Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.578000
Title: Investigating the role of inflammation-modulated microRNAs and signalling pathways in blood-barrier dysfunction
Author: Lopez-Ramirez, Miguel Alejandro
Awarding Body: Open University
Current Institution: Open University
Date of Award: 2012
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Abstract:
Cerebral endothelial cells (CEC) constitute a key cellular element of the blood- brain barrier (BBB) due to their bidirectional selective permeability that contributes to central nervous system (CNS) homeostasis. However, their specialized barrier function is altered during neuroinflammatory disorders such as multiple sclerosis (MS). Yet, the molecular mechanisms implicated in the loss of brain endothelial barrier function remain to be fully elucidated. To address this question, we first established an in vitro BBB inflammation model using the human cerebral microvascular endothelial cell line, hCMEC/D3 cell, we then determined the mRNA signature of hCMEC/D3 cells in the absence and presence of pro-inflammatory cytokines and compared results to previously published data on mouse CEC transcriptome analysis. We classified genes expressed at the mRNA level into inter-brain endothelial junctions, transporter systems, cytoskeletal associated molecules, integrin-focal adhesions complexes, cell adhesion molecules and chemokines. We report that long term exposure of hCMEC/D3 cells to TNFa/IFNy results in a new CEC gene expression pattern associated with barrier dysfunction. We then investigated the signalling pathways implicated in cytokine-induced increase in paracellular permeability. We propose that during neuroinflammation the concentration of cytokines in the CNS micfoenviroment to which CECs are exposed determines the extent of caspase-mediated barrier permeability changes which may be generalized, as a result of brain endothelial apoptosis, or more subtle, as a result of alterations in the organization of junctional complex molecules. In addition, we found that the PKC-JNK axis might be an important "control node" to regulate the paracellular pathway of CECs during inflammation. In the third chapter, we have investigated whether the BBB-signature of CECs might be in part regulated by microRNAs (miRNAs). MiRNAs are endogenous non-coding small RNAs that suppress gene expression at a post-transcriptional level and have been shown to modulate several biological processes. As a first approach we determined the changes in miRNA levels induced by pro-inflammatory cytokines in hCMEC/D3 cells. We then confirmed the deregulation of brain endothelial miRNAs in MS brain tissue and in experimental auto immune encephalomyelitis, an animal model of MS. Interestingly, we observed a temporal-pattern of miRNA expression that might correspond to pro- and anti- inflammatory miRNAs that fine tune gene expression in cerebral endothelium during an inflammatory response. In the fourth chapter, we investigated the role of miR-l55 on brain endothelial barrier integrity. MiR-l55 was shown to be rapidly and highly increased in hCMEC/D3 cells after cytokine stimulation. Our findings showed that increased levels of miR-l55 in brain endothelium might participate in cell activation during inflammation resulting in a moderate increase in leukocyte adhesion and a robust effect on modulating brain endothelial paracellular permeability. We propose that the paracellular permeability effect of miR-l55 involves targeting a set of interrelated genes that regulate cell-cell junctions and focal adhesions. In addition, we characterized the cytokine-induced signalling pathways mediating upregulation of miR-155 in cultured CECs. Our results suggest that miRNAs modulate key features of CECs and may constitute major players in the pathogenesis of CNS inflammatory disorders that affect the BBB. In summary, we have determined the role of some key brain endothelial miRNAs and signalling pathways in a critical pathogenic feature of neuroinflammation, BBB dysfunction, which may have important consequences for the rational development of therapies for CNS inflammatory disorders.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.578000  DOI: Not available
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