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Reactive oxygen species mediate Rac-induced loss of cell-cell adhesion in primary human endothelial cells

¹ Department of Experimental Immunohematology, CLB and Laboratory for Clinical and Experimental Immunology, Academic Medical Center, Plesmanlaan 125 1066 CX, Amsterdam
² Division of Cell Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands

View larger version (152K): [in a new window]   Fig. 1. Transduction of Tat-RacV12 in fibroblasts and epithelial cells. Tat-PTD (control, a) or Tat-RacV12 (b) was transduced into NIH3T3 fibroblasts for 30 minutes. Next the cells were fixed and permeabilized, (immuno)stained for actin (red) and vinculin (green) and analysed by confocal microscopy. Tat-RacV12 induced rapid formation of actin stress fibers and induced pronounced vinculin-containing focal adhesions and lamellipodia along the cells' periphery (inset). Bar, 10 µm (inset: bar, 5 µm). Ras-transformed epithelial MDCK cells were transduced with the Tat-PTD (c) or Tat-RacV12 (d), and the distribution of F-actin and ß-catenin was analysed by immunocytochemistry after 3 hours. Tat-RacV12 did not induce stress fibers but recruited F-actin to the cellular cortex. In addition, ß-catenin was found to be more pronounced at the sites of cell-cell contact, which is indicative of increased cell-cell adhesion. Bar, 10 µm.

View larger version (109K): [in a new window]   Fig. 2. Effect of Tat-RacV12 on HUVEC. (A) GFP-actin-expressing immortalized HUVEC were transduced with Tat-RacV12 while the cells were monitored by time-lapse confocal microscopy (phase contrast, top row; GFP-actin, bottom row). Tat-RacV12 induced a rapid contractile response (asterisks) followed by extensive induction of lamellipodia formation (arrows). Bar, 10 µm. (B) Primary HUVEC were cultured on fibronectin-coated coverslips to near confluency and transduced for 15 (Bb) or 30 minutes (Bc) with Tat-RacV12 or for 30 minutes with Tat-RacN17 (b,d). Next, the cells were fixed and immunostained for F-actin (red) and VE-cadherin (green). Bar, 20 µm. (C) Primary HUVEC were cultured at different densities (high, a,b; middle, c,d; low, e,f) and next transduced with Tat-RacV12 for 30 minutes. Next, the cells were fixed and immunostained for F-actin (red) and VE-cadherin (green). Bars, 20 µm.

View larger version (129K): [in a new window]   Fig. 3. Role of Rho in the control of endothelial cell-cell adhesion. (A) Primary HUVECs on fibronectin-coated coverslips were pretreated or not with the Rho-inactivating C3 exoenzyme (10 µg/ml) and subsequently transduced with the Tat PTD or Tat-RacV12 (50 nM) for 30 minutes. The cells were fixed and stained for F-actin (red) and VE-cadherin (green). Bar, 20 µm. (B) Primary HUVEC were transduced with Tat-RhoV14 or Tat-RhoN19 for 30 minutes, fixed and stained for F-actin (red) and vinculin or VE-cadherin (green). Bars, 20 µm.

View larger version (96K): [in a new window]   Fig. 4. Effect of Tat-RacV12 and Tat-RhoV14 on endothelial barrier function. (A) Primary HUVECs were cultured on fibronectin-coated transwell-filters until confluent, and monolayers were pretreated for 30 minutes with Tat-RacV12 and TAT-RhoV14 (both 50 nM) or an antibody against VE-cadherin that was used as a positive control. Permeability was assessed 2 hours later as described in the Materials and Methods. (B) Endothelial cells, which were cultured on fibronectin-coated transwell filters, were treated with Tat-RacV12 for 30 minutes, and cells were then stained and fixed for F-actin (red) and VE-cadherin (green). Bar, 10 µm.

View larger version (82K): [in a new window]   Fig. 5. Role for ROS in Tat-RacV12-mediated loss of cell-cell adhesion in pHUVEC. (A) Tat-RacV12-induced production of reactive oxygen species (ROS). Endothelial cells were loaded with the H2O2-sensitive dye DHR, washed, transduced with the TAT-peptide ([UNK]) or Tat-RacV12 (, 50 nM) and monitored for DHR fluorescence using time-lapse microscopy. Images showing DHR fluorescence at t=0 minutes and t=60 minutes are shown in the panels on the left. Bar, 50 µm. The graph depicts the % increase over basal values. (B) The role of ROS in the Tat-RacV12-induced loss of cell-cell adhesion. pHUVEC were cultured in the absence (a,b) or presence (c) of 5 mM N-AC prior to transduction with Tat-RacV12 for 30 minutes (b,c). Fixed cells were stained for F-actin (red) and VE-cadherin (green). Bar, 20 µm.

View larger version (82K): [in a new window]   Fig. 6. Tat-RacV12 induces tyrosine phosphorylation of junctional proteins. (A) Primary HUVECs were treated with Tat-peptide (control) or Tat-RacV12 for 30 minutes, fixed and stained for F-actin (red) and phosphotyrosine (PY, green). Arrows indicate increased phosphorylation at cell borders and in between cells. Bar, 20 µm. (B) Primary HUVECs were treated with Tat-RacV12 for 30 minutes, fixed and stained for ß-catenin (a) and phosphotyrosine (b). (c) shows the merged picture; colocalization as determined using specific software (identifying pixels with equal intensities of red and green) is indicated in white. Bar, 5 µm. (C) Primary HUVECs were treated with Tat-RacV12 for the various time periods indicated, after which VE-cadherin was immunoprecipitated. Western blots for phosphotyrosine (upper panel) and for VE-cadherin (lower panel, migrating at a molecular weight of approximately 130 kDa) are shown. VE-cadherin itself (130 kDa) is not phosphorylated upon Tat-RacV12 treatment. (D) Immunoprecipitation of -catenin followed by western blotting for phosphotyrosine (upper panel) following Tat-RacV12 treatment for the time periods indicated with or without pretreatment with N-AC. The lower panel indicates blotting for -catenin to control for equal loading.

View larger version (12K): [in a new window]   Fig. 7. Role for ROS in the migration of endothelial cells. pHUVEC, pretreated or not with 5 mM N-AC, were allowed to migrate for 5 hours towards 0% (), 5% ([UNK]) and 10% () (v/v) FCS in medium with or without the N-AC present during the assay, after which the cells were fixed, stained and counted by fluorescence microscopy as described in the Materials and Methods.