiol injected into adult ovariectomized rats increases c-jun, jun-B and jun-D gene transcription in the uterus. Kinetics and amplitude of response are different for each gene, since c-jun is activated first, within 30 min after injection, followed by jun-D and jun-B, 60 and 90 min after injection, respectively. Maximal activation of jun-B marks a drop in transcription of all the jun genes. Furthermore, transcriptional activation by 17 beta-estradiol of the growth-regulated beta- and gamma-cytoskeletal actin genes is prevented by an inhibitor of protein synthesis, indicating that it is a secondary response to the hormone. These data support the hypothesis that during growth stimulation of target cells the estrogen receptor induces transcription of regulatory genes, triggering in this way a cascade of gene regulation events that results in progression through the cell cycle.
Assuntos
Actinas/genética , Núcleo Celular/metabolismo , Estradiol/farmacologia , Regulação da Expressão Gênica , Genes jun , Transcrição Gênica , Útero/metabolismo , Animais , Núcleo Celular/efeitos dos fármacos , Sondas de DNA , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Genes fos , Genes jun/efeitos dos fármacos , Genes myc , Cinética , Ovariectomia , Poli A/genética , Poli A/isolamento & purificação , RNA/genética , RNA/isolamento & purificação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos , Transcrição Gênica/efeitos dos fármacos , Útero/efeitos dos fármacosRESUMO
AIM: To investigate protease activated receptor 2 (PAR-2) expression in human coronary atherosclerotic lesions because PAR-2 is involved in the modulation of inflammatory events and vascular function. METHODS: An immunohistochemical analysis was performed on serial arterial sections, using the following antibodies: MDA2, a murine monoclonal antibody against malondialdehyde lysine epitopes of oxidised low density lipoprotein (oxLDL); HAM-56, a monoclonal antibody against human macrophages/foam cells; B5, a rabbit polyclonal antibody against PAR-2; and SAM11, a mouse monoclonal antibody against human PAR-2. Sections containing at least one lesion showing substantial immunostaining were counted as positive, and results were expressed as per cent of all sections of the same artery. RESULTS: PAR-2 expression was enhanced in human coronary atherosclerotic lesions. This phenomenon correlated with an increase in oxLDL epitopes in the coronary artery. CONCLUSION: This study shows for the first time that PAR-2 expression is enhanced in human coronary atherosclerotic lesions, and suggests that PAR-2 dependent cellular trafficking may be one of the regulatory signalling responses to vascular injury. Further pharmacological studies will establish whether modulation (and in which direction) of PAR-2 represents a possible therapeutic target for controlling the vascular response to injury.
Assuntos
Doença da Artéria Coronariana/metabolismo , Receptor PAR-2/metabolismo , Adulto , Idoso , Doença da Artéria Coronariana/patologia , Humanos , Técnicas Imunoenzimáticas , Lipoproteínas LDL/metabolismo , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de DoençaRESUMO
A protein that binds polycyclic aromatic hydrocarbons (PAHs) with high affinity and sediments in a sucrose gradient at 4 S has been described in rat liver cytosol. This "4 S" PAH binding protein precipitates at a 40-60% ammonium sulfate saturation. This partial purification procedure allows assay of this protein by using purified 3H-benzo(a)pyrene (3H-BaP) as radioactive ligand and dextran-coated charcoal as adsorbent for unreacted 3H-BaP. The 3H-BaP binding activity measured as a function of pH shows its maximum activity between pH 7.3 and 10.5. The "4 S" PAH binding protein is stable up to 42 degrees C even in the absence of the ligand. At 65 degrees C the binding sites for 3H-BaP are destroyed. The binding activity assayed as a function of protein concentration is linear between 0.4 and 2 mg/ml at 0 degrees C, whereas at 37 degrees C higher protein concentrations (4 mg/ml) can be reached. Exposure to guanidine X HCl (3 M) and urea (5 M) for 20 min at 4 degrees C inhibits the PAH binding completely to the "4 S" protein. Quick dilution or dialysis does not restore the binding activity. The dissociation rate of the "4 S" PAH binding protein measured in the presence of an excess of unlabeled ligand at 0 degrees C is biphasic and shows a two-step, first-order kinetic pattern. At 37 degrees C the dissociation rate is linear and faster, and is complete after 5 min of incubation. The association rate shows the same behavior: the binding is complete after 10 min at 0 degrees C, whereas at 37 degrees C the reaction is 10 times as fast. The dissociation equilibrium constants at 0 degrees C and 37 degrees C are respectively 2.45 X 10(-9) M and 1.09 X 10(-9) M. The high rates of association and dissociation of BaP to "4 S" PAH binding protein were used to set up an assay to exchange radioactive 3H-BaP with cold BaP.
Assuntos
Compostos Policíclicos , Animais , Citosol/metabolismo , Feminino , Guanidina , Guanidinas/farmacologia , Concentração de Íons de Hidrogênio , Cinética , Fígado/metabolismo , Ligação Proteica/efeitos dos fármacos , Ratos , Ratos Endogâmicos , Temperatura , Ureia/farmacologiaRESUMO
A macromolecule binding 3H-methylcholanthrene (3H-MCA) and 3H-benzo(a)pyrene (3H-BaP) and sedimenting in the 4-5 S region of sucrose gradient (4.5 S) was identified in rat liver cytosol. The binding was displaced by 100-fold molar excess unlabeled ligands whereas 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) was ineffective. The dissociation constant for both polycyclic aromatic hydrocarbons (PAHs) was of the order of 10(-8) M or lower. Both 3H-MCA and 3H-BaP bound to 4.5 S in a non covalent manner, since 92% of the bound radioactivity was extractable with ethyl ether. Furthermore the binding was strongly reduced by urea 8 M and by guanidine. HCl 4 M (99 and 70% respectively). Thin layer chromatography of the ethyl ether-solubilized radioactivity showed a peak comigrating with PAHs used as standards. When chromatographed on Sephadex G-200, 4.5 S was eluted as a sharp peak with an apparent molecular weight of 50-60,000 daltons. Enzyme treatment of liver cytosol showed that the 4.5 S binding sites were destroyed by micrococcal nuclease (92% of inhibition). Papain and phosphodiesterase I and II reduced the binding to 50%, whereas DNase I, DNase II, RNase, phospholipase A2 and C and trypsin were ineffective. These data suggest that the PAHs binding macromolecule of rat liver cytosol is a protein associated with a polynucleotide. The binding of both PAHs was enhanced by increasing the incubation temperature, the maximum being reached after 20-30 min at 37 degrees C. After 2.5 min at 65 degrees C, binding sites were completely destroyed. The same temperature-induced "activation" was obtained also by prewarming the cytosol at 37 degrees C in the absence of ligands.
Assuntos
Proteínas de Transporte/análise , Nucleoproteínas/metabolismo , Compostos Policíclicos/metabolismo , Temperatura , Animais , Benzo(a)pireno/metabolismo , Cromatografia em Gel , Citosol/análise , Feminino , Metilcolantreno/metabolismo , Nucleoproteínas/análise , Dibenzodioxinas Policloradas/farmacologia , Ratos , Ratos Endogâmicos , TrítioRESUMO
DBA/2N is a genetically non responsive inbred strain of mice in which administration of polycyclic aromatic hydrocarbons (PAHs) does not induce microsomal monooxygenase activity. DBA/2N mouse liver cytosol contains a polycyclic aromatic hydrocarbon-binding protein that sediments, in a sucrose gradient, at 4S ("4S" PAH-BP). Its binding kinetic and physicochemical properties indicate that this protein is practically indistinguishable from the "4S" PAH-BP identified and characterized in liver cytosol of rats and other PAH responsive rodents including C57 B1/6J mice. "4S" PAH-BP was purified to homogeneity from DBA/2N mouse liver by ammonium sulfate fractionation of the cytosol, followed by Sephadex G-200 chromatography and, finally, affinity chromatography using 1-aminopyrene-Sepharose 6B. This procedure yielded about 50 micrograms of protein from 50-60 g of mouse liver, with a recovery of 18%. "4S" PAH-BP as a complex with 3H-(benzo-a-pyrene) was more than 99% pure. A single band was seen on polyacrylamide gel electrophoresis under non denaturing conditions. H-BaP comigrated with the protein band. 3H-BaP bound to the protein was displaced by PAHs with a specificity identical to that obtained using crude cytosol. On electrophoresis in SDS gels, the purified protein migrated as a single protein band with an apparent molecular weight of 40,000.
Assuntos
Proteínas de Transporte/isolamento & purificação , Fígado/análise , Animais , Benzo(a)pireno/metabolismo , Ligação Competitiva , Proteínas de Transporte/metabolismo , Centrifugação com Gradiente de Concentração , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Citosol/análise , Eletroforese em Gel de Poliacrilamida , Feminino , Camundongos , Camundongos Endogâmicos DBARESUMO
Rodent liver cytosol and other biological systems contain two proteins that bind polycyclic aromatic hydrocarbons (PAH) in a non covalent manner and that sediment at a different rate when centrifuged on sucrose gradient. The role of the smaller protein ("4S" PAH-BP) was studied. When DBA/2N mouse liver homogenate was incubated with 3H-BaP, most of the radioactivity was found in the microsomal subcellular fraction. The cytosol binding activity apparently decreased but reincubation of the cytosol with the radioactive ligand completely restored "4S" PAH-BP activity. The microsomal uptake of 3H-BaP can be studied in a reconstituted system in which microsomes are incubated with radioactive benzo(a)pyrene in the presence of crude cytosol. In these conditions the microsomal uptake rate of 3H-BaP increased with the temperature and at 37 degrees C ten minutes were required to reach the plateau. When cytosol was substituted by HEDG buffer, the amount of radioactivity found in the microsomes decreased drastically. 0.2 microM was the benzo(a)pyrene concentration required to saturate the microsomes. When microsomes were incubated with ammonium sulfate cytosolic fractions or with homogeneously purified "4S" PAH-BP, the 3H-BaP uptake was restored and reached the maximum with 3 micrograms/ml of purified protein. The radioactive benzo(a)pyrene bound to microsomes was oxidated in the presence of NADPH regenerating system. The oxidated products were discharged from microsomes only when "4S" PAH-BP was either present during the incubation or added at its end. Thus, this protein is able to transfer benzo(a)pyrene to the microsomal metabolization sites and to facilitate the release of oxidized products and, presumably, bind them.