Figure 6
HDAC2 regulates the expression of tight junction proteins in MCECs.
(a) siRNA-mediated silencing of HDAC2 increases the expression of tight junctions in primary cultured MCECs. (Left) Representative gel picture showing the gene expression of occludin and claudin-5. GAPDH was used as an internal control for RT-PCR. (Right) Representative Western blot for occludin and claudin-5. β-actin was used as an internal control for Western blot. After serum starvation for 24 hours, MCECs were transfected with scrambled siRNA or siRNA specific to HDAC2 for 48 hours. Results were similar from four independent experiments. (b) Overexpression of HDAC2 with plasmid encoding HDAC2 (pHDAC2) decreases the expression of tight junction proteins in MCECs. Immuocytochemical staining of MCECs performed with antibodies to occludin and claudin-5. Arrow heads denote disorganized tight junctions. Results were similar from four independent experiments. Scale bar = 50 μm. (c) Overexpression of HDAC2 with pHDAC2 increases vascular endothelial permeability in MCECs. Vascular endothelial permeability was determined by using the passage of rhodamine B isothiocyanate (RITC)-labeled dextran through monolayer of MCECs. Each bar depicts the mean ± SEM of four independent experiments. Mann-Whitney U-test was used for statistical analysis. *P <0.01 vs control group. (d) COMP-Ang1 protein (400 ng/ml) decreases HDAC2 expression and increases expression of occludin and claudin-5 in MCECs. Representative Western blot for HDAC2, occludin and claudin-5. Results were similar from four independent experiments. (e) COMP-Ang1 protein-mediated increase in tight junction expression is dependent on PI3K/Akt or MEK/ERK pathway. MCECs were pretreated with chemical inhibitor of PI3K/Akt pathway (LY 294002) or MEK/ERK pathway (U0126) and then treated with COMP-Ang1 protein (400 ng/ml). Results were similar from four independent experiments. MCECs, mouse cavernous endothelial cells.
