Fig 2: Target genes shared by RhoB and VEZF1 in BVECs and LVECs.(a) Relative mRNA levels of VEGF-R2, NRP1 and TIMP3 in BVECs and LVECs following silencing of either RhoB or VEZF1 as determined by QRT–PCR (performed in triplicate on two different cell isolation lots, mean±s.e.m., unpaired two-tailed Student’s t-test). (b) In vivo binding of VEZF1 and RhoB-Flag to the promoter region of target genes, that is, VEGF-R2, NRP1 and TIMP3, containing predicted in silico VEZF1 DNA binding sites (red triangle in schematic drawing of promoters) assessed by ChIP experiments. The start site of transcription is referred as +1. The position of primers used for QRT–PCR experiments is represented by blue arrows. The enrichment for each DNA fragment upon immunoprecipitation of VEZF1 and RhoB-Flag is illustrated as histograms based on % of input (QRT–PCR performed in triplicate on two different cell isolation lots, mean±s.e.m., unpaired two-tailed Student’s t-test).

Fig 3: Model for RhoB-mediated coordination of angiogenesis and lymphangiogenesis in dermal wounds.(a) After tissue challenge initiated by dermal wounding and in response to numerous extracellular signals that remain to be explored in vivo, the immediate early response gene RhoB is induced and its protein accumulates in both blood and lymphatic vascular endothelial cells. The GTP-bound form of RhoB (red star) partially localizes in the nucleus of both cell types, where its physical interaction within the VEZF1 transcriptional complex regulates the differential expression of specific direct target genes in BVECs and LVECs leading to an increase in BVEC proliferation and sprouting and a simultaneous decrease in LVEC proliferation and sprouting. Accordingly, RhoB null mice exhibit reduced angiogenesis versus earlier, augmented and abnormal lymphangiogenesis in this and other pathological scenarios. (b) We propose that the opposing roles served by RhoB in these two endothelial cell types contribute to the coordination of an early angiogenic response versus a delayed lymphangiogenic response previously observed in wound healing. In this setting, the early angiogenic response participates in the initial inflammatory phase by facilitating recruitment of inflammatory cells into the wound bed (day 5–7). The delayed lymphangiogenesis allows these cells time to initiate tissue repair. Thereafter, the new lymphatics support the resolution of the local oedema (small black arrows) by draining fluid excess, cells and debris (big black arrow) (day 9–14).

Fig 4: RhoB loss leads to enhanced and abnormal lymphangiogenesis in response to stress.(a) Intravital microlymphangiography by in vivo injection of FITC–dextran (MW 2,000 kDa) into lymphatics of ear skin 7 days after ear wounding in adult wt and RhoB−/− mice. Dextran-positive areas in isolated frames taken from the movies at early (2–10 s (s)), middle (30 s) and late (60 s) times following tracer injection were quantified (n=7 mice per genotype, mean±s.e.m., unpaired two-tailed Student’s t-test). (b) Confocal analysis following in vivo FITC–dextran injection in lymphatics and whole-mount staining of podoplanin expression in the granulation tissue present 7 days after ear wounding in adult wt and RhoB−/− mice. Arrowhead and star respectively highlight dextran leakage from an abnormal lymphatic vessel and tracer accumulation in the adjacent tissue of RhoB−/− mice in the merged image. (c) Induction of DTH reactions in the ear skin of adult wt and RhoB−/− mice. Ear swelling is expressed as the increase (Δ) over the original ear thickness in μm. Ear thickness was measured daily for 11 days following challenge using a Mitutoyo caliper (n=12 mice per genotype, mean±s.e.m., unpaired two-tailed Student’s t-test). (d) Intravital microlymphangiography by in vivo injection of FITC–dextran (MW 2,000 kDa) into lymphatics of ear skin 7 days after inflammation induced by oxazolone in adult wt and RhoB−/− mice. FITC–dextran-positive areas in isolated frames taken from movies at early (2–10 s (s)), middle (30 s) and late (60 s) times following tracer injection were quantified (n=6 mice per genotype, mean±s.e.m., unpaired two-tailed Student’s t-test). Scale bars: 1 mm (a,d) and 100 μm (b).

Fig 5: Activation of ROCK signaling by RHOA knockdown in SW948(A) Expression of RHOA, RHOB, and RHOC, and phosphorylation of MLC2 in SW948 treated with 1 nM of RHOA-siRNA. Proteins were harvested on days 1, 2, and 3 after siRNA treatment. The protein expression levels were detected using western blotting. (B) Actin stress fibers of SW948 treated with 1 nM of RHOA-siRNA. Actin stress fibers were stained with Rhodamine Phalloidin, and DAPI was used for nuclear staining. Stained cells were analyzed with confocal fluorescence microscopy. Representative images of three independent chambers are shown. Details of immunocytochemistry are described in Materials and Methods. Scale bar shows 10 μm. (C) Restoration of cell survival by a ROCK inhibitor, Y-27632. SW948 was cultivated for 7 days with 3 μM or 10 μM of Y-27632. The relative cell survival rate is shown as a percentage of that in the control-siRNA-treated SW948 that was not treated with Y-27632. Data are shown as mean ± SD (n = 3). Significance compared with the Y-27632 non-treated group between RHOA-siRNA-treated groups was determined by Student’s t-test. *p < 0.05. (D) Restoration of cell survival by RHOB/RHOC-siRNAs. SW948 was cultivated for 7 days with RHOA-siRNA (1 μM) and/or RHOB-siRNA (1 μM) and/or RHOC-siRNA (0.2 μM). Cell survival rate of obtained transfectants was evaluated as described in Figure 1. Data are shown as mean ± SD (n = 3). Significant differences between RHO-siRNA-treated groups were determined by Student’s t-test. *p < 0.05. (E) Protein expression of cells tested in (D).