RESUMO
The natural healing capacity of damaged articular cartilage is poor, rendering joint surface injuries a prime target for regenerative medicine. While autologous chondrocyte or mesenchymal stem cell (MSC) implantation can be applied to repair cartilage defects in young patients, no appropriate long-lasting treatment alternative is available for elderly patients with osteoarthritis (OA). Multipotent progenitor cells are reported to present in adult human articular cartilage, with a preponderance in OA cartilage. These facts led us to hypothesize the possible use of osteoarthritis-derived chondrocytes as a cell source for cartilage tissue engineering. We therefore analyzed chondrocyte- and stem cell-related markers, cell growth rate, and multipotency in OA chondrocytes (OACs) and bone marrow-derived MSCs, along with normal articular chondrocytes (ACs) as a control. OACs demonstrated similar phenotype and proliferation rate to MSCs. Furthermore, OACs exhibited multilineage differentiation ability with a greater chondrogenic differentiation ability than MSCs, which was equivalent to ACs. We conclude that chondrogenic capacity is not significantly affected by OA, and OACs could be a potential source of multipotent progenitor cells for cartilage tissue engineering.
Assuntos
Cartilagem Articular/citologia , Condrócitos/citologia , Osteoartrite , Células-Tronco/citologia , Engenharia Tecidual/métodos , Adipócitos/citologia , Idoso , Diferenciação Celular , Linhagem da Célula , Membrana Celular/metabolismo , Proliferação de Células , Condrogênese/genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Osteogênese , Fenótipo , Medicina Regenerativa/métodosRESUMO
OBJECTIVES: To evaluate whether Hypoxia-inducible factor-2α (HIF-2α) regulates expression of endochondral ossification-related molecules in human OA meniscus. METHODS: Expressions of HIF-2α, type X collagen (COL10), matrix metalloproteinase (MMP)-13, and vascular endothelial growth factor (VEGF) in non-OA and OA menisci were analyzed by real-time RT-PCR and immunohistochemistry (IHC). Meniscal cells from OA patients were treated with interleukin-1ß (IL-1ß) and gene expression was analyzed. After knockdown of HIF-2α in OA meniscal cells, COL10 and MMP-13 expression were analyzed by RT-PCR, western blotting, immunofluorescence and ELISA. RESULT: Histological analysis demonstrated weak staining of the superficial layer and large round cells in OA meniscus. RT-PCR analysis showed that HIF-2α, COL10, MMP-13, and VEGF mRNA expressions were higher in OA than non-OA meniscal cells. IHC showed a coordinated staining pattern of HIF-2α, COL10, and MMP-13 in OA meniscus. IL-1ß treatment increased HIF-2α, COL10, and MMP-13 expressions in OA meniscal cells, and knockdown of HIF-2α suppressed IL-1ß-mediated increase in COL10 and MMP-13 expression. CONCLUSIONS: These results suggested that HIF-2α may cause meniscal matrix degradation by transactivation of MMP-13. HIF-2α may be a therapeutic target for modulating matrix degradation in both articular cartilage and meniscus during knee OA progression.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Colágeno Tipo X/metabolismo , Metaloproteinase 13 da Matriz/metabolismo , Menisco/citologia , Osteoartrite/metabolismo , Idoso , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Cartilagem Articular/citologia , Cartilagem Articular/metabolismo , Células Cultivadas , Colágeno Tipo X/genética , Feminino , Humanos , Interleucina-1beta/farmacologia , Masculino , Metaloproteinase 13 da Matriz/genética , Menisco/metabolismo , Pessoa de Meia-Idade , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: The healing mechanism of ruptured or injured tendons is poorly understood. To date, some lineage-specific factors, such as scleraxis and tenomodulin, have been reported as markers of tenocyte differentiation. Because few studies have focused on tenocyte lineage-related factors with respect to the repaired tissue of healing tendons, the aim of this study was to investigate their expression during the tendon healing process. METHODS: Defects were created in the patellar tendons of rats, and the patellae and patellar tendons were harvested at 3 days and at 1, 2, 3, 6, 12, and 20 weeks after surgery. They were studied using micro-computed tomography, and paraffin-embedded sections were then prepared for histological evaluation. Reverse transcription-polymerase chain reactions were performed to analyze the expression of genes related to the tenocyte lineage, chondrogenesis, and ossification. RESULTS: Repaired tissue became increasingly fibrous over time and contained a greater number of vessels than normal tendons, even in the later period. Safranin O staining revealed the existence of proteoglycan at 1 week and its persistence through 20 weeks. Ossification was detected in all tendons at 12 weeks. The expression of tenocyte lineage-related genes was high at 1 and 2 weeks. Chondrogenic genes were up-regulated until 6 weeks. Runt-related transcription factor 2, an osteogenic gene, was up-regulated at 20 weeks. CONCLUSIONS: In our tendon defect model, cells participating in the tendon healing process appeared to differentiate toward tenocyte lineage only in the early phase, and chondrogenesis seemed to occur from the early phase onward. To improve tendon repair, it will be necessary to promote and maintain tenogenesis and to inhibit chondrogenesis, especially in the early phase, in order to avoid erroneous differentiation of stem cells.
Assuntos
Tendões/citologia , Tendões/fisiologia , Animais , Fatores Biológicos/biossíntese , Diferenciação Celular , Masculino , Ratos , Ratos Sprague-Dawley , Tendões/irrigação sanguínea , CicatrizaçãoRESUMO
S100A12 is a member of the S100 protein family, which are intracellular calcium-binding proteins. Although there are many reports on the involvement of S100A12 in inflammatory diseases, its presence in osteoarthritic cartilage has not been reported. The purpose of this study was to investigate the expression of S100A12 in human articular cartilage in osteoarthritis (OA) and to evaluate the role of S100A12 in human OA chondrocytes. We analyzed S100A12 expression by immunohistochemical staining of cartilage samples obtained from OA and non-OA patients. In addition, chondrocytes were isolated from knee cartilage of OA patients and treated with recombinant human S100A12. Real-time RT-PCR was performed to analyze mRNA expression. Protein production of matrix metalloproteinase 13 (MMP-13) and vascular endothelial growth factor (VEGF) in the culture medium were measured by ELISA. Immunohistochemical analyses revealed that S100A12 expression was markedly increased in OA cartilages. Protein production and mRNA expression of MMP-13 and VEGF in cultured OA chondrocytes were significantly increased by treatment with exogenous S100A12. These increases in mRNA expression and protein production were suppressed by administration of soluble receptor for advanced glycation end products (RAGE). Both p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) inhibitors also suppressed the increases in mRNA expression and protein production of MMP-13 and VEGF. We demonstrated marked up-regulation of S100A12 expression in human OA cartilages. Exogenous S100A12 increased the production of MMP-13 and VEGF in human OA chondrocytes. Our data indicate the possible involvement of S100A12 in the development of OA by up-regulating MMP-13 and VEGF via p38 MAPK and NF-κB pathways.
Assuntos
Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Osteoartrite/metabolismo , Proteínas S100/biossíntese , Células Cultivadas , Condrócitos/efeitos dos fármacos , Humanos , Metaloproteinase 13 da Matriz/biossíntese , Metaloproteinase 13 da Matriz/genética , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , RNA Mensageiro/biossíntese , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/metabolismo , Proteínas S100/genética , Proteínas S100/farmacologia , Proteína S100A12 , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/genética , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
HIF-1 (hypoxia-inducible factor 1) is a master regulator of cellular adaptive responses to hypoxia. The expression and transcriptional activity of the HIF-1alpha subunit is stringently controlled by intracellular oxygen tension through the action of prolyl and asparaginyl hydroxylases. In the present study we demonstrate that PG (n-propyl gallate) activates HIF-1 and expression of its downstream target genes under normoxic conditions in cultured cells and in mice. The stability and transcriptional activity of HIF-1alpha are increased by PG. PG treatment inhibits the interaction between HIF-1alpha and VHL (von Hippel-Lindau protein) and promotes the interaction between HIF-1alpha and p300, indicating that PG inhibits the activity of both prolyl and asparaginyl HIF-1alpha hydroxylases. We conclude that PG activates HIF-1 and enhances the resultant gene expression by directly affecting the intracellular oxygen sensing system in vitro and in vivo and that PG represents a lead compound for the development of a non-toxic activator of HIF-1.
Assuntos
Fator 1 Induzível por Hipóxia/metabolismo , Oxigênio/metabolismo , Galato de Propila/farmacologia , Animais , Linhagem Celular , Humanos , Camundongos , Transfecção , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
BACKGROUND: The noninvasive measurement of coronary flow velocity in the left anterior descending artery (LAD) has recently been realized by using the transthoracic Doppler echocardiography (TTDE). A couple of investigations demonstrated that the diastolic-to-systolic peak velocity ratio (DSVR) by TTDE is a simple and noninvasive method for the detection of severe stenosis in the elective settings. However, the usefulness of DSVR by TTDE in the emergency settings has not been evaluated. OBJECTIVE: The purpose of this study was to assess the clinical feasibility to document the LAD flow by TTDE in emergency patients who complained of chest pain. METHODS: We studied 49 consecutive patients with acute coronary syndrome who were going to undergo emergency coronary angiography (CAG) for the anatomical diagnosis and the facilitated percutaneous coronary intervention (PCI). Prior to CAG, we recorded the LAD flow by TTDE and measured the diastolic peak velocity (DVp), systolic peak velocity (SVp), and their ratio, DSVR (DVp/SVp) of LAD flow. RESULTS: By CAG, the culprit lesions actually resided in the proximal LAD in 36 patients. Among the 36 patients, we detected the Doppler LAD flow in 29. Five out of 7 patients who were unable to detect the LAD flow revealed total occlusions by CAG. DSVR of the LAD is significantly lower in 17 patients who showed severe stenoses (>90%) than those in the rest of 12 patients who did not show such critical stenoses (1.44 +/- 0.16 vs 2.10 +/- 0.26, P < 0.0001). CONCLUSION: In the emergency settings, a noninvasive assessment of the LAD flow by TTDE accurately estimates the critical stenotic lesions of the LAD.
Assuntos
Estenose das Carótidas/diagnóstico por imagem , Oclusão Coronária/diagnóstico por imagem , Ecocardiografia Doppler , Ecocardiografia , Medicina de Emergência , Idoso , Angiografia Coronária , Feminino , Humanos , Masculino , Estudos ProspectivosRESUMO
A prominent feature of various inflamed and diseased tissue is the presence of low oxygen tension (hypoxia). Effector cells of the innate immune system must maintain their viability and physiologic functions in a hypoxic microenvironment. Monocytes circulating in the bloodstream differentiate into macrophages. During this process, cells acquire the ability to exert effects at hypoxic sites of inflammation. The transcription factor hypoxia-inducible factor 1 (HIF-1) mediates adaptive responses to reduced oxygen availability. In this study, we demonstrated that lipopolysaccharide (LPS) induces HIF-1 activation by enhancing both HIF-1alpha protein expression through a translation-dependent pathway and HIF-1alpha transcriptional activity in THP-1 human myeloid cells that have undergone macrophage differentiation but not in undifferentiated monocytic THP-1 cells. LPS-induced HIF-1 activation was blocked by treatment with antioxidant (N-acetylcysteine or thioredoxin-1), NADPH oxidase inhibitor (diphenyleneiodonium), indicating that reactive oxygen species generated in response to LPS are essential in this process. LPS-mediated activation of HIF-1 was independent of NF-kappaB activity. LPS-induced ROS generation and HIF-1 activation required the expression of Toll-like receptor 4 or myeloid differentiation factor (MyD) 88, thus providing a molecular basis for the selective activation of HIF-1 in differentiated THP-1 cells.
Assuntos
Diferenciação Celular/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Lipopolissacarídeos/metabolismo , Macrófagos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/metabolismo , Linhagem Celular , Inibidores Enzimáticos/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Receptores de Lipopolissacarídeos/metabolismo , Macrófagos/citologia , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , NG-Nitroarginina Metil Éster/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismoRESUMO
INTRODUCTION: Small patella syndrome (SPS) is a rare skeletal dysplasia relating to the T-box protein 4 (TBX4) gene, which regulates the development of lower extremities. Patients typically present with recurrent patellar dislocation (RPD) in childhood or adolescence, leading to a diagnosis of SPS and subsequent treatment to improve activity levels. However, those with mild symptoms may not be diagnosed when young and present later after skeletal maturation, which might compromise treatment options. Further understanding of genetic mutations of SPS could possibly help early diagnosis and following adequate surgical treatment. In this case report, we present a surgically treated adult female case of RPD associated with SPS, carrying a novel heterozygous mutation in the TBX4 gene. CASE REPORT: A 19-year-old female presented with persistent right knee pain after an atraumatic episode ofpatellar dislocation during walking. The patient had a history of recurrent patella instability of the right knee with an onset at the age of 8 years due to a minor trauma. Patellar apprehension sign was positive bilaterally. There was radiological evidence of bilateral small patellae, hypoplastic femoral trochlea, and tibial tuberosity. A direct sequencing of the coding regions in the TBX4 gene had confirmed the diagnosis of SPS. A novel heterozygous mutation (p.L39PfsX35) was found in the patient and her father. Surgical treatment was indicated and the patient underwent an isolated medial patellofemoral ligament (MPFL) reconstruction while no distal realignment osteotomy was performed due to hypoplastic tibial tuberosity. Excellent subjective and objective outcomes were obtained at 1 year postoperatively. DISCUSSION: To the best of our knowledge, this is the first reported SPS case with a novel mutation in the TBX4 gene in an Asian population. While a satisfying short-term outcome was obtained by an isolated MPFL reconstruction, early genetic diagnosis in childhood with adequate surgical treatment (e.g., Roux-Goldthwait procedure) would be ideal considering the limited treatment options in skeletally matured patients. The reported case has added one mutation variant of the TBX4 gene, which may help prevent delays in diagnosis of SPS.
RESUMO
Hypoxia elicits a wide range of responses that occur at different organizational levels in the body. Hypoxia is not only a signal for energy conservation and metabolic change, but triggers expression of a select set of genes. The transcription factor hypoxia-inducible factor 1 (HIF-1) is now appreciated to be a master factor of the gene induction. Although knowledge on molecular mechanisms of HIF-1 activation in response to hypoxia is accumulating, the molecular mechanism of maintenance of HIF-1 activity under normoxic conditions remains to be elucidated. We demonstrate that the intravenous anesthetic propofol reversibly inhibits HIF-1 activity and the gene expression mediated by HIF-1 by blocking the synthesis of the HIF-1alpha subunit under 20% or 5% O2 conditions, but not under 1% O2 conditions.
Assuntos
Anestésicos Intravenosos/farmacologia , Hipóxia Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas Nucleares/efeitos dos fármacos , Oxigênio/metabolismo , Propofol/farmacologia , Fatores de Transcrição/efeitos dos fármacos , Anticorpos Monoclonais/metabolismo , Hipóxia Celular/genética , Linhagem Celular , Relação Dose-Resposta a Droga , Endotélio Vascular/citologia , Regulação da Expressão Gênica , Genes Reporter , Glutationa Transferase/metabolismo , Humanos , Fator 1 Induzível por Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Immunoblotting , Cinética , Luciferases/metabolismo , Plasmídeos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional , Veias Umbilicais/citologiaRESUMO
The modified maze procedure using radiofrequency devices has become an increasingly common surgical option for patients with atrial fibrillation. Several lesion sets have been proposed and tested, but it remains unclear which yields the best results. We studied 61 patients who underwent the modified maze procedure using radiofrequency devices from March 2005. The pulmonary veins were isolated separately on both sides, and a connecting lesion was made inferiorly in the early series of 30 patients (group 1). In 31 patients (group 2) treated from May 2007, we added a superior connecting lesion between both pulmonary veins (completing a box lesion), and also performed coronary sinus ablation from the epicardial side, using a monopolar device. At 6 months postoperatively, maintenance of sinus rhythm with and without antiarrhythmic medications was 70% and 63%, respectively in group 1, and 94% and 90% in group 2 (both p<0.05). Multivariate analysis indicated that the box lesion with coronary sinus ablation was an independent predictor of the maintenance of sinus rhythm at 6 months. These 2 lesions should not be eliminated from the modified maze procedure.
Assuntos
Fibrilação Atrial/cirurgia , Ablação por Cateter/instrumentação , Idoso , Antiarrítmicos/uso terapêutico , Fibrilação Atrial/fisiopatologia , Ablação por Cateter/efeitos adversos , Distribuição de Qui-Quadrado , Seio Coronário/fisiopatologia , Seio Coronário/cirurgia , Desenho de Equipamento , Feminino , Humanos , Japão , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Veias Pulmonares/fisiopatologia , Veias Pulmonares/cirurgia , Estudos Retrospectivos , Medição de Risco , Fatores de Risco , Fatores de Tempo , Resultado do TratamentoRESUMO
Calcium ion is one of the most important second messengers of cellular signal transduction including hypoxia-elicited signals. In this study, we investigated the effects of the L-type calcium channel blockers such as nifedipine, efonidipine cilnidipine, diltiazem, and verapamil, on the activity of hypoxia-inducible factor-1 (HIF-1), a key transcription factor in control of hypoxia-induced gene expression. Using the lung carcinoma cell line A549 cells, human aortic smooth muscle cells, and human umbilical vein endothelial cells, we demonstrated that cilnidipine exclusively suppressed HIF-1 activity and the expressions of downstream genes in a cell-type specific manner. We also demonstrated that cilnidipine blocked the synthesis of the HIF-1alpha protein not by affecting activity of the intracellular hypoxia-sensing element prolyl hydroxylases but inhibiting activity of Akt and mitogen-activated protein kinase and that the inhibition is not dependent on the effect on calcium homeostasis.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Di-Hidropiridinas/farmacologia , Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Fator 1 Induzível por Hipóxia/metabolismo , Cálcio/metabolismo , Linhagem Celular Tumoral , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Oxigênio/metabolismo , Estresse Fisiológico/efeitos dos fármacosRESUMO
BACKGROUND: Macrophage migration inhibitory factor (MIF) is not only a cytokine which has a critical role in several inflammatory conditions but also has endocrine and enzymatic functions. MIF is identified as an intracellular signaling molecule and is implicated in the process of tumor progression, and also strongly enhances neovascularization. Overexpression of MIF has been observed in tumors from various organs. MIF is one of the genes induced by hypoxia in an hypoxia-inducible factor 1 (HIF-1)-dependent manner. METHODS/PRINCIPAL FINDINGS: The effect of MIF on HIF-1 activity was investigated in human breast cancer MCF-7 and MDA-MB-231 cells, and osteosarcoma Saos-2 cells. We demonstrate that intracellular overexpression or extracellular administration of MIF enhances activation of HIF-1 under hypoxic conditions in MCF-7 cells. Mutagenesis analysis of MIF and knockdown of 53 demonstrates that the activation is not dependent on redox activity of MIF but on wild-type p53. We also indicate that the MIF receptor CD74 is involved in HIF-1 activation by MIF at least when MIF is administrated extracellularly. CONCLUSION/SIGNIFICANCE: MIF regulates HIF-1 activity in a p53-dependent manner. In addition to MIF's potent effects on the immune system, MIF is linked to fundamental processes conferring cell proliferation, cell survival, angiogenesis, and tumor invasiveness. This functional interdependence between MIF and HIF-1alpha protein stabilization and transactivation activity provide a molecular mechanism for promotion of tumorigenesis by MIF.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Fatores Inibidores da Migração de Macrófagos/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Sequência de Bases , Linhagem Celular Tumoral , Regulação da Expressão Gênica/fisiologia , Humanos , RNA Interferente PequenoRESUMO
BACKGROUND: Previous studies reported that a coronary flow velocity (FV) pattern with a rapid diastolic deceleration time (DDT) immediately after percutaneous coronary intervention implies advanced microvascular damage in patients who have experienced an acute myocardial infarction (AMI). METHODS: Using transthoracic echocardiography, we recorded the coronary FV in the left anterior descending coronary artery (LAD) and the FV in the intramyocardial artery 2 days after successful percutaneous coronary intervention in 24 patients who had experienced an anterior AMI. We measured the DDT of the LAD and the intramyocardial artery. DDT of the LAD and the intramyocardial artery was detected in the anteroseptal lesion, the wall motion of which revealed severe hypokinesis or akinesis. We performed echocardiography during both the acute phase and 6 months after the AMI. RESULTS: Patients were divided into two groups (group A: DDT of the LAD < or = 600 milliseconds [n = 10], group B: DDT of the LAD > or = 600 milliseconds [n = 14]). DDT of the LAD and the intramyocardial artery was significantly shorter for group A than group B (373 +/- 223 vs 786 +/- 105 milliseconds, P < .0001). In the acute phase, there were no significant differences in left ventricular (LV) wall-motion score index (WMSI), LV end-diastolic volume (EDV), or ejection fraction (WMSI: 2.38 +/- 0.24 vs 2.08 +/- 0.58, P = .20; LV EDV: 160 +/- 41 vs 154 +/- 34 mL; ejection fraction: 45 +/- 11 vs 46 +/- 5%). However, WMSI and LV EDV in group A were significantly greater than in group B (WMSI: 2.47 +/- 0.16 vs 1.84 +/- 0.57, P = .01; LV EDV: 198 +/- 28 vs 132 +/- 37 mL, P = .0004) and the ejection fraction in group A was significantly lower than in group B (38 +/- 9 vs 55 +/- 10%, P = .001) during the chronic phase. CONCLUSIONS: In patients who had experienced an anterior AMI, we could predict wall-motion recovery of the infarcted area by using the coronary FV of the LAD and FV of the intramyocardial artery.
Assuntos
Estenose Coronária/diagnóstico por imagem , Estenose Coronária/cirurgia , Vasos Coronários/diagnóstico por imagem , Infarto do Miocárdio/diagnóstico por imagem , Infarto do Miocárdio/cirurgia , Disfunção Ventricular Esquerda/diagnóstico por imagem , Disfunção Ventricular Esquerda/cirurgia , Angioplastia Coronária com Balão , Estenose Coronária/complicações , Ecocardiografia Doppler/métodos , Feminino , Humanos , Interpretação de Imagem Assistida por Computador/métodos , Masculino , Infarto do Miocárdio/complicações , Prognóstico , Resultado do Tratamento , Disfunção Ventricular Esquerda/etiologia , Remodelação VentricularRESUMO
Monocytes/macrophages of the myeloid lineage are the main cellular effectors of innate immunity. Hypoxia-inducible factor 1 (HIF-1) is essential for myeloid cell activation in response to inflammatory stimuli. However, it has not been established whether HIF-1 activity is induced during differentiation from monocyte to macrophage. We demonstrate that macrophage differentiation of THP-1 cells or monocytes from peripheral blood induces increased expression of both HIF-1alpha and HIF-1beta as well as increased HIF-1 transcriptional activity leading to increased expression of HIF-1 target genes. The increased HIF-1 activity in differentiated THP-1 cells resulted from the combined effect of increased HIF-1alpha mRNA levels and increased HIF-1alpha protein synthesis. Differentiation-induced HIF-1alpha protein and mRNA and HIF-1-dependent gene expression was blocked by treating cells with an inhibitor of the protein kinase C or MAP kinase signaling pathway. THP-1 cell differentiation was also associated with increased phosphorylation of the translational regulatory proteins p70 S6 kinase, S6 ribosomal protein, eukaryotic initiation factor 4E, and 4E binding protein 1, thus providing a possible mechanism for the modulation of HIF-1alpha protein synthesis. RNA interference studies demonstrated that HIF-1alpha is dispensable for macrophage differentiation but is required for functional maturation.
Assuntos
Diferenciação Celular/fisiologia , Fator 1 Induzível por Hipóxia/metabolismo , Macrófagos/metabolismo , Processamento Alternativo/fisiologia , Linhagem Celular Tumoral , Células Cultivadas , Regulação da Expressão Gênica/fisiologia , Humanos , Fator 1 Induzível por Hipóxia/genética , Macrófagos/citologia , Interferência de RNA/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ativação Transcricional/fisiologiaRESUMO
Hypoxia induces a series of adaptive physiological responses including gene inductions. Hypoxia-inducible factor 1 (HIF-1) is a master transcription factor that regulates hypoxia-induced gene expression to maintain homeostasis in the living body. Opioids are potent analgesic agents that are widely used in clinical practice. Therefore, we investigated the effect of opioids on HIF-1 activity. SH-SY5Y human neuronal cells, which express opioid receptors intrinsically, were cultured under 1% or 20% O2 conditions with or without treatment by DAGO, DPDPE, or U-50488, which are the selective agonists of mu-, delta-, and kappa-opioid receptors, respectively. Expression of subunits of HIF-1, HIF-1alpha, and HIF-1beta were examined by Western blot using specific antibodies. Expression of the HIF-1-dependent gene were investigated by reporter assay. None of the selective agonists of opioid receptors tested affected HIF-1 activation by hypoxia. Therefore, it is suggested that opioid receptor-mediated signals do not affect HIF-1-dependent cellular hypoxia-induced gene responses.
Assuntos
Hipóxia Celular/genética , Proteínas de Ligação a DNA/genética , Expressão Gênica/efeitos dos fármacos , Proteínas Nucleares/genética , Receptores Opioides/agonistas , Fatores de Transcrição/genética , (trans)-Isômero de 3,4-dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclo-hexil)-benzenoacetamida/farmacologia , Analgésicos não Narcóticos/farmacologia , Analgésicos Opioides/farmacologia , Linhagem Celular , Proteínas de Ligação a DNA/biossíntese , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , D-Penicilina (2,5)-Encefalina/farmacologia , Humanos , Fator 1 Induzível por Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Proteínas Nucleares/biossíntese , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Estimulação Química , Fatores de Transcrição/biossínteseRESUMO
Adaptation to hypoxia and maintenance of O(2) homeostasis involve a wide range of responses that occur at different organizational levels in the body. One of the most important transcription factors that activate the expression of O(2)-regulated genes is hypoxia-inducible factor 1 (HIF-1). Nitric oxide (NO) mediates a variety of biological effects including relaxation of blood vessels and cytotoxicity of activated macrophages. We investigated the effect of the clinically used nitrates nitroglycerin (NTG), isosorbide dinitrate (ISDN), and sodium nitroprusside (SNP) on HIF-1-mediated transcriptional responses to hypoxia. We demonstrate that among the three nitrates, only SNP inhibits HIF-1 activation in response to hypoxia. In contrast, NTG or ISDN does not affect HIF-1 activity. SNP inhibits the accumulation of HIF-1alpha, the regulatory subunit of HIF-1, and the transcriptional activation of HIF-1alpha via a mechanism that is not dependent on either NO or soluble guanylate cyclase.