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1.
FASEB J ; 29(12): 4893-900, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26276131

RESUMO

Altered immune function has been demonstrated in astronauts during spaceflights dating back to Apollo and Skylab; this could be a major barrier to long-term space exploration. We tested the hypothesis that spaceflight causes changes in microRNA (miRNA) expression. Human leukocytes were stimulated with mitogens on board the International Space Station using an onboard normal gravity control. Bioinformatics showed that miR-21 was significantly up-regulated 2-fold during early T-cell activation in normal gravity, and gene expression was suppressed under microgravity. This was confirmed using quantitative real-time PCR (n = 4). This is the first report that spaceflight regulates miRNA expression. Global microarray analysis showed significant (P < 0.05) suppression of 85 genes under microgravity conditions compared to normal gravity samples. EGR3, FASLG, BTG2, SPRY2, and TAGAP are biologically confirmed targets and are co-up-regulated with miR-21. These genes share common promoter regions with pre-mir-21; as the miR-21 matures and accumulates, it most likely will inhibit translation of its target genes and limit the immune response. These data suggest that gravity regulates T-cell activation not only by transcription promotion but also by blocking translation via noncoding RNA mechanisms. Moreover, this study suggests that T-cell activation itself may induce a sequence of gene expressions that is self-limited by miR-21.


Assuntos
Regulação da Expressão Gênica , Ativação Linfocitária , MicroRNAs/genética , Voo Espacial , Linfócitos T/imunologia , Regiões 3' não Traduzidas , Adulto , Humanos , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Ausência de Peso , Adulto Jovem
2.
J Am Heart Assoc ; 4(8): e002034, 2015 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-26296857

RESUMO

BACKGROUND: Patients with peripheral artery disease (PAD) experience significant morbidity and mortality. The OMEGA-PAD I Trial, a randomized, double-blinded, placebo-controlled trial, addressed the hypothesis that short-duration, high-dose n-3 polyunsaturated fatty acids (n-3 PUFA) oral supplementation improves endothelial function and inflammation in PAD. METHODS AND RESULTS: Eighty patients with stable claudication received 4.4 g of fish oil or placebo for 1 month. The primary end point was endothelial function as measured by brachial artery flow-mediated vasodilation. Secondary end points included biomarkers of inflammation, n-3 polyunsaturated fatty acids metabolome changes, lipid profile, and walking impairment questionnaires. Although there was a significant increase in FMD in the fish oil group following treatment (0.7±1.8% increase from baseline, P=0.04), this response was not different then the placebo group (0.6±2.5% increase from baseline, P=0.18; between-group P=0.86) leading to a negative finding for the primary endpoint. There was, however, a significant reduction in triglycerides (fish oil: -34±46 mg/dL, P<0.001; placebo -10±43 mg/dL, P=0.20; between-group differential P-value: 0.02), and an increase in the omega-3 index of 4±1% (P<0.001) in the fish oil group (placebo 0.1±0.9%, P=0.49; between-group P<0.0001). We observed a significant increase in the production of pathway markers of specialized pro-resolving mediators generated from n-3 polyunsaturated fatty acids in the fish oil group. CONCLUSIONS: High-dose, short-duration fish oil supplementation did not lead to a different response in the primary end point of endothelial function between the treatment and placebo group, but improved serum triglycerides and increased the production of downstream n-3 polyunsaturated fatty acids-derived products and mediators in patients with PAD. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov/. Unique identifier: NCT01310270.


Assuntos
Suplementos Nutricionais , Ácidos Graxos Ômega-3/administração & dosagem , Óleos de Peixe/administração & dosagem , Doença Arterial Periférica/tratamento farmacológico , Administração Oral , Idoso , Biomarcadores/sangue , Artéria Braquial/efeitos dos fármacos , Artéria Braquial/fisiopatologia , Método Duplo-Cego , Tolerância ao Exercício/efeitos dos fármacos , Ácidos Graxos Ômega-3/sangue , Feminino , Óleos de Peixe/sangue , Humanos , Mediadores da Inflamação/sangue , Masculino , Pessoa de Meia-Idade , Doença Arterial Periférica/sangue , Doença Arterial Periférica/diagnóstico , Doença Arterial Periférica/fisiopatologia , São Francisco , Inquéritos e Questionários , Fatores de Tempo , Resultado do Tratamento , Triglicerídeos/sangue , Vasodilatação/efeitos dos fármacos
3.
FASEB J ; 29(10): 4122-32, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26085131

RESUMO

The health risks of a dysregulated immune response during spaceflight are important to understand as plans emerge for humans to embark on long-term space travel to Mars. In this first-of-its-kind study, we used adoptive transfer of T-cell receptor transgenic OT-II CD4 T cells to track an in vivo antigen-specific immune response that was induced during the course of spaceflight. Experimental mice destined for spaceflight and mice that remained on the ground received transferred OT-II cells and cognate peptide stimulation with ovalbumin (OVA) 323-339 plus the inflammatory adjuvant, monophosphoryl lipid A. Control mice in both flight and ground cohorts received monophosphoryl lipid A alone without additional OVA stimulation. Numbers of OT-II cells in flight mice treated with OVA were significantly increased by 2-fold compared with ground mice treated with OVA, suggesting that tolerance induction was impaired by spaceflight. Production of proinflammatory cytokines were significantly increased in flight compared with ground mice, including a 5-fold increase in IFN-γ and a 10-fold increase in IL-17. This study is the first to show that immune tolerance may be impaired in spaceflight, leading to excessive inflammatory responses.


Assuntos
Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Citocinas/imunologia , Tolerância Imunológica/imunologia , Mediadores da Inflamação/imunologia , Voo Espacial , Adjuvantes Imunológicos/farmacologia , Transferência Adotiva , Animais , Antígenos/farmacologia , Linfócitos T CD4-Positivos/metabolismo , Divisão Celular/efeitos dos fármacos , Divisão Celular/imunologia , Células Cultivadas , Citocinas/metabolismo , Feminino , Citometria de Fluxo , Tolerância Imunológica/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-17/imunologia , Interleucina-17/metabolismo , Lipídeo A/análogos & derivados , Lipídeo A/imunologia , Lipídeo A/farmacologia , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Ovalbumina/farmacologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Fatores de Tempo
4.
Am J Physiol Regul Integr Comp Physiol ; 308(6): R480-8, 2015 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-25568077

RESUMO

Healthy immune function depends on precise regulation of lymphocyte activation. During the National Aeronautics and Space Administration (NASA) Apollo and Shuttle eras, multiple spaceflight studies showed depressed lymphocyte activity under microgravity (µg) conditions. Scientists on the ground use two models of simulated µg (sµg): 1) the rotating wall vessel (RWV) and 2) the random positioning machine (RPM), to study the effects of altered gravity on cell function before advancing research to the true µg when spaceflight opportunities become available on the International Space Station (ISS). The objective of this study is to compare the effects of true µg and sµg on the expression of key early T-cell activation genes in mouse splenocytes from spaceflight and ground animals. For the first time, we compared all three conditions of microgravity spaceflight, RPM, and RWV during immune gene activation of Il2, Il2rα, Ifnγ, and Tagap; moreover, we confirm two new early T-cell activation genes, Iigp1 and Slamf1. Gene expression for all samples was analyzed using quantitative real-time PCR (qRT-PCR). Our results demonstrate significantly increased gene expression in activated ground samples with suppression of mouse immune function in spaceflight, RPM, and RWV samples. These findings indicate that sµg models provide an excellent test bed for scientists to develop baseline studies and augment true µg in spaceflight experiments. Ultimately, sµg and spaceflight studies in lymphocytes may provide insight into novel regulatory pathways, benefiting both future astronauts and those here on earth suffering from immune disorders.


Assuntos
Tolerância Imunológica/genética , Ativação Linfocitária/genética , Voo Espacial , Baço/imunologia , Linfócitos T/imunologia , Simulação de Ausência de Peso , Animais , Técnicas de Cultura de Células , Células Cultivadas , Regulação para Baixo , Feminino , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-2/genética , Interleucina-2/metabolismo , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/citologia , Baço/metabolismo , Linfócitos T/metabolismo
5.
Biomaterials ; 35(7): 2162-71, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24332390

RESUMO

Three-dimensional (3D) culture of hepatocytes leads to improved and prolonged synthetic and metabolic functions, but the underlying molecular mechanisms are unknown. In order to investigate the role of 3D cell-cell interactions in maintaining hepatocyte differentiated functions ex vivo, primary mouse hepatocytes were cultured either as monolayers on tissue culture dishes (TCD) or as 3D aggregates in rotating wall vessel (RWV) bioreactors. Global gene expression analyses revealed that genes upregulated in 3D culture were distinct from those upregulated during liver development and liver regeneration. Instead, they represented a diverse array of hepatocyte-specific functional genes with significant over-representation of hepatocyte nuclear factor 4α (Hnf4a) binding sites in their promoters. Expression of Hnf4a and many of its downstream target genes were significantly increased in RWV cultures as compared to TCD. Conversely, there was concomitant suppression of mesenchymal and cytoskeletal genes in RWV cultures that were induced in TCDs. These findings illustrate the importance of 3D cell-cell interactions in maintaining fundamental molecular pathways of hepatocyte function and serve as a basis for rational design of biomaterials that aim to optimize hepatocyte functions ex vivo for biomedical applications.


Assuntos
Hepatócitos/fisiologia , Animais , Sequência de Bases , Primers do DNA , Perfilação da Expressão Gênica , Hepatócitos/metabolismo , Camundongos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
6.
Vasc Med ; 18(5): 263-74, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24052491

RESUMO

Despite current consensus guidelines recommending intensive cardiovascular risk factor management for peripheral artery disease (PAD), patients suffering from PAD continue to experience significant morbidity and mortality. This excess morbid burden is at least partially related to impaired vascular function and systemic inflammation. Interventions bridging this gap are critical. Dietary supplementation of n-3 polyunsaturated fatty acids (n-3 PUFA) has been shown to improve endothelial function and reduce inflammation in different cohorts, as well as to decrease cardiovascular events in secondary prevention trials in patients with coronary artery disease. Their effects in the PAD population are, however, less well understood. The OMEGA-PAD trial is a double-blinded, randomized, placebo-controlled trial that examines the impact of a high-dose, short-duration dietary oral supplementation of n-3 PUFA on vascular function and inflammation in patients with established PAD. The purpose of this article is to provide a detailed description of the design and methods of the OMEGA-PAD trial, and a summary of baseline characteristics of the cohort.


Assuntos
Suplementos Nutricionais , Ácidos Graxos Ômega-3/administração & dosagem , Doença Arterial Periférica/dietoterapia , Idoso , Protocolos Clínicos , Estudos de Coortes , Método Duplo-Cego , Endotélio Vascular/fisiopatologia , Feminino , Humanos , Inflamação/prevenção & controle , Masculino , Pessoa de Meia-Idade , Doença Arterial Periférica/fisiopatologia
7.
Sci Rep ; 3: 1494, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23511048

RESUMO

Mechanical forces including gravity affect endothelial cell (ECs) function, and have been implicated in vascular disease as well as physiologic changes associated with low gravity environments. The goal of this study was to investigate the impact of gravitational mechanical unloading on ECs phenotype as determined by patterns of gene expression. Human umbilical vascular endothelial cells were exposed to 1-gravity environment or mechanical unloading (MU) for 24 hours, with or without periods of mechanical loading (ML). MU led to a significant decrease in gene expression of several adhesion molecules and pro-inflammatory cytokines. On the contrary, eNOS, Caveolin-1 and -2 expression were significantly increased with MU. There was a decrease in the length and width of the cells with MU. Addition of ML during the MU period was sufficient to reverse the changes triggered by MU. Our results suggest that gravitational loading could dramatically affect vascular endothelial cell function.


Assuntos
Caveolinas/metabolismo , Moléculas de Adesão Celular/metabolismo , Gravitação , Elevação dos Membros Posteriores , Células Endoteliais da Veia Umbilical Humana/patologia , Inflamação/patologia , Estresse Mecânico , Animais , Aorta/patologia , Fenômenos Biomecânicos , Moléculas de Adesão Celular/genética , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Imunofluorescência , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Camundongos , Modelos Animais , Modelos Biológicos , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase Tipo III/metabolismo , Nitritos/metabolismo
9.
J Leukoc Biol ; 92(6): 1133-45, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22750545

RESUMO

This study tested the hypothesis that transcription of immediate early genes is inhibited in T cells activated in µg. Immunosuppression during spaceflight is a major barrier to safe, long-term human space habitation and travel. The goals of these experiments were to prove that µg was the cause of impaired T cell activation during spaceflight, as well as understand the mechanisms controlling early T cell activation. T cells from four human donors were stimulated with Con A and anti-CD28 on board the ISS. An on-board centrifuge was used to generate a 1g simultaneous control to isolate the effects of µg from other variables of spaceflight. Microarray expression analysis after 1.5 h of activation demonstrated that µg- and 1g-activated T cells had distinct patterns of global gene expression and identified 47 genes that were significantly, differentially down-regulated in µg. Importantly, several key immediate early genes were inhibited in µg. In particular, transactivation of Rel/NF-κB, CREB, and SRF gene targets were down-regulated. Expression of cREL gene targets were significantly inhibited, and transcription of cREL itself was reduced significantly in µg and upon anti-CD3/anti-CD28 stimulation in simulated µg. Analysis of gene connectivity indicated that the TNF pathway is a major early downstream effector pathway inhibited in µg and may lead to ineffective proinflammatory host defenses against infectious pathogens during spaceflight. Results from these experiments indicate that µg was the causative factor for impaired T cell activation during spaceflight by inhibiting transactivation of key immediate early genes.


Assuntos
Genes Precoces , Ativação Linfocitária/genética , NF-kappa B/metabolismo , Linfócitos T/metabolismo , Fator de Transcrição RelA/metabolismo , Transcrição Gênica , Ausência de Peso , Antígenos CD28/metabolismo , Complexo CD3/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Análise por Conglomerados , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo
10.
J Surg Res ; 177(1): e35-43, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22572621

RESUMO

BACKGROUND: Diet is known to have an important impact on cardiovascular health. n-3 Fatty acids (FAs), found in high quantity in fish oil, have demonstrated beneficial effects in patients with coronary artery disease. The role of n-6 FAs remains more controversial. The objective of this study was to examine the effect of arachidonic acid (AA), an n-6 FA, and eicosapentanoic acid (EPA), an n-3 FA, on the interaction between monocytes and endothelial cells (ECs). DESIGN: We used a cellular model of ECs (EA.hy.926) and monocytes (human leukemic myelomonocytic U937). Confluent ECs were treated with AA or EPA, in the presence of tumor necrosis factor-alpha (TNF-α) or vehicle alone for either 4 or 24h. Adhesion of monocytes to the endothelial monolayer was performed. For gene expression, reverse transcription, followed by real-time quantitative polymerase chain reaction, was performed. RESULTS: There was a significant increase in adhesion of monocytes to the endothelial monolayer in the presence of n-6 FAs, both in the presence and in the absence of TNF-α at 4 and 24h. The adhesion of monocytes to the endothelial monolayer was decreased with n-3 FAs at 24h. Intercellular adhesion molecule 1, vascular cell adhesion molecule 1, E-Selectin, Interleukin 6, and TNF-α were significantly increased in ECs treated with n-6 FAs. CONCLUSIONS: We conclude that AA increases inflammation and enhances the ability of ECs to bind monocytes in vitro. EPA leads to a decrease in the ability of EA.hy.926 to bind monocytes, although the effect appears more modest. Taken together, these data indicate that the n-6 FA AA could potentiate inflammation and early events of atherosclerosis.


Assuntos
Moléculas de Adesão Celular/metabolismo , Células Endoteliais/efeitos dos fármacos , Ácidos Graxos Ômega-3/farmacologia , Ácidos Graxos Ômega-6/farmacologia , Monócitos/efeitos dos fármacos , Ácido Araquidônico/farmacologia , Linhagem Celular Tumoral , Ácido Eicosapentaenoico/farmacologia , Humanos , Prostaglandina-Endoperóxido Sintases/metabolismo , Transdução de Sinais
11.
Vasc Med ; 17(1): 51-63, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22363018

RESUMO

There is substantial evidence that polyunsaturated fatty acids (PUFAs) such as n-3 and n-6 fatty acids (FAs) play an important role in prevention of atherosclerosis. In vitro and in vivo studies focusing on the interactions between monocytes and endothelial cells have explored the molecular effects of FAs on these interactions. Epidemiological surveys, followed by large, randomized, control trials have demonstrated a reduction in major cardiovascular events with supplementation of n-3 FAs in secondary prevention settings. The evidence of beneficial effects specific to patients with peripheral artery disease (PAD) remains elusive, and is the focus of this review.


Assuntos
Suplementos Nutricionais , Ácidos Graxos Ômega-3/metabolismo , Ácidos Graxos Ômega-3/uso terapêutico , Ácidos Graxos Insaturados/metabolismo , Doença Arterial Periférica/metabolismo , Doença Arterial Periférica/prevenção & controle , Doenças Cardiovasculares/epidemiologia , Doenças Cardiovasculares/prevenção & controle , Causalidade , Comorbidade , Células Endoteliais/metabolismo , Ácidos Graxos Ômega-6/metabolismo , Óleos de Peixe/metabolismo , Humanos , Monócitos/metabolismo , Doença Arterial Periférica/epidemiologia , Prevenção Secundária
13.
PLoS One ; 6(9): e24004, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21912659

RESUMO

BACKGROUND: Studies suggest that micronutrients may modify the risk or delay progression of prostate cancer; however, the molecular mechanisms involved are poorly understood. We examined the effects of lycopene and fish oil on prostate gene expression in a double-blind placebo-controlled randomized clinical trial. METHODS: Eighty-four men with low risk prostate cancer were stratified based on self-reported dietary consumption of fish and tomatoes and then randomly assigned to a 3-month intervention of lycopene (n = 29) or fish oil (n = 27) supplementation or placebo (n = 28). Gene expression in morphologically normal prostate tissue was studied at baseline and at 3 months via cDNA microarray analysis. Differential gene expression and pathway analyses were performed to identify genes and pathways modulated by these micronutrients. RESULTS: Global gene expression analysis revealed no significant individual genes that were associated with high intake of fish or tomato at baseline or after 3 months of supplementation with lycopene or fish oil. However, exploratory pathway analyses of rank-ordered genes (based on p-values not corrected for multiple comparisons) revealed the modulation of androgen and estrogen metabolism in men who routinely consumed more fish (p = 0.029) and tomato (p = 0.008) compared to men who ate less. In addition, modulation of arachidonic acid metabolism (p = 0.01) was observed after 3 months of fish oil supplementation compared with the placebo group; and modulation of nuclear factor (erythroid derived-2) factor 2 or Nrf2-mediated oxidative stress response for either supplement versus placebo (fish oil: p = 0.01, lycopene: p = 0.001). CONCLUSIONS: We did not detect significant individual genes associated with dietary intake and supplementation of lycopene and fish oil. However, exploratory analyses revealed candidate in vivo pathways that may be modulated by these micronutrients. TRIAL REGISTRATION: ClinicalTrials.gov NCT00402285.


Assuntos
Carotenoides/farmacologia , Suplementos Nutricionais , Óleos de Peixe/farmacologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Carotenoides/uso terapêutico , Dieta , Método Duplo-Cego , Óleos de Peixe/uso terapêutico , Humanos , Licopeno , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Placebos , Neoplasias da Próstata/tratamento farmacológico
14.
J Orthop Surg Res ; 6: 8, 2011 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-21306641

RESUMO

INTRODUCTION: The difficulty in re-growing and mineralizing new bone after severe fracture can result in loss of ambulation or limb. Here we describe the sequential roles of FGF-2 in inducing gene expression, cell growth and BMP-2 in gene expression and mineralization of bone. MATERIALS AND METHODS: The regulation of gene expression was determined using real-time RTPCR (qRTPCR) and cell proliferation was measured by thymidine incorporation or fluorescent analysis of DNA content in MC3T3E1 osteoblast-like cells. Photomicroscopy was used to identify newly mineralized tissue and fluorescence was used to quantify mineralization. RESULTS: Fibroblast growth factor-2 (FGF-2) had the greatest ability to induce proliferation after 24 hours of treatment when compared to transforming growth factor beta (TGFß, insulin-like growth factor-1 (IGF-1), bone morphogenic protein (BMP-2), platelet derived growth factor (PDGF) or prostaglandin E2 (PGE2). We found that FGF-2 caused the most significant induction of expression of early growth response-1 (egr-1), fgf-2, cyclo-oxygenase-2 (cox-2), tgfß and matrix metalloproteinase-3 (mmp-3) associated with proliferation and expression of angiogenic genes like vascular endothelial growth factor A (vegfA) and its receptor vegfr1. We found that FGF-2 significantly reduced gene expression associated with mineralization, e.g. collagen type-1 (col1a1), fibronectin (fn), osteocalcin (oc), IGF-1, noggin, bone morphogenic protein (bmp-2) and alkaline phosphatase (alp). In contrast, BMP-2 significantly stimulated expression of the mineralization associated genes but had little or no effect on gene expression associated with growth. CONCLUSIONS: The ability of FGF-2 to re-program a mineralizing gene expression profile to one of proliferation suggests that FGF-2 plays a critical role of osteoblast growth in early fracture repair while BMP-2 is instrumental in stimulating mineralization.


Assuntos
Proteína Morfogenética Óssea 2/fisiologia , Calcificação Fisiológica/fisiologia , Proliferação de Células , Fator 2 de Crescimento de Fibroblastos/fisiologia , Regulação da Expressão Gênica/fisiologia , Osteoblastos/citologia , Osteoblastos/fisiologia , Animais , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/farmacologia , Calcificação Fisiológica/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Dinoprostona/farmacologia , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas In Vitro , Fator de Crescimento Insulin-Like I/farmacologia , Camundongos , Osteoblastos/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/farmacologia , Fatores de Tempo , Fator de Crescimento Transformador beta/farmacologia
15.
Cancer Causes Control ; 22(1): 141-50, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21103921

RESUMO

BACKGROUND: Nutritional factors are associated with reduced risk of prostate cancer progression, yet mechanisms remain unclear. We examined the effects of lycopene and fish oil supplements versus placebo on the normal prostate microenvironment, among men pursuing active surveillance for low-burden prostate cancer. We hypothesized that lycopene or fish oil supplements would down-regulate insulin-like growth factor-1 (IGF-1) and cyclooxygenase 2 (COX-2) gene expression, respectively, reflecting putative proliferation (IGF-1) and inflammatory (COX-2) pathways relevant to carcinogenesis. METHODS: We conducted a 3-month randomized, double-blinded, clinical trial comparing prostate tissue gene expression profiles (assessed by qRT-PCR) among men with favorable-risk prostate cancer receiving either 30 mg/day lycopene, 3 g/day fish oil (including 1,098 mg eicosapentaenoic and 549 mg docosahexaenoic fatty acids) or placebo. RESULTS: Among 69 men (22 assigned to lycopene, 21 to fish, and 26 to placebo), there was no difference in the change from baseline to the 3 months in IGF-1 expression level between the placebo and lycopene arms (p = 0.93) nor in COX-2 expression between the placebo and fish arms (p = 0.99). CONCLUSION: Compared to placebo, 3-month intervention with lycopene or fish oil did not significantly change IGF-1 and COX-2 gene expression in the normal prostate microenvironment in men with low-burden prostate cancer. Further analysis of global gene expression profiles may shed light on the bioactivity and relevance of these nutrients in prostate cancer.


Assuntos
Anti-Inflamatórios/uso terapêutico , Carotenoides/uso terapêutico , Ciclo-Oxigenase 2/biossíntese , Suplementos Nutricionais , Óleos de Peixe/uso terapêutico , Fator de Crescimento Insulin-Like I/biossíntese , Neoplasias da Próstata/metabolismo , Método Duplo-Cego , Perfilação da Expressão Gênica , Humanos , Licopeno , Masculino , Pessoa de Meia-Idade , Neoplasias da Próstata/dietoterapia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Conduta Expectante
16.
Tissue Eng Part A ; 15(3): 559-67, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18724832

RESUMO

Understanding cell biology of three-dimensional (3D) biological structures is important for more complete appreciation of in vivo tissue function and advancing ex vivo organ engineering efforts. To elucidate how 3D structure may affect hepatocyte cellular responses, we compared global gene expression of human liver hepatocellular carcinoma cell line (HepG2) cells cultured as monolayers on tissue culture dishes (TCDs) or as spheroids within rotating wall vessel (RWV) bioreactors. HepG2 cells grown in RWVs form spheroids up to 100 mum in diameter within 72 h and up to 1 mm with long-term culture. The actin cytoskeleton in monolayer cells show stress fiber formation while spheroids have cortical actin organization. Global gene expression analysis demonstrates upregulation of structural genes such as extracellular matrix, cytoskeletal, and adhesion molecules in monolayers, whereas RWV spheroids show upregulation of metabolic and synthetic genes, suggesting functional differences. Indeed, liver-specific functions of cytochrome P450 activity and albumin production are higher in the spheroids. Enhanced liver functions require maintenance of 3D structure and environment, because transfer of spheroids to a TCD results in spheroid disintegration and subsequent loss of function. These findings illustrate the importance of physical environment on cellular organization and its effects on hepatocyte processes.


Assuntos
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Esferoides Celulares/metabolismo , Actinas/metabolismo , Proliferação de Células , Genes Neoplásicos , Humanos , Fígado/metabolismo , Fígado/patologia , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Regulação para Cima
17.
FEBS Lett ; 580(28-29): 6533-6, 2006 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-17113084

RESUMO

Cyclooxygenase-2 (COX-2) catalyzes the rate-limiting step in the prostanoid biosynthesis pathway, converting arachidonic acid into prostaglandin H(2). COX-2 exists as 72 and 74kDa glycoforms, the latter resulting from an additional oligosaccharide chain at residue Asn(580). In this study, Asn(580) was mutated to determine the biological significance of this variable glycosylation. COS-1 cells transfected with the mutant gene were unable to express the 74kDa glycoform and were found to accumulate more COX-2 protein and have five times greater COX-2 activity than cells expressing both glycoforms. Thus, COX-2 turnover appears to depend upon glycosylation of the 72kDa glycoform.


Assuntos
Ciclo-Oxigenase 2/metabolismo , Proteínas de Membrana/metabolismo , Animais , Ácido Araquidônico/farmacologia , Asparagina/genética , Células COS , Chlorocebus aethiops , Glicosilação/efeitos dos fármacos , Humanos , Isoenzimas/metabolismo , Mutagênese/efeitos dos fármacos
18.
J Bone Miner Res ; 21(6): 946-55, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16753025

RESUMO

UNLABELLED: Fifteen minutes of physiological MS induces FGF-2 in osteogenic cells. Here, we show that MS induced proliferation in both MC3T3-E1 and BMOp cells isolated from Fgf2(+/+) mice; Fgf2(-/-) BMOp cells required exogenous FGF-2 for a normal proliferation response. The induction of fgf-2 is mediated by PKA and ERK pathways. INTRODUCTION: Mechanical stress (MS) induces gene expression and proliferation of osteogenic MC3T3-E1 cells. We have previously shown that physiological levels of MS in MC3T3-E1 cells causes extracellular signal-regulated kinase (ERK)1/2 phosphorylation. Here we evaluate the induction and importance of fibroblast growth factor-2 (FGF-2) for MS-induced proliferation. MATERIALS AND METHODS: We characterized the MS induction of fgf-2 using a 15-minute pulse of 120 mustrain and studied the stability of fgf-2 message using actinomycin D. Bone marrow stromal cells (BMOp) isolated from Fgf2(-/-) and Fgf2(+/+) mice were used to study the importance of FGF-2 in MS-induced proliferation. RESULTS: We found that the induction of fgf-2 by MS is dependent on both protein kinase A (PKA) and ERK pathways. MS transiently induces fgf-2 within 30 minutes. FGF-2 receptor (FGFR2) was also significantly increased within 1 h. All three isoforms of FGF-2 (24, 22, and 18 kDa) were significantly increased by MS. The MS-mediated increase of fgf-2 mRNA was caused by new synthesis and not stabilization. Pretreatment of MC3T3-E1 cells with cycloheximide showed that the induction of fgf-2 did not require new protein synthesis. Pretreating MC3T3-E1 cells with the mitogen-activated protein kinase (MAPK)/ERK kinase 1/2 (MEK1/2) inhibitor, U0126, or H-89, a PKA inhibitor, significantly inhibited the induction of fgf-2, showing that mechanical induction of fgf-2 is dependent on ERK and PKA signaling pathways. The downstream consequence of a single 15-minute stress pulse was a 3.5-fold increase in cell number in MC3T3-E1 compared with growth in nonstressed control cells. In studies using bone marrow osteoprogenitor cells (BMOp) isolated from Fgf2(+/+)and Fgf2(-/-) mice, we found that FGF-2 was necessary for a full proliferative response to MS. CONCLUSIONS: These studies show that FGF-2 is an immediate-early gene induced by MS, and its expression is mediated by both the PKA and MAPK signal transduction pathways. FGF-2 was required for a full proliferative response.


Assuntos
Fator 2 de Crescimento de Fibroblastos/metabolismo , Genes Precoces/fisiologia , Osteoblastos/metabolismo , Osteogênese/fisiologia , Animais , Proliferação de Células , Células Cultivadas , Ciclo-Oxigenase 2/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Fator 2 de Crescimento de Fibroblastos/genética , Camundongos , Camundongos Knockout , Osteoblastos/citologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/biossíntese , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Estresse Mecânico , Regulação para Cima
19.
J Cell Biochem ; 99(2): 435-49, 2006 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-16619267

RESUMO

Bone loss has been repeatedly documented in astronauts after flight, yet little is known about the mechanism of bone loss in space flight. Osteoblasts were activated during space flight in microgravity (microg) with and without a 1 gravity (1 g) field and 24 genes were analyzed for early induction. Induction of proliferating cell nuclear antigen (PCNA), transforming growth factor beta (TGFbeta), cyclo-oxygenase-2 (cox-2), cpla2, osteocalcin (OC), c-myc, fibroblast growth factor-2 (fgf-2), bcl2, bax, and fgf-2 message as well as FGF-2 protein were significantly depressed in microg when compared to ground (gr). Artificial onboard gravity normalized the induction of c-myc, cox-2, TGFbeta, bax, bcl2, and fgf-2 message as well as FGF-2 protein synthesis in spaceflight samples. In normal gravity, FGF-2 induces bcl2 expression; we found that bcl2 expression was significantly reduced in microgravity conditions. Since nuclear shape is known to elongate in the absence of mitogens like FGF-2, we used high-resolution image-based morphometry to characterize changes in osteoblast nuclear architecture under microgravity, 1 g flight, and ground conditions. Besides changes in cell shape (roundish/elliptic), other high-resolution analyses show clear influences of gravity on the inner nuclear structure. These changes occur in the texture, arrangement, and contrast of nuclear particles and mathematical modeling defines the single cell classification of the osteoblasts. Changes in nuclear structure were evident as early as 24 h after exposure to microgravity. This documented alteration in nuclear architecture may be a direct result of decreased expression of autocrine and cell cycle genes, suggesting an inhibition of anabolic response in microg. Life on this planet has evolved in a normal gravity field and these data suggest that gravity plays a significant role in regulation of osteoblast transcription.


Assuntos
Anabolizantes/metabolismo , Núcleo Celular/ultraestrutura , Osteoblastos/metabolismo , Osteoblastos/ultraestrutura , Ausência de Peso/efeitos adversos , Células 3T3 , Animais , Reabsorção Óssea/etiologia , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Primers do DNA/genética , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Expressão Gênica , Humanos , Camundongos , Microscopia de Fluorescência , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Voo Espacial
20.
Cancer Res ; 66(3): 1427-33, 2006 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-16452198

RESUMO

Essential fatty acids are not only energy-rich molecules; they are also an important component of the membrane bilayer and recently have been implicated in induction of fatty acid synthase and other genes. Using gene chip analysis, we have found that arachidonic acid, an omega-6 fatty acid, induced 11 genes that are regulated by nuclear factor-kappaB (NF-kappaB). We verified gene induction by omega-6 fatty acid, including COX-2, IkappaBalpha, NF-kappaB, GM-CSF, IL-1beta, CXCL-1, TNF-alpha, IL-6, LTA, IL-8, PPARgamma, and ICAM-1, using quantitative reverse transcription-PCR. Prostaglandin E(2) (PGE(2)) synthesis was increased within 5 minutes of addition of arachidonic acid. Analysis of upstream signal transduction showed that within 5 minutes of fatty acid addition, phosphatidylinositol 3-kinase (PI3K) was significantly activated followed by activation of Akt at 30 minutes. Extracellular signal-regulated kinase 1 and 2, p38 and stress-activated protein kinase/c-Jun-NH(2)-kinase were not phosphorylated after omega-6 fatty acid addition. Thirty minutes after fatty acid addition, we found a significant 3-fold increase in translocation of NF-kappaB transcription factor to the nucleus. Addition of a nonsteroidal anti-inflammatory drug (NSAID) caused a decrease in COX-2 protein synthesis, PGE(2) synthesis, as well as inhibition of PI3K activation. We have previously shown that NSAIDs cause an inhibition of arachidonic acid-induced proliferation; here, we have shown that arachidonic acid-induced proliferation is also blocked (P < 0.001) by PI3K inhibitor LY294002. LY294002 also significantly inhibited the arachidonic acid-induced gene expression of COX-2, IL-1beta, GM-CSF, and ICAM1. Taken together, the data suggest that arachidonic acid via conversion to PGE(2) plays an important role in stimulation of growth-related genes and proliferation via PI3K signaling and NF-kappaB translocation to the nucleus.


Assuntos
Ácido Araquidônico/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/genética , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/genética , Cromonas/farmacologia , Ciclo-Oxigenase 2/biossíntese , Ciclo-Oxigenase 2/genética , Inibidores de Ciclo-Oxigenase 2/farmacologia , Ativação Enzimática/efeitos dos fármacos , Flurbiprofeno/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , Molécula 1 de Adesão Intercelular/genética , Interleucina-1/biossíntese , Interleucina-1/genética , Masculino , Morfolinas/farmacologia , NF-kappa B/genética , NF-kappa B/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Ativação Transcricional
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