Figure 5.
KRIT-1 regulates endothelial cell permeability. (A) Treatment of BAECs with 2 U/ml thrombin increased endothelial permeability to HRP, whereas treatment with 8-pCPT-2′-O-Me-cAMP inhibited thrombin-stimulated permeability, as did overexpression of recombinant KRIT-1 (in control siRNA–treated cells). Knockdown of KRIT-1 expression by KRIT-1 siRNA 530 caused an approximately twofold increase in permeability. Thrombin treatment further increased permeability in KRIT-1–depleted cells. However, 8-pCPT-2′-O-Me-cAMP had little effect on permeability in these cells. The increased permeability was almost completely rescued by reexpression of recombinant KRIT-1. Negative control siRNA had no effect on permeability. Data shown is the mean increase in permeability ± SEM; n = 5. *, P < 0.05 compared with control siRNA plus vehicle. (B) KRIT-1 overexpression reverses Rap1GAP-induced increased endothelial permeability. Data shown is the mean increase in permeability ± SEM; n = 4. *, P < 0.05 compared with vector control. (bottom) A representative blot of Rap1GAP expression in the cells used for permeability experiments. (C) Depletion of KRIT-1 increases stress fiber formation. The actin cytoskeleton of anti–KRIT-1 siRNA–treated and HA–KRIT-1–reconstituted cells was stained with rhodamine-phalloidin to assess changes in the distribution of F-actin as a consequence of KRIT-1 depletion. Epifluorescence images are representative; n = 3. Bar, 50 μm.