Defective ECM organization increases cellular tension and decreases barrier function of naive HUVECs. (A) ICAP-1-, CCM1-, or CCM2-depleted HUVECs were seeded at confluency on FN-coated slides. Cells were removed 48 h later by a triton/ammonium hydroxide treatment. Naive HUVECs were seeded on these decellularized remodeled matrices. (B and C) FN and ColIV staining of decellularized matrices remodeled in the absence (B) or presence of FUD (C). Note the linearity of FN fibers and ColIV in ICAP-1- and CCM1/2-depleted conditions. Bars, 1 µm. (D) Quantification of the percentage of naive HUVECs with central β1 integrin focal adhesions after 1 h of spreading. (E) Quantification of the percentage of naive HUVECs with transversal stress fibers after 4 h of spreading. (F) β-catenin and actin staining of confluent naive HUVECs on the remodeled matrices after 24 h. Bars, 5 µm. (G) Quantification of β-catenin staining thickness showing thinner junctions of CCM KD matrices. (H) Time-course measurement of the electrical impedance of a monolayer of naive HUVECs on these matrices. The data shown are from a single representative experiment out of three repeats. (I) Normalized impedance to that on CT matrix showing a significant decrease of monolayer resistance barrier on CCM KD matrices due to structural changes in FN remodeling. Error bars are ± SEM (n = 3). *, P < 0.05; **, P < 0.005; ***, P < 0.0005 using GLMM with Tukey’s test.