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RhoB controls endothelial barrier recovery by inhibiting Rac1 trafficking to the cell border

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Rho/ROCK signaling is essential to maintain the integrity of the endothelial barrier, but the contributions of specific Rho GTPase family members are unclear. Here, Marcos-Ramiro et al. show that RhoB specifically regulates intracellular trafficking of the Rho GTPase Rac1 and thereby controls endothelial barrier restoration during inflammation.

No MeSH data available.


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RhoB regulates cell area recovery after acute contraction independently of cell–cell contacts. (A) HA-RhoBV14 has no effect on endothelial cell–cell junctions. HA-RhoBV14 was expressed for 48 h in confluent HUVECs. Bottom images show enlargements of squared areas from HA-RhoBV14 transfected (1) and untransfected cells (2). Bar, 20 µm. (B) Time-lapse microscopy of subconfluent HUVECs stimulated with thrombin. Cell areas were quantified from individual frames taken at different times (right graph). Graph shows the mean + SEM of at least 10 cells per experiment from three different experiments. Bar, 20 µm. (C) siRNA-transfected, TNF-pretreated HUVECs were stimulated with thrombin for the indicated times, and then fixed and stained for F-actin and RhoB. Right graph quantifies cell area. Mean + SEM from three different experiments. 50 cells per experiment. RhoB-depleted cells recover their initial area (respreading) at 60 min poststimulation. *, P < 0.04; **, P < 0.03. (D) HUVECs expressing GFP or HA-RhoBV14 for 24 h were stimulated with thrombin for 60 min, stained for F-actin and HA-RhoBV14, and the cell area quantified. Graph shows the mean + SEM of at least 10 cells per experiment from three different experiments. **, P = 0.001. (E) siRNA-transfected, TNF-pretreated HDMVECs were stimulated with thrombin for 2.5 h (respreading), and then fixed and stained for F-actin and RhoB. Graph shows the mean + SEM from at least 50 cells per experiment, from three different experiments. *, P ≤ 0.05; **, P < 0.01. Bar, 10 µm.
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fig5: RhoB regulates cell area recovery after acute contraction independently of cell–cell contacts. (A) HA-RhoBV14 has no effect on endothelial cell–cell junctions. HA-RhoBV14 was expressed for 48 h in confluent HUVECs. Bottom images show enlargements of squared areas from HA-RhoBV14 transfected (1) and untransfected cells (2). Bar, 20 µm. (B) Time-lapse microscopy of subconfluent HUVECs stimulated with thrombin. Cell areas were quantified from individual frames taken at different times (right graph). Graph shows the mean + SEM of at least 10 cells per experiment from three different experiments. Bar, 20 µm. (C) siRNA-transfected, TNF-pretreated HUVECs were stimulated with thrombin for the indicated times, and then fixed and stained for F-actin and RhoB. Right graph quantifies cell area. Mean + SEM from three different experiments. 50 cells per experiment. RhoB-depleted cells recover their initial area (respreading) at 60 min poststimulation. *, P < 0.04; **, P < 0.03. (D) HUVECs expressing GFP or HA-RhoBV14 for 24 h were stimulated with thrombin for 60 min, stained for F-actin and HA-RhoBV14, and the cell area quantified. Graph shows the mean + SEM of at least 10 cells per experiment from three different experiments. **, P = 0.001. (E) siRNA-transfected, TNF-pretreated HDMVECs were stimulated with thrombin for 2.5 h (respreading), and then fixed and stained for F-actin and RhoB. Graph shows the mean + SEM from at least 50 cells per experiment, from three different experiments. *, P ≤ 0.05; **, P < 0.01. Bar, 10 µm.

Mentions: We observed that the expression of a constitutive active mutant RhoB-V14 had no effect on cell–cell junction integrity even though cell-spread area was significantly reduced (Fig. 5 A). This led us to hypothesize that the role of RhoB in thrombin-mediated endothelial remodeling may be independent of the reannealing of cell–cell junctions. Indeed, the kinetics of thrombin-induced contraction and subsequent spreading in TNF-pretreated subconfluent HUVECs were consistent with those of barrier disruption and reformation in confluent cells, indicating that cell contraction and recovery are independent of the cell–cell junctions (Figs. 3 C and 5 B). Confocal analysis revealed that RhoB knockdown slightly increased cell area of subconfluent HUVECs before thrombin stimulation and clearly accelerated cell respreading after thrombin-mediated contraction (Fig. 5 C). 60 min after thrombin stimulation, RhoB-depleted cells recovered their initial spreading area, whereas siControl cells had only 60% of their area before stimulation (Fig. 5 C). In contrast, RhoB-V14 expression reduced cell respreading (Fig. 5 D), suggesting that RhoB negatively regulates the membrane extension that follows acute contraction. A similar increase in cell area was observed in RhoB-depleted subconfluent HDMVECs at the time of cell respreading (Fig. 5 E).


RhoB controls endothelial barrier recovery by inhibiting Rac1 trafficking to the cell border
RhoB regulates cell area recovery after acute contraction independently of cell–cell contacts. (A) HA-RhoBV14 has no effect on endothelial cell–cell junctions. HA-RhoBV14 was expressed for 48 h in confluent HUVECs. Bottom images show enlargements of squared areas from HA-RhoBV14 transfected (1) and untransfected cells (2). Bar, 20 µm. (B) Time-lapse microscopy of subconfluent HUVECs stimulated with thrombin. Cell areas were quantified from individual frames taken at different times (right graph). Graph shows the mean + SEM of at least 10 cells per experiment from three different experiments. Bar, 20 µm. (C) siRNA-transfected, TNF-pretreated HUVECs were stimulated with thrombin for the indicated times, and then fixed and stained for F-actin and RhoB. Right graph quantifies cell area. Mean + SEM from three different experiments. 50 cells per experiment. RhoB-depleted cells recover their initial area (respreading) at 60 min poststimulation. *, P < 0.04; **, P < 0.03. (D) HUVECs expressing GFP or HA-RhoBV14 for 24 h were stimulated with thrombin for 60 min, stained for F-actin and HA-RhoBV14, and the cell area quantified. Graph shows the mean + SEM of at least 10 cells per experiment from three different experiments. **, P = 0.001. (E) siRNA-transfected, TNF-pretreated HDMVECs were stimulated with thrombin for 2.5 h (respreading), and then fixed and stained for F-actin and RhoB. Graph shows the mean + SEM from at least 50 cells per experiment, from three different experiments. *, P ≤ 0.05; **, P < 0.01. Bar, 10 µm.
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fig5: RhoB regulates cell area recovery after acute contraction independently of cell–cell contacts. (A) HA-RhoBV14 has no effect on endothelial cell–cell junctions. HA-RhoBV14 was expressed for 48 h in confluent HUVECs. Bottom images show enlargements of squared areas from HA-RhoBV14 transfected (1) and untransfected cells (2). Bar, 20 µm. (B) Time-lapse microscopy of subconfluent HUVECs stimulated with thrombin. Cell areas were quantified from individual frames taken at different times (right graph). Graph shows the mean + SEM of at least 10 cells per experiment from three different experiments. Bar, 20 µm. (C) siRNA-transfected, TNF-pretreated HUVECs were stimulated with thrombin for the indicated times, and then fixed and stained for F-actin and RhoB. Right graph quantifies cell area. Mean + SEM from three different experiments. 50 cells per experiment. RhoB-depleted cells recover their initial area (respreading) at 60 min poststimulation. *, P < 0.04; **, P < 0.03. (D) HUVECs expressing GFP or HA-RhoBV14 for 24 h were stimulated with thrombin for 60 min, stained for F-actin and HA-RhoBV14, and the cell area quantified. Graph shows the mean + SEM of at least 10 cells per experiment from three different experiments. **, P = 0.001. (E) siRNA-transfected, TNF-pretreated HDMVECs were stimulated with thrombin for 2.5 h (respreading), and then fixed and stained for F-actin and RhoB. Graph shows the mean + SEM from at least 50 cells per experiment, from three different experiments. *, P ≤ 0.05; **, P < 0.01. Bar, 10 µm.
Mentions: We observed that the expression of a constitutive active mutant RhoB-V14 had no effect on cell–cell junction integrity even though cell-spread area was significantly reduced (Fig. 5 A). This led us to hypothesize that the role of RhoB in thrombin-mediated endothelial remodeling may be independent of the reannealing of cell–cell junctions. Indeed, the kinetics of thrombin-induced contraction and subsequent spreading in TNF-pretreated subconfluent HUVECs were consistent with those of barrier disruption and reformation in confluent cells, indicating that cell contraction and recovery are independent of the cell–cell junctions (Figs. 3 C and 5 B). Confocal analysis revealed that RhoB knockdown slightly increased cell area of subconfluent HUVECs before thrombin stimulation and clearly accelerated cell respreading after thrombin-mediated contraction (Fig. 5 C). 60 min after thrombin stimulation, RhoB-depleted cells recovered their initial spreading area, whereas siControl cells had only 60% of their area before stimulation (Fig. 5 C). In contrast, RhoB-V14 expression reduced cell respreading (Fig. 5 D), suggesting that RhoB negatively regulates the membrane extension that follows acute contraction. A similar increase in cell area was observed in RhoB-depleted subconfluent HDMVECs at the time of cell respreading (Fig. 5 E).

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Rho/ROCK signaling is essential to maintain the integrity of the endothelial barrier, but the contributions of specific Rho GTPase family members are unclear. Here, Marcos-Ramiro et al. show that RhoB specifically regulates intracellular trafficking of the Rho GTPase Rac1 and thereby controls endothelial barrier restoration during inflammation.

No MeSH data available.


Related in: MedlinePlus