Fig. 4.

Influence of angiopoietin (ANG)2/ANG1 imbalance on endothelial hyper-permeability ex vivo. Human pulmonary microvascular endothelial cells were seeded at high density on gelatin-coated permeable supports and cultured to form a confluent monolayer. After pre-incubation with filtered and diluted (7.5 %) patient serum (preoperative (preop) or 24 h, respectively) for 30 minutes, fluorescein isothiocyanate (FITC)-conjugated dextran (70 kDa, final concentration 1 mg/ml) was added to the upper compartment and the inserts were set on a magnetic stirrer. Thrombin (final concentration 1 IU/ml) was added to induce endothelial permeability. Samples (of 50 μl) were taken from the lower compartment at the indicated time points. Dextran concentration was determined using a fluorescent plate reader. Thrombin-induced hyper-permeability is expressed as fold of the average basal flux during the initial 15-minute equilibration period before thrombin addition. Left, change in endothelial cell (EC) permeability after pre-incubation with serum samples with a mild shift in the ANG2/ANG1 ratio upon coronary artery bypass graft (CABG) (6-fold from baseline to 24 h). Right, change of EC permeability after pre-incubation with serum samples with pronounced shift in the ANG2/ANG1 ratio upon CABG (18-fold from baseline to 24 h). N = 8–10, mean ± standard error of the mean, *p <0.05, **p <0.01. Pat patient