Abstract 49: Deletion of Mirna 17-92 in Cerebral Endothelial Cells Induces Disruption of BBB

Abstract

Background: The MicroRNA 17-92 (miR17-92) cluster regulates endothelial homeostasis. However, the effect of the miR17-92 cluster in cerebral endothelial cells on blood brain barrier (BBB) has not been investigated. We examined BBB integrity in a transgenic mouse line in which miR17-92 cluster is ablated in endothelial cells. Methods and Results: Adult Tie2-Cre:miR17-92 flox/flox (Tie2/miR17-92) mice and Tie2-Cre:Tomato (Tie2/red) reporter mice were used. Cerebral vessels in Tie2/red reporter mice exhibited red fluorescence, indicating specific expression of Tie2 in cerebral endothelial cells. RT-PCR analysis revealed ~70% reduction of miRNA levels of individual members of the miR17-92 cluster in endothelial cells harvested from Tie2/miR17-92 mouse brain, confirming deletion of the miR17-92 cluster. Time-lapse microscopic analysis of cultured endothelial cells showed a substantial reduction of capillary formation by miR17-92 cluster knockout endothelial cells compared to the cells harvested from Tie2/red reporter mice. In vivo, analysis of 3D cerebral microvessels showed that ablation of the miR17-92 markedly (P<0.05) increased capillary diameter in the cortex (7.1±0.4μm vs 5.1±0.3μm in reporter, n=4 mice/group), corpus callosum (CC, 6.6±0.4 vs 4.7±0.3), and striatum (6.7±0.4 vs 5.0±0.3). Triple immunofluorescent staining showed that the absence of this cluster substantially (p<0.05) reduced pericytes identified by platelet-derived growth factor-β positive cells (32±6 cells vs 80±6 cells in cortex, 34±7 vs 77±6 in CC, and 36±6 vs 79±5 in striatum) and decreased astrocyte end-foot processes identified by aquaporin 4 positive cells (42±6 cells vs 72±6 cells in cortex, 40±10 vs 70±6 in CC, and 36±6 vs 65±8 in striatum). These data suggest that cerebral endothelil cells lacking miR17-92 causes disruption of vascular coverage by pericytes and astrocyte end-foot processes. Deletion of the miR17-92 cluster also increased extravasation of Evans blue dye into the parenchyma and reduced the number of doublecortin positive neuroblasts in the subventricular zone. Conclusion: Our data indicate that ablation of the miR17-92 cluster in cerebral endothelial cells disrupts BBB integrity, which could exacerbate ischemic damage.