Background: Insulin like growth factor (IGF1) treatment to middle aged female rats post stroke reduces infarct volume, and this is preceded by reduced permeability of the blood brain barrier (BBB) at 4h post stroke. Analysis of ischemic tissue at 4h showed that IGF1 treatment significantly reduced expression of 8 miRNA. To determine if these miRNA are mechanistically linked to barrier function, we tested the impact of these miRNA on barrier function using human brain microvascular endothelial cells (hBMECs) under ischemic conditions. Specifically, we mimicked the actions of IGF1 by using antagomirs to miR-29c, miR-33a and miR-92b.
Methods: hBMECs were grown to confluence on collagen-coated dishes (5% CO2, 95% air) and exposed to acute oxygen-glucose deprivation (6h OGD, 1% O2 % and 5 % CO2) in the presence of IGF1 or vehicle. In addition, cultures were also treated with IGF1+JB-1 (IGFR antagonist), IGF1+ LY294002 (Akt inhibitor) and rapamycin (mTOR inhibitor), to establish IGF1 mediated pathways on barrier function. In parallel, cultures were exposed to antagomirs for mir29c, mir 33a and mir92b or scrambled oligo controls. Integrity of the cell monolayer was assessed by tomato-lectin staining and LDH assays for cell death. Lectin stained cell cultures (3 experimental replicates) were photographed, coded and analyzed for continuity of monolayer using a novel algorithm to determine intercellular ‘spaces’.
Results: OGD caused significant retraction (11 fold increase, p<0.02) and discontinuous aggregates of cells, while IGF1 preserved the integrity of the endothelial monolayer and were no different from normoxic condition. JB-1 (13 fold increase,p<0.01) and Akt inhibitor (3.3 fold, p<0.02) abrogated the effects of IGF-1. Antagomirs to mir92b and mir33a also maintained cell integrity after OGD, (7 fold,p<0.03) while mir29c and scrambled oligos (10 fold damage, p<0.04) were ineffective. LDH assay was not significantly different in any of the treatment conditions, indicating that necrotic cell death was not altered.
Conclusions: These findings provide direct evidence that IGF-1 actions at the endothelial barrier are replicated by specific miRNA and raise the possibility that IGF-1’s actions can be replicated by targeted suppression of miRNAs.