Figure 3.
Figure 3. RhoB negatively regulates endothelial barrier recovery after acute contraction human endothelial cells. (A and B) Single knockdown of RhoA, RhoB, and RhoC has no effect on the barrier function of TNF-stimulated HUVECs. (A) Absolute TEER of HUVECs transfected with the indicated siRNA oligonucleotides for 72 h or incubated with TNF for 7 h. Control cells were incubated with 5 µm Y-27632 for 30 min before TNF stimulation when specified. Discontinuous line marks the basal resistance values in empty electrodes. *, P < 0.5. RhoBp, RhoB pool. (B) Permeability to dextran-FITC of siRNA-transfected HUVECs plated on transwells for 48 h and stimulated with TNF. No statistically significant differences were found in permeability values between siControl and siRho-transfected cells. (C) Left graph shows changes in TEER during thrombin-induced acute contraction in cells pretreated with TNF for 7 h as in A. Mean ± SEM from at least three independent experiments. (right graph) Percentage of maximum contraction with respect to siControl cells during acute contraction (0.5 h of thrombin stimulation) and barrier recovery (1.2 h of thrombin stimulation). A total of 5 µM Y-27632 was added 30 min before thrombin stimulation when indicated. Mean + SEM from at least three independent experiments. *, P < 0.02; **, P < 0.007; ***, P < 10−3. (D) Percentage of maximum contraction with respect to siControl cells during acute contraction (C.) and barrier recovery (R.) in HUVECs pretreated with TNF between 18 and 20 h. ***, P < 2.2 × 10−5. (E) Permeability to dextran-FITC was performed as in B but analyzed during the time of barrier recovery after acute contraction induced by thrombin. (F) Staining for β-catenin and F-actin in TNF-pretreated HUVECs stimulated during the recovery phase after thrombin. Empty spaces between cells were identified by automated image analysis (mask). β-Catenin staining at cell borders was quantified by semiautomated image processing in five different experiments. *, P ≤ 0.046. Bar, 20 µm. (G) Percentage of maximum contraction with respect to siControl cells during acute contraction and the subsequent recovery induced with thrombin in HUVECs not previously stimulated with TNF. *, P = 0.047; **, P = 0.009; ***, P = 9.4 × 10−5. (H) Changes in TEER during thrombin-induced acute contraction in HDMVECs pretreated with TNF for 7 h (left). Mean ± SEM from at least three independent experiments. Percentage of maximum contraction with respect to siControl1 during contraction and barrier recovery in HDMVECs (right). *, P ≤ 0.05; **, P < 0.01; ***, P < 0.005. (I) TNF-pretreated HDMVECs were fixed after 2.5 h of thrombin stimulation (recovery phase) and stained for β-catenin and F-actin. β-Catenin staining at cell borders was quantified as in F. *, P < 0.05; **, P < 0.01. Bar, 10 µm. Mean + SEM of at least three experiments.

RhoB negatively regulates endothelial barrier recovery after acute contraction human endothelial cells. (A and B) Single knockdown of RhoA, RhoB, and RhoC has no effect on the barrier function of TNF-stimulated HUVECs. (A) Absolute TEER of HUVECs transfected with the indicated siRNA oligonucleotides for 72 h or incubated with TNF for 7 h. Control cells were incubated with 5 µm Y-27632 for 30 min before TNF stimulation when specified. Discontinuous line marks the basal resistance values in empty electrodes. *, P < 0.5. RhoBp, RhoB pool. (B) Permeability to dextran-FITC of siRNA-transfected HUVECs plated on transwells for 48 h and stimulated with TNF. No statistically significant differences were found in permeability values between siControl and siRho-transfected cells. (C) Left graph shows changes in TEER during thrombin-induced acute contraction in cells pretreated with TNF for 7 h as in A. Mean ± SEM from at least three independent experiments. (right graph) Percentage of maximum contraction with respect to siControl cells during acute contraction (0.5 h of thrombin stimulation) and barrier recovery (1.2 h of thrombin stimulation). A total of 5 µM Y-27632 was added 30 min before thrombin stimulation when indicated. Mean + SEM from at least three independent experiments. *, P < 0.02; **, P < 0.007; ***, P < 10−3. (D) Percentage of maximum contraction with respect to siControl cells during acute contraction (C.) and barrier recovery (R.) in HUVECs pretreated with TNF between 18 and 20 h. ***, P < 2.2 × 10−5. (E) Permeability to dextran-FITC was performed as in B but analyzed during the time of barrier recovery after acute contraction induced by thrombin. (F) Staining for β-catenin and F-actin in TNF-pretreated HUVECs stimulated during the recovery phase after thrombin. Empty spaces between cells were identified by automated image analysis (mask). β-Catenin staining at cell borders was quantified by semiautomated image processing in five different experiments. *, P ≤ 0.046. Bar, 20 µm. (G) Percentage of maximum contraction with respect to siControl cells during acute contraction and the subsequent recovery induced with thrombin in HUVECs not previously stimulated with TNF. *, P = 0.047; **, P = 0.009; ***, P = 9.4 × 10−5. (H) Changes in TEER during thrombin-induced acute contraction in HDMVECs pretreated with TNF for 7 h (left). Mean ± SEM from at least three independent experiments. Percentage of maximum contraction with respect to siControl1 during contraction and barrier recovery in HDMVECs (right). *, P ≤ 0.05; **, P < 0.01; ***, P < 0.005. (I) TNF-pretreated HDMVECs were fixed after 2.5 h of thrombin stimulation (recovery phase) and stained for β-catenin and F-actin. β-Catenin staining at cell borders was quantified as in F. *, P < 0.05; **, P < 0.01. Bar, 10 µm. Mean + SEM of at least three experiments.

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