During the last decade it has been recognized that blood brain barrier (BBB) dysfunction exists in most neurological diseases, including multiple sclerosis, stroke, Alzheimer's and Parkinson's diseases, brain infections and epilepsy. Inflammation plays significant role in BBB injury, secondary to pro-inflammatory factors produced in the brain or blood and leukocyte engagement of brain endothelium. Brain microvascular endothelial cells (BMVEC) are active participants and regulators of inflammatory processes at a site of inflammation. Inflammatory responses in brain endothelium involve hundreds of genes which expression requires fine-tuned regulation. microRNAs (miRNAs) recently emerged as major regulators of gene expression. Very limited information exists about their participation in inflammatory responses in brain endothelial cells. We propose to investigate the role of miRNAs in brain endothelium using well-established in vitro systems for functional studies of BBB and in vivo imaging of brain microvasculature in the model of neuroinflammation. Based on our studies of BBB dysfunction during neuroinflammation, we propose that barrier protection is best achieved when the intervening agents possess anti-inflammatory properties and can stabilize tight junctions as we have shown via inhibition of glycogen synthase kinase (GSK3? To identify a set of relevant miRNAs, we activated primary human BMVEC with the inflammatory stimulus, TNF?identified 137 down-regulated miRNAs and 180 up-regulated miRNAs. In replicate BMVEC, we inhibited GSK3?n BMVEC during TNF?timulation and detected several miRNAs with expression patterns that were regulated by GSK3?The same miRNAs were changed in brain microvessels isolated from mice exposed to TNF?miRNA overexpression prevented secretion of inflammatory mediators in BMVEC. In our proposed study, we will test the overexpression or inhibition of selected miRNAs on BBB tightness and the effects on monocyte-endothelial cell engagement (adhesion/migration). Using bioinformatics, we will identify other targets for miRNAs. Next, we will perform miRNA transfection in vivo and monitor how this will change leukocyte adhesion/migration in an animal model of neuroinflammation. Proposed experiments will provide identification and functional assessment of miRNAs in brain endothelium.
Endothelial dysfunction is perhaps the earliest event in the initiation of vascular disease, caused by inflammation. In this work we propose to use miRNA machinery as a therapeutic tool to prevent deleterious effects of endothelial dysfunction on neuroinflammation. Being able to avoid blood brain barrier from increased permeability would prevent vascular malfunction, which is a most common features of several central nervous system disorders, such as Alzheimer's and Parkinson's diseases, multiple sclerosis, HIV-1-associated neurocognitive disorder, stroke, atherosclerosis and traumatic brain injury.
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