Fig. 8
Cortistatin recovers the integrity and function of brain endothelial cells after ischemic-like conditions. a Endothelial permeability to EBA was represented as the tracer permeability fold change vs the permeability in OGD-R wild-type BECs (set at 1). N = 5–8 cultures/group. b Immunofluorescence analysis of cellular distribution for tight/adherens-junctions and stress fibers in wild-type and cortistatin-deficient BECs after OGD-R incubated with cortistatin. Asterisks indicate continuous membrane expression of ZO-1 (red), claudin-5 (red), and VE-cadherin (green), reduced intracellular claudin-1 (green), and random cytosolic F-actin organization (red), when compared to BECs exposed to OGD-R without cortistatin addition (as shown in Fig. 4b–d; schematic images with injury hallmarks represented in the top: Ref-BECs). Scale bar: 20 µm. c Quantification of location (ZO-1) and expression (claudin-1, claudin-5, VE-cadherin, F-actin) by fluorescence intensity analysed from 25–50 ROIs in 4 independent fields. N = 6 cultures/group. d Levels of inflammatory mediators determined in culture supernatants. N = 8 cultures/group. e Top, representation of transendothelial migration assay. Wild-type mice immune cells were incubated for 24 h at the top of Cort+/+, Cort+/−, and Cort−/− BECs covered inserts, previously activated with TNF-α (10 ng/ml, 24 h). The migration assay was performed using MCP-1 (50 μg/ml) or IP-10 (50 μg/ml), as macrophages and T-cell chemoattractants, respectively. CST (100 nM) was applied when indicated. Bottom, data represented the percentage of migrated immune cells vs the control (empty-coated insert). N = 4–6 cultures/group. For data in c, d, FC represents fold change vs reference values based on OGD-R wild-type BECs quantification (set at 1). Cort+/+ BECs in NX/NG are also used as a basal condition reference (dashed line). Data are the mean ± SEM with dots representing individual values of independent cultures. Cells in each culture were derived from 4 pooled brains. *vs Cort+/+ BECs exposure to OGD-R + CST; #vs BECs of corresponding genotype (Cort+/+, Cort+/−, Cort−/−) exposure to OGD-R.*/#p ≤ 0.05, **/##p ≤ 0.01, ***/###p ≤ 0.001, ****/####p ≤ 0.0001