It has been recognized that blood brain barrier (BBB) dysfunction exists in most neurological diseases,
including stroke, multiple sclerosis, Alzheimer's and Parkinson's diseases, brain infections and epilepsy.
Inflammation plays a 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 whose 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 caused by ischemia/reperfusion (I/R) events 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 the BBB and imaging of brain microvasculature in an in vivo 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.
Recently we have identified highly modified miRNAs, belonging to the let-7 miRNA family, which are important
for endothelial maintenance. let-7 and miR-98 were predicted to target the inflammatory molecules, CCL2,
CCL5, IL8 and IP10/CXCL10. Overexpression of let-7 and miR-98 in vitro and in vivo resulted in reduced
leukocyte adhesion to and migration across brain endothelium and diminished expression of pro-inflammatory
cytokines. In oxygen glucose deprivation (OGD) followed by reperfusion (OGD/R), an in vitro I/R model,
overexpression of these miRNAs led to increased BBB tightness, thereby attenuating barrier `leakiness'.
Overexpression of these miRNAs resulted in decreased infarct volume and neutrophil infiltration in the brain in
tMCAO, an in vivo I/R stroke animal model. In our proposed study, we will test the overexpression or inhibition
of selected miRNAs on BBB tightness and leukocyte-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 the presence of miRNAs will change leukocyte adhesion/migration in an animal model of stroke.
Proposed experiments will provide identification and functional assessment of miRNAs in brain endothelium,
and lead to future therapeutic developments for prevention of deleterious effects of ischemia/reperfusion on the