Rac1 and Tumor Angiogenesis injected at days 5 and 10 (after initial inoculation) with pSico-Con (1, white) or pSico- b3 (2, black) and harvested at day 14. Left bar graph shows mean tumor volume in mm3 (+ s.e.m). P,0.05. N = 5 animals per group. Right bar graph shows mean number of endomucin positive vessels per tumor area per mm2 (+ s.e.m.). P,0.001. N = 5 animals per group. doi:10.1371/journal.pone.0009766.g004 10 ng/ml VEGF-A164 in combination with either pSico-Con, pSico-Rac1 or pSico-Flk-1 lentiviral suspensions. After 15-days, angiogenic responses were assayed histologically. Results showed that, as expected, VEGF-mediated neovascularization was elevat- ed in b3-null/Tie1-Cre+ pSico-Con treated mice when compared with similarly treated wild-type/Tie1-Cre+ controls. pSico-Flk-1 treatment inhibited significantly VEGF-induced angiogenesis in both genotypes. However, pSico-Rac1 treatment had no effect in wild-type/Tie1-Cre+ mice, but did inhibit neovascularization in b3-null/Tie1-Cre+ mice when compared with pSico-Con controls (Figure 6A). Additionally, using a second approach, VEGF- induced angiogenesis in scrambled control-siRNA (Con siRNA) injected sponges in b3-null mice was elevated when compared with Con siRNA treated sponges in wild-type mice, and Rac1- specific-siRNA injection into sponges inhibited significantly angiogenic responses in b3-null, but not wild-type, mice (Figure 6B). Taken together these data suggest that Rac1 expression in endothelial cells is not essential for VEGF-mediated angiogenesis in wild-type mice but is involved in such responses in b3-null mice in vivo. Further analysis of the role of Rac1 in VEGF-induced neovascularization responses was carried out using ex vivo aortic ring assays. Thoracic aortic rings from wild-type and b3-null mice were transfected with either a scrambled control (Con) or Rac1- specific siRNA. Rac1 mRNA and protein levels were reduced in aortic rings from both genotypes by approximately 85% after transfection with Rac1-specific siRNA (Figure 6C). These rings were embedded in Matrigel in the presence of VEGF alone or VEGF plus DC101, an antibody that blocks Flk-1 activity [27]. Quantitation of microvessel outgrowth showed that Rac1- depletion did not alter wild-type microvessel sprouting, but Figure 5. Postanatal retinal vascularization is not affected by endothelial Rac1 deletion. Wild-type PDGFB-iCreER and PDGFB- iCreER; Rac1 flox/flox newborn pups were treated daily with tamoxifen and their retinas were examined at P12. The vasculature was visualised by Interleukin b4 staining (ILB4 - green) and Claudin 5 (red). No obvious differences in vascularization were observed between genotypes. Scale bar: 10 mm upper micrographs; 100 mm lower micrographs. doi:10.1371/journal.pone.0009766.g005 inhibited significantly the enhanced numbers of microvessel sprouts from b3-null aortas (Figure 6D). In addition, DC101 treatment significantly affected this process in Con- and Rac1- siRNA transfected aortic rings from both genotypes (P,0.01, Figure 6D). Rac1-depletion does not affect the levels or activity of other tested Rho GTPases in either wild-type or b3-null endothelial cells To begin to understand the mechanisms responsible for the biological effects of Rac1-depletion in wild-type and b3-null endothelial cells, we examined first the levels and activity of Rac2, Rac3, Cdc42 and Rho. Rac1-specific siRNA treatment of microvascular endothelial cells isolated from wild-type or b3-null mice reduced significantly Rac1 mRNA levels (Figure 7A), total protein levels (Figure 7B), and active Rac1 levels (Figure 1A). In contrast, transfection of endothelial cells using Con siRNA had no effect on Rac1 expression in either genotype. Importantly, no compensation by expression or activity of other tested Rho-related GTPases (Rac2, Rac3, Cdc42 and RhoA) were observed in either wild-type- or b3-null-Rac1-depleted endothelial cells (Figure 7B). These results suggest that the lack of an inhibitory response to Rac1 loss in wild-type endothelial cells is not due to compensation by either Rac2, Rac3, Cdc42 or RhoA. In addition, the dependency of b3-null endothelial cells on Rac1 is not due to any intrinsic change in these Rho GTPases. Rac1 is not essential for VEGF-mediated endothelial cell proliferation, scratch closure or tube formation in 2- dimensions Given that Rac1 has been implicated in regulating positively cell migration, proliferation and tube formation, at least in vitro, we decided to examine also whether Rac1-depletion in b3-null endothelial cells affects these processes. Analysis of endothelial cell growth by seeding equal numbers of Mock, Con- or Rac1-siRNA transfected wild-type or b3-null endothelial cells and counting the total number of cells per condition between 0 and 3 days showed no significant differences between Rac1-depleted and control cells in either genotype despite the increased growth rates of b3-null endothelial cells (Figure 8A). In addition, Trypan blue exclusion showed that the percentage of cells that were viable did not change after Rac1-depletion between 1-3 days after seeding in either genotype (Figure 8B). These data suggest that Rac1-knockdown does not inhibit the proliferation or viability of wild-type or b3-null endothelial cells in culture. In contrast, Rac1-depletion inhibited VEGF-mediated 2-dimensional (2D) scratch closure (Figure 8C) and cord formation (Figure 8D) when compared with Mock and Con siRNA controls without affecting cell viability (Figure 8D). These results confirm the reported role of Rac1 in promoting VEGF-mediated endothelial cell migration and cord formation conducted in standard 2D tissue culture conditions. However, given that the requirement for Rac1 in 3-dimensional (3D) versus 2D migration may be different [30,31], and that our in vivo data did not show a requirement for Rac1 in wild-type angiogenesis, the effect of Rac1-depletion in endothelial cell migration was examined further in a 3D-modified Boyden chamber assay. Importantly, knockdown of Rac1 had no effect on VEGF-induced 3D chemotactic migration of wild-type PLoS ONE | www.plosone.org 7 March 2010 | Volume 5 | Issue 3 | e9766