RESUMO
BACKGROUND: Altered gene expression is an important feature of ischemic cerebral injury and affects proteins of many functional classes. We have used microarrays to investigate the changes in gene expression at various times after middle cerebral artery occlusion in human and rat brain. RESULTS: Our results demonstrated a significant difference in the number of genes affected and the time-course of expression between the two cases. The total number of deregulated genes in the rat was 335 versus 126 in the human, while, of 393 overlapping genes between the two array sets, 184 were changed only in the rat and 36 in the human with a total of 41 genes deregulated in both cases. Interestingly, the mean fold changes were much higher in the human. The expression of novel genes, including p21-activated kinase 1 (PAK1), matrix metalloproteinase 11 (MMP11) and integrase interactor 1, was further analyzed by RT-PCR, Western blotting and immunohistochemistry. Strong neuronal staining was seen for PAK1 and MMP11. CONCLUSION: Our findings confirmed previous studies reporting that gene expression screening can detect known and unknown transcriptional features of stroke and highlight the importance of research using human brain tissue in the search for novel therapeutic agents.
Assuntos
Encéfalo/metabolismo , Regulação da Expressão Gênica/fisiologia , Infarto da Artéria Cerebral Média/patologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Acidente Vascular Cerebral , Idoso , Idoso de 80 Anos ou mais , Animais , Encéfalo/patologia , Células Cultivadas , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Feto , Glucose/deficiência , Humanos , Hipóxia/etiologia , Hipóxia/mortalidade , Infarto da Artéria Cerebral Média/genética , Infarto da Artéria Cerebral Média/metabolismo , Masculino , Metaloproteinase 11 da Matriz/genética , Metaloproteinase 11 da Matriz/metabolismo , Pessoa de Meia-Idade , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Proteína SMARCB1 , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/metabolismoRESUMO
PAX3 encodes a transcription factor, which with Zic1 is necessary for induction of the neural crest during early embryonic development. There are 7 human PAX3 isoforms (a-h). PAX3e is the full length isoform comprising 10 exons. PAX3c comprises 8 exons plus 5 codons of intron 8, while PAX3g has a truncated transactivation domain. Previous studies by us indicated that these isoforms have different activities in melanocytes in vitro. In this study, a mouse gene oligo array ( approximately 7.5 k oligos), from the Human Genome Mapping Project (HGMP) Resource Centre, was used to screen for alterations in downstream gene expression in PAX3c, PAX3e and PAX3g melanocyte transfectants, compared with empty vector controls. The data analyses identified 109 genes up or downregulated, at least 2-fold, and involved in cell differentiation, proliferation, migration, adhesion, apoptosis and angiogenesis. Semi-quantitative RT-PCR and Western blotting confirmed the changes identified by microarrays for several putative targets of PAX3, including Met, MyoD and Muc18, and previously undescribed targets, including Dhh, Fgf17, Kitl and Rac1. Thus, our data reveal that PAX3 isoforms regulate distinct but overlapping sets of genes in melanocytes in vitro.
Assuntos
Regulação da Expressão Gênica , Melanócitos/metabolismo , Fatores de Transcrição Box Pareados/fisiologia , Animais , Apoptose/genética , Diferenciação Celular/genética , Movimento Celular/genética , Perfilação da Expressão Gênica , Humanos , Melanócitos/química , Camundongos , Neovascularização Fisiológica/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , TransfecçãoRESUMO
Our previous work has demonstrated that angiogenesis occurs in the damaged brain tissue of patients surviving acute ischaemic stroke and increased microvessel density in the penumbra is associated with longer patient survival. The brain is one of the richest sources of FGF-2 and several studies have noted its angiogenic and neuroprotective effects in the nervous system. These findings led us to investigate the expression and localisation of both FGF-2 mRNA and protein in brain tissue collected within 12 h of death from 10 patients who survived for between 24 h and 43 days after acute stroke caused by thrombosis or embolus. Western blot analysis demonstrated increased FGF-2 protein expression in both grey and white matter in the infarcted core and the penumbra region compared to the normal contralateral hemisphere of all 10 patients studied. Using indirect immunoperoxidase staining of paraffin embedded sections, we observed the presence of FGF-2 in neurones, astrocytes, macrophages and endothelial cells. In situ hybridisation was used to localise and quantify mRNA expression in ischaemic brain tissue of the same 10 patients. The expression of FGF-2 in the penumbra of all patients was significantly raised compared with infarcted tissue and normal-looking contralateral hemisphere. In addition, serum FGF-2 was significantly increased between 1 and 14 days (P<0.001) in many patients with both ischaemic stroke (n=28) and intra-cerebral haemorrhage (n=16) compared with age-matched control subjects undergoing routine medical examinations (n=20). We suggest that up-regulation of FGF-2 is one of the mechanisms that leads to angiogenesis and neuro-protection in the penumbra region after acute stroke in man.