Rac1 and Tumor Angiogenesis Figure 6. VEGF-mediated angiogenesis is dependent on endothelial-Rac1 expression in b3-null mice but not wild-type mice. A. Representative images of 14-day-old VEGF-impregnated sponges in wild-type/Tie1-Cre+ and b3-null/Tie1-Cre+ mice after treatment with pSico-Con, pSico-Rac1 and pSico-Flk-1. Endomucin-positive staining (blue) identified microvessels. Scale bar: 50 mm. Bar graph represents mean number of microvessels per area of sponge (+ s.e.m.). Blood vessel density was reduced significantly in pSico-Flk-1-treated mice of both genotypes (*P,0.05), and in pSico-Rac1-treated b3-null/Tie1-Cre+ mice, but not pSico-Rac1-treated wild-type/Tie1-Cre+ mice (n.s.d, no significant differences). N = 6 sponges per group. B. Subcutaneous sponges implanted into wild-type mice were injected with either PBS or VEGF in the presence (Rac1 siRNA) or absence (Con siRNA) of Rac1-specific siRNA. Bar graph shows mean number of laminin-positive vessels per area of sponge per mm2 (+ s.e.m). Blood vessel density was increased significantly in VEGF alone-treated (*P,0.01) when compared with PBS controls. Rac1 siRNA-treatment had no significant effect (n.s.d, no significant differences). N = 6–10 sponges per group. C. Semi-quantitative RT-PCR (left) and Western blot (right) analyzes from wild-type and b3-null aortic explants transfected with either Con or Rac1 specific-siRNA. Rac1 siRNA reduced significantly the expression of both Rac1 mRNA (*P,0.01) and protein (*P,0.001) levels. RT-PCR for Actin and Western blotting for Hsc-70 provided mRNA and protein loading controls, respectively. Bar graphs represent mean Rac1 mRNA and Rac1 protein levels (+ s.e.m.). N = 3 independent experiments. D. Ex vivo mouse aortic ring assays. Representative high-power light micrographs of VEGF-mediated microvessel sprouting from wild-type and b3-null mouse aortic rings treated with Con or Rac1-specific siRNA and Con siRNA plus DC101. Bar graphs represent quantification of microvessel numbers from 5-day-old VEGF- stimulated aortic ring cultures transfected with Mock, Con or Rac1 siRNA in the presence or absence of DC101. Sprouting angiogenesis was reduced significantly after DC101 treatment of aortas and in Rac1-specific siRNA transfected b3-null, but not wild-type, aortas. Bar graph represents mean number microvessel sprouts/aortic ring (+ s.e.m.).*P,0.01; n.s.d, no significant differences. N = 3-5 independent experiments. doi:10.1371/journal.pone.0009766.g006 endothelial cells, but did inhibit this mode of migration in b3-null endothelial cells. As controls wild-type endothelial cells transfected with Con siRNA and treated with DC101 showed baseline migration and this was further inhibited in similarly treated b3- cells (P,0.05, Figure 8E). These results suggest that the role of Rac1 in 3D and 2D is different. Rac1 depletion does not affect VEGF-receptor2 expression Since we have shown previously that the loss of b3-integrin can elevate VEGF-mediated responses, VEGFR2/Flk-1 levels and Flk- 1 activity [12,27] we sought to determine the effect of Rac1- depletion on Flk-1 levels in wild-type and b3-null endothelial cells. Expression of Flk-1 was analyzed in primary wild-type and b3-null endothelial cells after transfection with either scrambled-control or Rac1-specific siRNA. Western blot analysis recapitulated our previous work showing that b3-null endothelial cells express higher levels of Flk-1 than wild-type controls. However, Rac1-depletion did not affect Flk-1 levels in either genotype (Figure 9A). This suggested that the elevated Rac1 activity that occurs in b3-null endothelial cells is downstream of changes in VEGFR2. Although Rac1 appears dispensable to normal in vivo angiogen- esis, the question remained as to whether elevated Rac1-activity could influence angiogenesis. To test whether elevated Rac1- activity influences 3D tube formation in vitro, both WT Rac1 and constitutively active Rac1 (Rac1 QL) were over-expressed in human umbilical endothelial cells (HUVEC) plated on a confluent layer of fibroblasts [32]. Tubules were visualized by PECAM-1 staining after 5 days in culture. The number of branch points in HUVEC overexpressing either form of Rac1 was significantly higher when compared to vector only transfected controls (P,0.02, Figure 9B), demonstrating that elevated Rac1 levels or activity can enhance tube formation. Overall, our findings suggest that normally (in adult wild-type mice) angiogenesis does not depend on Rac1. However, when b3- integrin levels are altered, tumor growth, tumor angiogenesis and VEGF-driven angiogenesis are enhanced and dependent on Rac1. Discussion Here we describe the role of Rac1 in tumor growth, tumor angiogenesis and VEGF-induced angiogenesis in vivo. Importantly, none of these processes were affected when Rac1 was depleted or deleted in the tumor endothelium of adult wild-type mice. These findings reveal that, unexpectedly, Rac1 expression in adult endothelial cells is not essential for tumor angiogenesis. However, we report that active levels of Rac1 are increased in b3-null endothelial cells and that the enhanced tumor growth, tumor angiogenesis and VEGF-mediated responses in b3-null mice were all dependent on endothelial Rac1 expression since Rac1- depletion in b3-null endothelial cells reduced significantly these responses. These data demonstrate that the requirement for endothelial-Rac1 becomes important when vascular b3-integrin is absent. Endothelial cell proliferation, migration and tube formation are essential processes during tumor angiogenesis and Rac1 activity is believed to be important for these cellular processes in vitro implying a possible requirement for this molecule in angiogenesis in vivo. However our data demonstrating that tumor angiogenesis and VEGF-induced angiogenesis in vivo were not affected in either pSico-Rac1-infected wild-type/Tie1-Cre+ mice or tamoxifen- treated Rac1 flox/flox PDGFB-iCreER mice, imply that angio- genic processes such as proliferation, migration and tube formation in vivo are not affected by the induced-loss of Rac1 in adult endothelial cells. In contrast, recent studies have suggested a positive role for Rac1 in endothelial cell function. For example, a 50% loss of Rac1 in Tie2-Cre mice results in reduced neovascularization in the hind-limb ischemia model [24]. In addition, Rac1-deletion in Tie2-Cre embryos results in early lethality due to severe heart and vasculogenic defects by E8.5 [25]. The apparent discrepancies between our results and those reported in Tie2-Cre mice may be due to several reasons. For example, the haploinsufficiency or deletion of Rac1 in these systems is from early embryogenesis and this has been shown to induce changes in blood pressure and vasodilation [24] that, in turn, are known to affect neovascularization indirectly [33] and therefore it is likely that such changes in blood pressure would actually affect angiogenesis in the Rac1-haploinsufficient-Tie2-Cre mice. In contrast, using the pSico or PDGFB-iCreER-inducible systems we have deleted Rac1, predominantly, in adult tumor or VEGF-induced neo-endothelial cells themselves, thus reducing any side effects on the whole vasculature. In addition, Cre activity in Tie2-Cre mice, as well as being present in endothelial cells, is also apparent in approximately 80% of hematopoietic cells [34,35], and it is tempting to speculate that the haploinsufficiency or deletion of Rac1 in this compartment may well affect angiogenesis. In contrast, in the pSico models that we have used, it is unlikely that the hematopoietic compartment is subject to Rac1-deletion and thus these cells are unlikely to affect angiogenesis in our system. Regarding proliferation, our results corroborate the findings that increased Rac1 activity correlates with enhanced endothelial proliferation, since the elevated Rac1-activity in b3-null endothe- lial cells show elevated proliferative capacity [36]. However, our PLoS ONE | www.plosone.org 9 March 2010 | Volume 5 | Issue 3 | e9766