RESUMO
OBJECTIVE: The discovery of IL-7R(alpha) polymorphisms implicated in the pathogenesis of multiple sclerosis has highlighted the importance of interleukin 7 (IL-7) in central nervous system diseases. Hypoxia affects neurological disease states in part by modulating expression of many early and late response genes. The present work used cultured PC12 cells to investigate the effect of hypoxia on IL-7 expression. METHOD: PC12 cells were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 medium. RNA was isolated and reverse transcriptase-polymerase chain reaction (RT-PCR) was run to quantify messenger RNA (mRNA) change. Western blots were used to assess IL-7 protein change in the medium. Extracellular free Ca(2+) was removed by using Ca(2+)-free DMEM/F12 with 1 mM ethylene glycol tetraacetic acid for 45 minutes before the start of hypoxia. RESULTS: Exposure of PC12 cells to 1% oxygen for 6 hours decreased IL-7 mRNA by 77% using RT-PCR (p<0.01). Exposure to 1% oxygen for 24 hours decreased IL-7 protein in the medium by 21% (p<0.05). As hypoxia duration increased (2, 4, 6 and 24 hours) or oxygen concentrations decreased (10%, 5% and 1%), IL-7 mRNA expression progressively decreased. Removal of extracellular free Ca(2+) completely prevented these hypoxia-induced decreases of IL-7 mRNA. DISCUSSION: Since IL-7 exhibits trophic properties in developing brain, down-regulation of IL-7 by hypoxia may contribute to hypoxia-induced injury to neural cells.
Assuntos
Cálcio/farmacologia , Hipóxia Celular/genética , Interleucina-7/metabolismo , Animais , Western Blotting , Regulação para Baixo , Oxigênio/farmacologia , Células PC12 , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Heme oxygenase-1 (HO-1) is an inducible and cytoprotective enzyme that provides a defense against oxidant damage. The present study screened 137 HO-1/interacting proteins using a profound co-immunoprecipitation (Co-IP) coupled with proteomics, and profiled the global HO-1 interactome network, including oxidative phosphorylation, endoplasmic reticulum and transport vesicle functions. Among these molecules, we observed that a novel interactor, emopamil-binding protein (EBP), is closely related to the cholesterol metabolism process. This study demonstrated that cholesterol promotes excessive oxidative stress and alters the energy metabolism in cardiomyocytes, further triggering numerous cardiovascular diseases. We observed that cholesterol caused the overexpression of EBP and HO-1 by the activation of AKT and Nrf2/mTOR pathways. In addition, HO-1 and EBP performed a myocardial protective function. The overexpression of HO-1 alleviated the cholesterol-induced excessive oxidative stress status by inhibition of the carbohydrate metabolism. Notably, we also confirmed that the loss of partial HO-1 activity aggravated the oxidative damage and cardiac systolic function induced by a high-fat diet in HO-1 heterozygous (HO-1+/-) mice. These findings indicate that the HO-1/EBP interaction plays a protective role in alleviating the dysfunction of oxidative stress and cardiac systolic function induced by cholesterol stimulation.
Assuntos
Proteínas de Transporte/metabolismo , Colesterol/metabolismo , Heme Oxigenase-1/metabolismo , Hipóxia/metabolismo , Miócitos Cardíacos/metabolismo , Transdução de Sinais , Esteroide Isomerases/metabolismo , Animais , Metabolismo dos Carboidratos , Linhagem Celular , Hipóxia/patologia , Masculino , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/patologia , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Mapas de Interação de Proteínas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
After intracerebral hemorrhage (ICH), many changes of gene transcription occur that may be important because they will contribute to understanding mechanisms of injury and recovery. Therefore, gene expression was assessed using Affymetrix microarrays in the striatum and the overlying cortex at 24 h after intracranial infusions of blood into the striatum of adult rats. Intracerebral hemorrhage regulated 369 of 8,740 transcripts as compared with saline-injected controls, with 104 regulated genes shared by the striatum and cortex. There were 108 upregulated and 126 downregulated genes in striatum, and 170 upregulated and 69 downregulated genes in the cortex. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) confirmed upregulation of IL-1-beta, Lipcortin 1 (annexin) and metallothionein 1,2, and downregulation of potassium voltage-gated channel, shaker-related subfamily, beta member 2 (Kcnab2). Of the functional groups of genes modulated by ICH, many metabolism and signal-transduction-related genes decreased in striatum but increased in adjacent cortex. In contrast, most enzyme, cytokine, chemokine, and immune response genes were upregulated in both striatum and in the cortex after ICH, likely in response to foreign proteins from the blood. A number of these genes may contribute to brain edema and cellular apoptosis caused by ICH. In addition, downregulation of growth factor pathways and the phosphatidylinositol 3-kinase (PI3K)/Akt pathway could also contribute to perihematoma cell death/apoptosis. Intracerebral hemorrhage-related downregulation of GABA-related genes and potassium channels might contribute to perihematoma cellular excitability and increased risk of post-ICH seizures. These genomic responses to ICH potentially provide new therapeutic targets for treatment.
Assuntos
Encéfalo/fisiologia , Hemorragia Cerebral/genética , Perfilação da Expressão Gênica , Genômica , Animais , Anexina A1/genética , Anexina A1/metabolismo , Encéfalo/metabolismo , Análise por Conglomerados , Modelos Animais de Doenças , Regulação para Baixo , Substâncias de Crescimento/genética , Substâncias de Crescimento/metabolismo , Interleucina-1/genética , Interleucina-1/metabolismo , Masculino , Metalotioneína/genética , Metalotioneína/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transcrição Gênica , Regulação para Cima , Ácido gama-Aminobutírico/genética , Ácido gama-Aminobutírico/metabolismoRESUMO
Ischemic brain and peripheral white blood cells release cytokines, chemokines and other molecules that activate the peripheral white blood cells after stroke. To assess gene expression in these peripheral white blood cells, whole blood was examined using oligonucleotide microarrays in 15 patients at 2.4+/-0.5, 5 and 24 h after onset of ischemic stroke and compared with control blood samples. The 2.4-h blood samples were drawn before patients were treated either with tissue-type plasminogen activator (tPA) alone or with tPA plus Eptifibatide (the Combination approach to Lysis utilizing Eptifibatide And Recombinant tPA trial). Most genes induced in whole blood at 2 to 3 h were also induced at 5 and 24 h. Separate studies showed that the genes induced at 2 to 24 h after stroke were expressed mainly by polymorphonuclear leukocytes and to a lesser degree by monocytes. These genes included: matrix metalloproteinase 9; S100 calcium-binding proteins P, A12 and A9; coagulation factor V; arginase I; carbonic anhydrase IV; lymphocyte antigen 96 (cluster of differentiation (CD)96); monocarboxylic acid transporter (6); ets-2 (erythroblastosis virus E26 oncogene homolog 2); homeobox gene Hox 1.11; cytoskeleton-associated protein 4; N-formylpeptide receptor; ribonuclease-2; N-acetylneuraminate pyruvate lyase; BCL6; glycogen phosphorylase. The fold change of these genes varied from 1.6 to 6.8 and these 18 genes correctly classified 10/15 patients at 2.4 h, 13/15 patients at 5 h and 15/15 patients at 24 h after stroke. These data provide insights into the inflammatory responses after stroke in humans, and should be helpful in diagnosis, understanding etiology and pathogenesis, and guiding acute treatment and development of new treatments for stroke.
Assuntos
Isquemia Encefálica/sangue , Regulação da Expressão Gênica , Monócitos/metabolismo , Neutrófilos/metabolismo , Acidente Vascular Cerebral/sangue , Adulto , Idoso , Isquemia Encefálica/tratamento farmacológico , Quimioterapia Combinada , Eptifibatida , Feminino , Fibrinolíticos/uso terapêutico , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/sangue , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Peptídeos/uso terapêutico , Inibidores da Agregação Plaquetária/uso terapêutico , Acidente Vascular Cerebral/tratamento farmacológico , Fatores de Tempo , Ativador de Plasminogênio Tecidual/uso terapêuticoRESUMO
Sequencing of the human genome and new microarray technology make it possible to assess all genes on a single chip or array. Recent studies show different patterns of gene expression related to different tissues and diseases, and these patterns of gene expression are beginning to be used for diagnosis and treatment decisions in various types of lymphoid and solid malignancies. Because of obvious problems obtaining brain tissue, progress in genomics of neurological diseases has been slow. To address this, we demonstrated that different types of acute injury in rodent brain produced different patterns of gene expression in peripheral blood. These animal studies have now been extended to human studies. Two groups have shown that there are specific genomic profiles in the blood of patients after ischemic stroke that are highly sensitive and specific for predicting stroke. Other recent studies demonstrate specific genomic profiles in the blood of patients with Down syndrome, neurofibromatosis, tuberous sclerosis, Huntington disease, multiple sclerosis, Tourette syndrome, and others. In addition, data demonstrate specific profiles of gene expression in the blood related to different drugs, toxins, and infections. Although all of these studies are still preliminary basic scientific endeavors, they suggest that this approach will have clinical applications to neurological diseases in humans.
Assuntos
Perfilação da Expressão Gênica/métodos , Genômica/métodos , Doenças do Sistema Nervoso/sangue , Doenças do Sistema Nervoso/genética , Fatores Etários , Animais , Humanos , Análise em Microsséries/métodos , Fatores SexuaisRESUMO
Bcl-xL is a well characterized death-suppressing molecule of the Bcl-2 family. Bcl-xL is expressed in embryonic and adult neurons of the CNS and may play a critical role in preventing neuronal apoptosis that occurs during brain development or results from diverse pathologic stimuli, including cerebral ischemia. In this study, we used a novel approach to study the potential neuroprotective effect of Bcl-xL as a therapeutic agent in the murine model of focal ischemia/reperfusion. We created a Bcl-xL fusion protein, designated as PTD-HA-Bcl-xL, which contains the protein transduction domain (PTD) derived from the human immunodeficiency TAT protein. We demonstrated that this fusion protein is highly efficient in transducing into primary neurons in cultures and potently inhibited staurosporin-induced neuronal apoptosis. Furthermore, intraperitoneal injection of PTD-HA-Bcl-xL into mice resulted in robust protein transduction in neurons in various brain regions within 1-2 hr, and decreased cerebral infarction (up to approximately 40%) in a dose-dependent manner, as determined at 3 d after 90 min of focal ischemia. PTD-HA-Bcl-xL was effective even when it was administered after the completion of ischemia (up to 45 min), and the protective effect was independent of the changes in cerebral blood flow or other physiological parameters. Finally, as shown by immunohistochemistry, Western blotting, and substrate-cleavage assays, PTD-HA-Bcl-xL attenuated ischemia-induced caspase-3 activation in ischemic neurons. These results thus confirm the neuroprotective effect of Bcl-xL against ischemic brain injury and provide the first evidence that the PTD can be used to efficiently transduce a biologically active neuroprotectant in experimental cerebral ischemia.
Assuntos
Apoptose , Produtos do Gene tat/química , Ataque Isquêmico Transitório/prevenção & controle , Neurônios/patologia , Proteínas Proto-Oncogênicas c-bcl-2/uso terapêutico , Animais , Encéfalo/patologia , Células Cultivadas , Relação Dose-Resposta a Droga , Injeções , Ataque Isquêmico Transitório/patologia , Cinética , Camundongos , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/uso terapêutico , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-bcl-2/administração & dosagem , Proteínas Proto-Oncogênicas c-bcl-2/genética , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/uso terapêutico , Proteína bcl-XRESUMO
BACKGROUND AND PURPOSE: Intracerebral hemorrhage has no effective treatment. The delayed appearance of edema, apoptosis, and inflammation in perihematomal brain suggests that these events may be targets for therapeutic intervention. To develop successful treatments, we must learn more about the effects of hemorrhage on brain tissue. In this study, we investigated the acute metabolic effects of intrastriatal hemorrhage in rat brain. METHODS: Lysed blood or saline (50 microL each) was injected into the striatum of male Sprague-Dawley rats. The rats recovered for 1 to 72 hours before injection of [14C]-2-deoxyglucose (intraperitoneally) 30 minutes before decapitation. Animals were pretreated with the N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor antagonists dizolcilpine maleate (MK-801; 1 mg/kg) or 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[f]quinoxaline (NBQX; 30 mg/kg), or saline vehicle. Additional animals received intrastriatal injections of glutamate (1.0 mmol/L), NMDA (1.0 mmol/L), or AMPA (0.1 mmol/L) in the place of blood. Semiquantitative autoradiographs from the brains were analyzed to determine the effects of hemorrhage on relative glucose metabolism. RESULTS: We found an acute phase of increased [14C]-2-deoxyglucose uptake in the perihematomal region that peaks 3 hours after lysed blood injection. Saline injections had no effect on striatal glucose utilization. The increased [14C]-2-deoxyglucose uptake produced by the hemorrhages was blocked by pretreatment with MK-801 and NBQX. Glutamate injections alone had no effect on striatal metabolism, whereas NMDA and AMPA injections increased [14C]-2-deoxyglucose uptake. CONCLUSIONS: The data imply that glutamate activation of NMDA or AMPA receptors increases glucose metabolism in perihematomal brain at early times after intracerebral hemorrhage. This may provide a possible target for the treatment of intracerebral hemorrhage.
Assuntos
Hemorragia Cerebral/metabolismo , Glucose/metabolismo , Receptores de Glutamato/metabolismo , Animais , Autorradiografia , Encéfalo/metabolismo , Circulação Cerebrovascular , Modelos Animais de Doenças , Ácido Glutâmico/metabolismo , Hematoma , Masculino , Ratos , Ratos Sprague-Dawley , Receptores de AMPA , Receptores de N-Metil-D-AspartatoRESUMO
Understanding transcriptional changes in brain after ischemia may provide therapeutic targets for treating stroke and promoting recovery. To study these changes on a genomic scale, oligonucleotide arrays were used to assess RNA samples from periinfarction cortex of adult Sprague-Dawley rats 24 h after permanent middle cerebral artery occlusions. Of the 328 regulated transcripts in ischemia compared with sham-operated animals, 264 were upregulated, 64 were downregulated, and 163 (49.7%) had not been reported in stroke. Of the functional groups modulated by ischemia: G-protein-related genes were the least reported; and cytokines, chemokines, stress proteins, and cell adhesion and immune molecules were the most highly expressed. Quantitative reverse transcription polymerase chain reaction of 20 selected genes at 2, 4, and 24 h after ischemia showed early upregulated genes (2 h) including Narp, Rad, G33A, HYCP2, Pim-3, Cpg21, JAK2, CELF, Tenascin, and DAF. Late upregulated genes (24 h) included Cathepsin C, Cip-26, Cystatin B, PHAS-I, TBFII, Spr, PRG1, and LPS-binding protein. Glycerol 3-phosphate dehydrogenase, which is involved in mitochondrial reoxidation of glycolysis derived NADH, was regulated more than 60-fold. Plasticity-related transcripts were regulated, including Narp, agrin, and Cpg21. A newly reported lung pathway was also regulated in ischemic brain: C/EBP induction of Egr-1 (NGFI-A) with downstream induction of PAI-1, VEGF, ICAM, IL1, and MIP1. Genes regulated acutely after stroke may modulate cell survival and death; also, late regulated genes may be related to tissue repair and functional recovery.
Assuntos
Isquemia Encefálica/fisiopatologia , Córtex Cerebral/fisiopatologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Animais , Isquemia Encefálica/patologia , Córtex Cerebral/patologia , Genômica , Infarto da Artéria Cerebral Média , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , RNA/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Estradiol reduces brain injury from many diseases, including stroke and trauma. To investigate the molecular mechanisms of this protection, the effects of 17-beta-estradiol on heat shock protein (HSP) expression were studied in normal male and female rats and in male gerbils after global ischemia. 17-beta-estradiol was given intraperitoneally (46 or 460 ng/kg, or 4.6 microg/kg) and Western blots performed for HSPs. 17-beta-estradiol increased hemeoxygenase-1, HSP25/27, and HSP70 in the brain of male and female rats. Six hours after the administration of 17-beta-estradiol, hemeoxygenase-1 increased 3.9-fold (460 ng/kg) and 5.4-fold (4.6 microg/kg), HSP25/27 increased 2.1-fold (4.6 microg/kg), and Hsp70 increased 2.3-fold (460 ng/kg). Immunocytochemistry showed that hemeoxygenase-1, HSP25/27,and HSP70 induction was localized to cerebral arteries in male rats, possibly in vascular smooth muscle cells. 17-beta-estradiol was injected intraperitoneally 20 minutes before transient occlusion of both carotids in adult gerbils. Six hours after global cerebral ischemia, 17-beta-estradiol (460 ng/kg) increased levels of hemeoxygenase-1 protein 2.4-fold compared with ischemia alone, and HSP25/27 levels increased 1.8-fold compared with ischemia alone. Hemeoxygenase-1 was induced in striatal oligodendrocytes and hippocampal neurons, and HSP25/27 levels increased in striatal astrocytes and hippocampal neurons. Finally, Western blot analysis confirmed that estrogen induced heat shock factor-1, providing a possible mechanism by which estrogen induces HSPs in brain and other tissues. The induction of HSPs may be an important mechanism for estrogen protection against cerebral ischemia and other types of injury.
Assuntos
Isquemia Encefálica/metabolismo , Encéfalo/metabolismo , Artérias Cerebrais/metabolismo , Estradiol/farmacologia , Proteínas de Choque Térmico/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Feminino , Fatores de Transcrição de Choque Térmico , Heme Oxigenase (Desciclizante)/metabolismo , Heme Oxigenase-1 , Masculino , Ratos , Ratos Sprague-Dawley , Fatores de TranscriçãoRESUMO
This study determined whether stroke and other types of insults produced a gene expression profile in blood that correlated with the presence of neuronal injury. Adult rats were subjected to ischemic stroke, intracerebral hemorrhage, status epilepticus, and insulin-induced hypoglycemia and compared with untouched, sham surgery, and hypoxia animals that had no brain injury. One day later, microarray analyses showed that 117 genes were upregulated and 80 genes were downregulated in mononuclear blood cells of the "injury" (n = 12) compared with the "no injury" (n = 9) animals. A second experiment examined the whole blood genomic response of adult rats after global ischemia and kainate seizures. Animals with no brain injury were compared with those with brain injury documented by TUNEL and PANT staining. One day later, microarray analyses showed that 37 genes were upregulated and 67 genes were downregulated in whole blood of the injury (n = 4) animals compared with the no-injury (n = 4) animals. Quantitative reverse transcription-polymerase chain reaction confirmed that the vesicular monoamine transporter-2 increased 2.3- and 1.6-fold in animals with severe and mild brain injury, respectively, compared with no-injury animals. Vascular tyrosine phosphatase-1 increased 2.0-fold after severe injury compared with no injury. The data support the hypothesis that there is a peripheral blood genomic response to neuronal injury, and that this blood response is associated with a specific blood mRNA gene expression profile that can be used as a marker of the neuronal damage.
Assuntos
Encefalopatias/sangue , Encefalopatias/genética , Perfilação da Expressão Gênica , Genoma , Proteínas de Membrana Transportadoras , Neuropeptídeos , Animais , Fenômenos Fisiológicos Sanguíneos , Regulação para Baixo , Ataque Isquêmico Transitório/patologia , Ataque Isquêmico Transitório/fisiopatologia , Ácido Caínico , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Monócitos/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteína Fosfatase 1 , Proteínas Tirosina Fosfatases/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Convulsões/induzido quimicamente , Convulsões/patologia , Convulsões/fisiopatologia , Proteínas Vesiculares de Transporte de Aminas Biogênicas , Proteínas Vesiculares de Transporte de MonoaminaRESUMO
Although wild-type Hsp70 (Hsp70WT) inhibits procaspase-3 processing by preventing apoptosome formation, Hsp70WT does not block active caspase-3. Because all caspase-3 inhibitors bear canonical DXXD caspase-3 recognition motifs, we determined whether mutated Hsp70s with caspase-binding motifs act as direct caspase-3 inhibitors. Based on Hsp70 molecular modeling, the DNQP, DEVQ, and EEVD regions localized on the surface of Hsp70WT were chosen, allowing us to design mutants while trying to avoid disrupting the global fold of the molecule and losing the possibility of protein-protein interactions. We replaced DNQP with DQMD, and DEVQ and EEVD with DEVD residues that should be optimal substrates for caspase-3. The resultant Hsp70 mutants directly interacted with active caspase-3 and blocked its proteolytic activity while retaining the ability to reverse protein denaturation and disrupt the interaction between Apaf-1 and procaspase-9. The Hsp70C-terminal mutants interacted with Apaf-1 and active caspase-3 significantly longer than Hsp70WT. The Hsp70 DXXD mutants protected neuron and teratocarcinoma (NT) cells against cell death much better than Hsp70WT whether given before or after serum withdrawal. Hsp70 mutants represent a possible approach to antiapoptotic biotherapeutics. Similar rational designs could be used to engineer inhibitors of additional caspase family members.
Assuntos
Apoptose/genética , Caspases/metabolismo , Proteínas de Choque Térmico HSP70/fisiologia , Mutação/genética , Motivos de Aminoácidos/genética , Fator Apoptótico 1 Ativador de Proteases , Sítios de Ligação/genética , Caspase 3 , Caspase 9 , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Meios de Cultura Livres de Soro/farmacologia , Desenho de Fármacos , Proteínas de Choque Térmico HSP70/genética , Humanos , Modelos Moleculares , Ligação Proteica/genética , Isoformas de Proteínas/genética , Proteínas/metabolismo , Transdução de Sinais/genética , Transdução Genética , Células Tumorais CultivadasRESUMO
Application of gene expression profiling to human diseases will be limited by availability of tissue samples. It was postulated that germline genetic defects affect blood cells to produce unique expression patterns. This hypothesis was addressed by using a test neurological disease-neurofibromatosis type 1 (NF1), an autosomal dominant genetic disease caused by mutations of the NF1 gene at chromosome 17q11.2. Oligonucleotide arrays were used to survey the blood gene expression pattern of 12 NF1 patients compared to 96 controls. A group of genes related to tissue remodeling, bone development and tumor suppression were down-regulated in NF1 blood samples. In addition, there were blood genomic patterns for gender and age: Y chromosome genes showing higher expression in males, indicating a gene-dosage effect; and genes related to lymphocyte functions showing higher expression in children. The results suggest that genetic mutations can be manifested at the transcriptional level in peripheral blood cells and blood gene expression profiling may be useful for studying phenotypic differences of human genetic diseases and possibly providing diagnostic and prognostic markers.
Assuntos
Fenômenos Fisiológicos Sanguíneos , Genômica , Neurofibromatose 1/sangue , Neurofibromatose 1/genética , Adolescente , Adulto , Fatores Etários , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores SexuaisRESUMO
In this study, we examine the effects of reperfusion on the activation of matrix metalloproteinase (MMP) and assess the relationship between MMP activation during reperfusion and neurovascular injury. Ischemia was produced using suture-induced middle cerebral artery occlusion in rats. The MMP activation was examined with in situ and gel zymography. Injury to cerebral endothelial cells and basal lamina was assessed using endothelial barrier antigen (EBA) and collagen IV immunohistochemistry. Injury to neurons and glial cells was assessed using Cresyl violet staining. These were examined at 3 h after reperfusion (8 h after initiation of ischemia) and compared with permanent ischemia at the same time points to assess the effects of reperfusion. A broad-spectrum MMP inhibitor, AHA (p-aminobenzoyl-Gly-Pro-D-Leu-D-Ala-hydroxamate, 50 mg/kg intravenously) was administered 30 min before reperfusion to assess the roles of MMPs in activating gelatinolytic enzymes and in reperfusion-induced injury. We found that reperfusion accelerated and potentiated MMP-9 and MMP-2 activation and injury to EBA and collagen IV immunopositive microvasculature and to neurons and glial cells in ischemic cortex and striatum relative to permanent ischemia. Administering AHA 30 min before reperfusion decreased MMP-9 activation and neurovascular injury in ischemic cerebral cortex.
Assuntos
Membrana Basal/enzimologia , Traumatismo Cerebrovascular/etiologia , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Traumatismo por Reperfusão/enzimologia , Reperfusão/efeitos adversos , Análise de Variância , Animais , Autoantígenos/metabolismo , Membrana Basal/efeitos dos fármacos , Membrana Basal/patologia , Colágeno Tipo IV/metabolismo , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Masculino , Fragmentos de Peptídeos/farmacologia , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/patologia , Fatores de TempoRESUMO
There are no biomarkers that differentiate cardioembolic from large-vessel atherosclerotic stroke, although the treatments differ for each and approximately 30% of strokes and transient ischemic attacks have undetermined etiologies using current clinical criteria. We aimed to define gene expression profiles in blood that differentiate cardioembolic from large-vessel atherosclerotic stroke. Peripheral blood samples were obtained from healthy controls and acute ischemic stroke patients (<3, 5, and 24 h). RNA was purified, labeled, and applied to Affymetrix Human U133 Plus 2.0 Arrays. Expression profiles in the blood of cardioembolic stroke patients are distinctive from those of large-vessel atherosclerotic stroke patients. Seventy-seven genes differ at least 1.5-fold between them, and a minimum number of 23 genes differentiate the two types of stroke with at least 95.2% specificity and 95.2% sensitivity for each. Genes regulated in large-vessel atherosclerotic stroke are expressed in platelets and monocytes and modulate hemostasis. Genes regulated in cardioembolic stroke are expressed in neutrophils and modulate immune responses to infectious stimuli. This new method can be used to predict whether a stroke of unknown etiology was because of cardioembolism or large-vessel atherosclerosis that would lead to different therapy. These results have wide ranging implications for similar disorders.
Assuntos
Aterosclerose/complicações , Células Sanguíneas , Embolia/complicações , Perfilação da Expressão Gênica , Acidente Vascular Cerebral/etiologia , Acidente Vascular Cerebral/genética , Biomarcadores/sangue , Plaquetas , Estudos de Casos e Controles , Diagnóstico Diferencial , Perfilação da Expressão Gênica/métodos , Perfilação da Expressão Gênica/normas , Humanos , Monócitos , Neutrófilos , Sensibilidade e Especificidade , Acidente Vascular Cerebral/diagnósticoRESUMO
A cDNA encoding a novel protein was cloned from ischemic rat brain and found to be homologous to testis Mea-2 Golgi-associated protein (Golga3). The sequence predicted a 165-kDa protein, and in vitro translated protein exhibited a molecular mass of 165-170 kDa. Because brain ischemia induced the mRNA, and the protein localized to the Golgi apparatus, this protein was designated Ischemia-Inducible Golgin Protein 165 (IIGP165). In HeLa cells, serum and glucose deprivation-induced caspase-dependent cleavage of the IIGP165 protein, after which the IIGP165 fragments translocated to the nucleus. The C-terminus of IIGP165, which contains a LXXLL motif, appears to function as a transcriptional co-regulator. Akt co-localizes with IIGP165 protein in the Golgi in vivo, and phosphorylates IIGP165 on serine residues 345 and 134. Though transfection of IIGP165 cDNA alone does not protect HeLa cells from serum deprivation or Brefeldin-A-triggered cell death, co-transfection of both Akt and IIGP165 cDNA or combined IIGP165-transfection with PDGF treatment significantly protects HeLa cells better than either treatment alone. These data show that Akt phosphorylation of IIGP165 protects against apoptotic cell death, and add to evidence that the Golgi apparatus also plays a role in regulating apoptosis.
Assuntos
Apoptose/fisiologia , Isquemia Encefálica/metabolismo , Encéfalo/metabolismo , Citoproteção/genética , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Motivos de Aminoácidos/fisiologia , Sequência de Aminoácidos/fisiologia , Animais , Autoantígenos/química , Autoantígenos/genética , Autoantígenos/metabolismo , Sequência de Bases , Encéfalo/fisiopatologia , Isquemia Encefálica/genética , Isquemia Encefálica/fisiopatologia , Células COS , Chlorocebus aethiops , Citoproteção/efeitos dos fármacos , DNA Complementar/farmacologia , Proteínas da Matriz do Complexo de Golgi , Células HeLa , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Dados de Sequência Molecular , Células PC12 , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Ratos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificação , Transfecção/métodosRESUMO
Exposure to moderate hypoxia alone does not cause neuronal death as long as blood pressure and cerebral blood flow are maintained in mammals. In neonatal and adult mammals including rats and mice, carotid occlusion in combination with hypoxia produces neuronal death and brain infarction. However, preexposure to 8% oxygen for 3 h protects the brain and likely other organs of neonatal and adult rats against combined hypoxia-ischemia 24 h later. In this paper, the possible mechanisms of this so-called hypoxia-induced tolerance to ischemia is discussed. One mechanism likely involves hypoxia-inducible factor-1alpha (HIF-1alpha). HIF-1alpha is a transcription factor that - during hypoxia - binds with a second protein (HIF-1beta) in the nucleus to promoter elements in hypoxia-responsive target genes. This causes upregulation of HIF target genes including VEGF, erythropoietin, iNOS, glucose transporter-1, glycolytic enzymes, and many other genes to protect the brain against ischemia 24 h later. In addition, non-HIF pathways including MTF-1, Egr-1 and others act directly or indirectly on other target genes to also promote hypoxia-induced preconditioning. Hypoxia preconditioning can be mimicked by iron chelators like desferrioxamine and transition metals like cobalt chloride that inhibit prolyl hydroxylases, increase HIF-1alpha levels in the brain, and produce protection of the brain against combined hypoxia-ischemia 24 h later. This hypoxia preconditioning has potential clinical usefulness in protecting high-risk newborns or to provide protection prior to surgery.
Assuntos
Encéfalo/irrigação sanguínea , Hipóxia-Isquemia Encefálica/fisiopatologia , Precondicionamento Isquêmico , Modelos Biológicos , Animais , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia , Camundongos , Ratos , Fatores de Transcrição/metabolismoRESUMO
Geldanamycin (GA), a benzoquinone ansamycin, binds Hsp90 in vitro, releases heat shock factor (HSF1) and induces heat shock proteins (Hsps). Because viral and transgenic overexpression of Hsps protects cells against ischemia in vitro, we hypothesized that GA would protect brain from focal ischemia by inducing Hsps in vivo. Adult male Sprague-Dawley rats were subjected to 2-hour middle cerebral artery occlusions (MCAO) using the suture technique followed by 22-h reperfusions. GA or vehicle was injected into the lateral cerebral ventricles (i.c.v) 24 h before ischemia. Geldanamycin at 1 microg/kg decreased infarct volumes by 55.7% (p < 0.01) and TUNEL-positive cells by 30% in cerebral cortex. GA also improved behavioral outcomes (p < 0.01) and reduced brain edema (p < 0.05). Western blots showed that the 1 microg/kg GA dose induced Hsp70 and Hsp25 protein 8.2-fold and 2.7-fold, respectively, by 48 h following administration. Immunocytochemistry showed that GA induced Hsp70 in neurons and Hsp25 in glia and arteries in cortex, hippocampus, hypothalamus, and other brain regions. GA reduced co-immunoprecipitation of HSF1 with Hsp90 in brain tissue homogenates, promoted HSE-binding of HSF in brain nuclear extracts using gel shift assays, and increased luciferase reporter gene transcription for the Hsp70 promoter in PC12 cells. The data show that geldanamycin protects brain from focal ischemia and that this may be due, at least in part, to geldanamycin stimulation of heat shock gene transcription.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Encéfalo/efeitos dos fármacos , Proteínas de Choque Térmico/metabolismo , Quinonas/farmacologia , Animais , Benzoquinonas , Western Blotting , Encéfalo/metabolismo , Encéfalo/patologia , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Proteínas de Choque Térmico HSP27 , Proteínas de Choque Térmico HSP70/metabolismo , Fatores de Transcrição de Choque Térmico , Imuno-Histoquímica , Injeções Intraventriculares , Lactamas Macrocíclicas , Masculino , Proteínas de Neoplasias/metabolismo , Ligação Proteica/efeitos dos fármacos , Ratos , Fatores de Transcrição , Ativação Transcricional/efeitos dos fármacosRESUMO
Animals exposed to brief periods of moderate hypoxia (8% to 10% oxygen for 3 hours) are protected against cerebral and cardiac ischemia between 1 and 2 days later. This hypoxia preconditioning requires new RNA and protein synthesis. The mechanism of this hypoxia-induced tolerance correlates with the induction of the hypoxia-inducible factor (HIF), a transcription factor heterodimeric complex composed of inducible HIF-1alpha and constitutive HIF-1beta proteins that bind to the hypoxia response elements in a number of HIF target genes. Our recent studies show that HIF-1alpha correlates with hypoxia induced tolerance in neonatal rat brain. HIF target genes, also induced following hypoxia-induced tolerance, include vascular endothelial growth factor, erythropoietin, glucose transporters, glycolytic enzymes, and many other genes. Some or all of these genes may contribute to hypoxia-induced protection against ischemia. HIF induction of the glycolytic enzymes accounts in part for the Pasteur effect in brain and other tissues. Hypoxia-induced tolerance is not likely to be equivalent to treatment with a single HIF target gene protein since other transcription factors including Egr-1 (NGFI-A) have been implicated in hypoxia regulation of gene expression. Understanding the mechanisms and genes involved in hypoxic tolerance may provide new therapeutic targets to treat ischemic injury and enhance recovery.
Assuntos
Isquemia Encefálica/fisiopatologia , Precondicionamento Isquêmico , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/fisiopatologia , Isquemia Encefálica/genética , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia , Ratos , Receptores de Hidrocarboneto Arílico/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The major heat shock protein, Hsp70, can protect against cell death by directly interfering with mitochondrial apoptosis pathways. However, Hsp70 also sensitizes cells to certain apoptotic stimuli like TNF. Little is known about how Hsp70 enhances apoptosis. We demonstrate here that Hsp70 promotes TNF killing by specifically binding the coiled-coil domain of I kappa B kinase gamma (IKK gamma) to inhibit IKK activity and consequently inhibit NF-kappa B-dependent antiapoptotic gene induction. An IKK gamma mutant, which interacts with Hsp70, competitively inhibits the Hsp70-IKK gamma interaction and relieves heat-mediated NF-kappa B suppression. Depletion of Hsp70 expression with RNA interference rescues TNF-mediated cell death. Although TNF may or may not be sufficient to trigger apoptosis on its own, TNF-triggered apoptosis was initiated or made worse when Hsp70 expression increased to high levels to disrupt NF-kappa B signaling. These results provide significant novel insights into the molecular mechanism for the pro-apoptotic behavior of Hsp70 in death-receptor-mediated cell death.