Figure 4.

8-CPT-AM prevents and reverses VEGF-induced tight junction disorganization. A, immunofluorescence staining of TJ proteins ZO-1, occludin, and claudin-5 was performed to assess their organization after the addition of 8-CPT-AM (1 μm) for 30 min and/or VEGF (50 ng/ml) for 1 h. Yellow arrows show loss of linear organization (invaginations), and yellow arrowheads show border breaks or gaps between adjacent cells. Histograms show scoring of TJ proteins as percent organization. ZO-1, occluding, and claudin-5 border staining were quantified by a semi-quantitative ranking score system based on a graded scale from 1 to 5: Grade 1 for near complete disorganization of border staining 0 to 25%; Grade 2 for 25 to 50% continuous border staining; Grade 3 for 50 to 75% continuous border staining; Grade 4 for 75% to 100% continuous border; and Grade 5 completely continuous border. B, changes in TJ total protein expression with VEGF or 8-CPT-AM were measured using Western blot analysis. C–E, no changes in ZO-1, occludin, and claudin-5 expression were observed in any of the experimental conditions. Results are expressed as the mean relative to the control ± S.D. with analysis by one-way analysis of variance and Bonferroni post hoc test: #, p < 0.01; ##, p < 0.005; ####, p < 0.0001, or ****, compared with VEGF.