Fig. 4
Dynamics of VEGFA-induced vascular leakage. a Point of leakage of circulating 2000 kDa FITC-Dextran and 400 kDa TRITC-Ficoll in a large observation area (left) and zoom-in (right). Flow direction (arrows) and leakage point in a capillary (dashed line), with leakage point boxed (solid line). Right panel, magnification of dashed box area. Capillary leakage point indicated by red arrow. Bar, 200 (left), 100 (right) and 10 μm (inset). b Leakage of 400 kDa Ficoll in response to VEGFA164 in the ear dermis of Cldn5(BAC)-GFP mice. Bar, 100 μm. c Leakage points (red arrow) in Cldn5(BAC)-GFP mouse dermis, in venule (upper) and capillary (lower), showing progressive leakage over time of 400 kDa TRITC-Ficoll in response to VEGFA164. Bar, 10 μm. d Quantification of leakage points/vessel length in the Cldn5(BAC)-GFP ear dermis. Capillaries with GFP expression (positive; pos), mixed (mix) or no GFP (negative; neg) expression were analyzed. Note that Cldn5-expressing capillaries do not leak. N.D.; not detected. n = 3 mice with 6 vessels of each type analyzed/mouse. e Representative leakage point captured by sXYT imaging at −1, 2.5, and 10 min after injection of VEGFA164 (upper and middle panels). Kymograph image (lower panel) for analysis of leakage dynamics of 2000 kDa FITC-Dextran and 70 kDa TRITC-Ficoll was made from the point indicated by the white arrow line in upper panels. Red arrow indicates the leakage point. Bar, 10 μm. f Leakage kinetics from venules (left) and capillaries (right) of 70 kDa TRITC-Ficoll (upper) and 2000 kDa FITC-Dextran (lower) with lag periods and leakage durations indicated. Gray area around solid black line indicates variability (S.D.). n = 7 mice with 10 capillaries and 10 venules analyzed. g Quantification of lag periods (min) for the different conditions. n = 7 mice and 10 vessels/condition as in f. No significant difference by Tukey–Kramer test. h Quantification of leakage duration (min) for the different conditions. n = 7 mice and 10 vessels/condition as in f. Tukey–Kramer test; *p < 0.05, **p < 0.01, ***p < 0.001
