Fig. 3
Role of cortistatin in the human brain endothelium. a Human brain-like endothelial cells (hBLECs) were obtained after 1 week of co-culture of CD34+-derived endothelial cells with pericytes. hBLECs were incubated 4 h under hypoxia followed by 20 h of reoxygenation (HPX-R) in the absence or presence of cortistatin (HPX-R + CST, 100 nM). b Endothelium integrity was evaluated and represented as the index (%) of EBA permeability vs an empty-coated insert. N = 3 cultures/group. Data are the mean ± SEM with dots representing individual values of independent cultures. c Left, expression of ZO-1 (green) was evaluated by immunofluorescence. Arrowheads indicate reduced and disrupted expression of ZO-1 after HPX-R. Cortistatin treatment protected the membrane from breakdown and recovered the tight pattern of intercellular ZO-1 (asterisks). Right, delocalization of ZO-1 was evaluated through the ratio of ZO-1 staining intensity in the membrane vs the cytosol. 25–50 selected ROIs in 4 independent fields were analysed. N = 6 cultures/group. d Cortistatin protein levels were quantified in hBLECs supernatants 20 h after exposure to NX/NG and HPX-R, as described. Results are normalized in ng protein/105 cells. N = 4 cultures/group. *p ≤ 0.05, **p ≤ 0.01. Scale bar: 20 µm