Vascular endothelial growth factor (VEGF) stimulation results in increased expression but not activity of RhoB protein in HUVEC. (A) RhoB protein levels are increased following VEGF stimulation. Cells were serum starved overnight in MCDB 131 with 0.25% FBS. Following this, media was replaced with MCDB 131 with 0.25% FBS and 20 ng/ml VEGF, and protein lysates were collected at the times indicated. Protein lysates were subjected to western blotting and total levels of RhoB protein were assessed. β-actin was used as a total protein loading control. (B) Levels of activated RhoB do not change following VEGF stimulation. Cells were serum starved overnight in MCDB 131 with 0.5% FBS, followed by stimulation with 20 ng/ml VEGF. At various times post-VEGF stimulation, protein lysates were collected and activated RhoB was assessed using G-LISA assays as described in Materials and Methods. Bars represent the mean ± SEM for duplicate measures in each of two independently performed experiments.

Depletion of RhoB inhibits in vitro vessel sprouting and capillary morphogenesis. (A) RhoB-depleted HUVEC have reduced ability to form vessel sprouts on collagen I gels. Cells were either mock transfected with PBS or transfected with 20 nM siRNAs (Control, RhoB siRNA 1, RhoB siRNA 2) as indicated and seeded onto collagen I gels at 24 h post transfection. At time zero (48 h post-siRNA transfection), cells were placed in EGM-2 growth media supplemented with 50 ng/ml VEGF to induce sprouting. Media supplemented with VEGF was refreshed every two days. Blinded counts were made from 10 random fields of view from each of duplicate plates, and compiled data are presented as mean ± SE of duplicate dishes from each of two independently performed experiments. Significance was determined by unpaired t-tests relative to control siRNA at each respective time point (* represents p < 0.05 and ** represents p < 0.0001) (B) Depletion of RhoB levels inhibit capillary morphogenesis. HUVEC were transfected with siRNAs as indicated and seeded onto polymerized basement membrane extract (BME) in EGM-2 growth media. Images were taken 24 h after cell seeding and capillary morphogenesis was determined by averaging the total number of cord-like structures in 4 fields of view as assessed with ImageJ software. The data is presented as the mean ± SE of two independently performed experiments. Significance was determined by ANOVA with Fisher's PLSD post hoc analysis (* represents p < 0.05 and ** represents p < 0.0001).

Inhibition of ROCK partially restores capillary morphogenesis in RhoB-depleted HUVEC. Cells were transfected with siRNAs as indicated and seeded onto BME in EGM-2 growth media in the absence or presence of (A) 10 μM Y-27632 or (B) 1 μM H-1152. In both cases, control media conditions contained EGM-2 growth media with equivalent volumes of DMSO as a vehicle control. Images were taken 24 h after cell seeding. (C, D) Analysis of capillary morphogenesis from (A, B). Capillary morphogenesis was determined by averaging the total number of cord-like structures in 4 fields of view as assessed with ImageJ software. Bars represent the mean ± SE of duplicate wells from each of two independently performed experiments (* represents p < 0.05 and ** represents p < 0.0001 as compared to the control siRNA sample using unpaired t-tests).

siRNA-mediated RhoB depletion inhibits endothelial cell migration but not viability. (A) Efficient depletion of RhoB protein levels was achieved in HUVEC with small interfering RNA treatment. HUVEC were either mock transfected with PBS (M) or transfected with 20 nM of the indicated siRNAs [Control (C), RhoB siRNA 1, RhoB siRNA 2]. Cellular total protein lysates were collected at 48 h post transfection and analyzed for levels of RhoB by western blot. Levels of β-actin served as a protein loading control. (B) Assessment of cell viability in RhoB-depleted HUVEC. Cells were either mock transfected with PBS or transfected with siRNAs, as indicated, and number of viable cells were assessed following trypan blue exclusion and counting with a Beckman Coulter Vi-Cell XR analyzer in triplicate wells at each time point. Data presented shows mean ± SD of two independently performed experiments. (C) RhoB-depletion inhibits cell migration. A scratch wound assay of transfected HUVEC was performed in MCDB 131, with 0.05% FBS and 50 ng/ml VEGF. Images depict wound size at time of wounding (Time = 0 h) and 24 h later. (D) Analysis of cell migration from (C). Bars represent the mean ± SD for percent wound closure, after 24 h for a representative experiment. P values were calculated using a two-tailed unpaired student's t test (* P < 0.05).

Depletion of RhoB modulates the activity of RhoC and RhoA, and the resulting increased RhoA activity contributes to the observed reduction in capillary morphogenesis. (A) G-LISA analysis indicating increased levels of activated RhoA in response to VEGF in cells with depleted RhoB expression. Cells were transfected with the indicated siRNAs, and 48 h post transfection were serum starved for 5 h in MCDB 131, and subsequently stimulated with 10 ng/ml VEGF in MCDB 131. At various times post-stimulation, protein lysates were generated and activated RhoA was detected by G-LISA assays as described in Materials and Methods. Bars represent the mean ± SE of a representative experiment performed in triplicate (* represents p < 0.05 as determined by unpaired t-tests when compared to Control siRNA samples at each time point). Similar trends were observed in independent experiments. (B) G-LISA analysis indicating reduced RhoC activity in cells treated with RhoB siRNAs. Cells were transfected with the indicated siRNAs (Control, RhoB siRNA 1, RhoB siRNA 2), followed by starvation overnight in MCDB 131 with reduced serum (0.5%), and for an additional 4 h in MCDB 131 (0% serum). Cells were subsequently stimulated with 50 ng/ml VEGF in MCDB 131 for 5 min and protein lysates were generated for analysis of RhoC activity (at 48 h post-siRNA transfection) by G-LISA as described in Materials and Methods. Bars indicate the mean ± SE for duplicate measures in each of two independently performed experiments. (C) Protein levels of RhoA and RhoC were assessed in RhoB siRNA-treated HUVEC. Cells were transfected with 20 nM of the indicated siRNAs and lysates collected at 48 h post transfection. Protein levels were analyzed by western blotting, with levels of β-actin serving as a control for protein loading. (D) Defective capillary morphogenesis in RhoB depleted HUVEC is restored upon inhibition of RhoA activity. HUVEC were transfected with siRNAs as indicated and seeded onto BME in EGM-2 growth media in the absence or presence of 0.25 μg/ml C3 transferase or DMSO as a vehicle control. Images are representative photos taken 24 h after cell seeding. (E) Analysis of capillary morphogenesis from (D). Capillary morphogenesis was determined by averaging the total number of cord-like structures in 4 fields of view in triplicate wells as assessed with ImageJ software. Bars represent the mean ± SE of duplicate wells from each of two independently performed experiments (* represents p < 0.05 and ** represents p < 0.0001 compared to control siRNA as determined by ANOVA with Fisher's PLSD post hoc testing).