Figure 5.

VCAM‐1 blockade reduces lymphatic permeability. (a, b) Representative FACS histograms (left panels) and staining quantification (right panels) of the classic VCAM‐1 receptors integrin α4 (Itga4), integrin α9 (Itga9) and integrin β1 (Itgb1) in 4T1‐1F8 cells (a) and cultured mouse LECs with or without TNF‐α treatment for 24 hr (b; n = 3). (c) Adhesion of fluorescently labeled 4T1‐1F8 cells to monolayers of cultured mouse LECs pretreated with TNF‐α or not and in the presence of control IgG or VCAM‐1 blocking antibodies (n = 3 wells/condition; 1 representative of 6 individual experiments is shown). (d) In vitro permeability of mouse LEC monolayers grown on transwell inserts (0.4 μm pore size) toward 70 kD FITC‐dextran. LEC monolayers were pretreated with TNF‐α overnight and subsequently incubated with control or 4T1 CM in presence of control IgG or anti‐VCAM‐1 antibodies (n = 3 wells/condition; one representative of three individual experiments is shown). Statistical test refers to 4T1‐1F8 CM—rat IgG1 compared to 4T1‐1F8 CM—anti‐VCAM‐1. (e) Representative images (left panels) and quantification (right panel) of VE‐cadherin staining in mouse LEC monolayers that were pretreated with TNF‐α overnight and subsequently incubated with control or 4T1 CM in presence of control IgG or anti‐VCAM‐1 antibodies (n = 10 random images/condition; *p < 0.05). [Color figure can be viewed at wileyonlinelibrary.com]