lack of an anti-tumor effect of many of the anti-avb3-inhibitors, except for in glioblastma, suggest that perhaps b3-integrin in the vasculature of some tumors is not significantly high or functional. Thus it is of importance to understand the molecular basis of b3- integrin low/deficient angiogenesis. Downstream signalling pathways of both integrins and growth factor receptors involve RhoA, Cdc42 and Rac1, all members of the family of Rho GTPases. RhoA, Cdc42 and Rac1 can be activated when GTP-bound, and inactivated, when GDP-bound. The activation of RhoA has been reported to be involved in the development of actin stress fibres, Cdc42 in filopodia formation and Rac1 in membrane-ruffling [20]. Rac1 has been shown to be required for the migration of cells, including endothelial cells, and also it appears to play a role in vascular tube formation and lumen formation, at least in vitro [21,22]. Its involvement in the function of endothelial cells has also been illustrated by the activation of Rac1 under flow and shear stress [23]. Recently, some studies have suggested a role for Rac1 in endothelial cells in vivo. For example, haploinsufficiency of Rac1 in Tie2-Cre mice results in the poor regulation of blood pressure, vasodilation and impaired hind limb ischemia-induced neovascularization, suggesting that a 50% loss of Rac1 in Tie2-Cre mice is sufficient to affect this process [24]. In addition, Rac1-deletion in Tie2-Cre mice has implied a role for Rac1 in developmental vasculogenesis, due, possibly, to its deletion both in Tie2-positive endothelial cells and in approximately 80% of hematopoetic cells which are also Tie2-positive [25]. However, since these embryos die at E9.5, before developmental angiogen- esis can proceed fully, the precise effect of endothelial-Rac1- deficiency in tumor angiogenesis in vivo is unknown. Rac1 and Tumor Angiogenesis Here we show that, surprisingly, the inducible-deletion of Rac1 in adult endothelial cells does not affect tumor angiogenesis or VEGF-mediated angiogenesis in adult mice. However, in the absence of b3-integrin, the dependency of these processes on Rac1 becomes apparent. Our data indicate that Rac1, in adult endothelial cells, is not normally required for the migration and capillary formation necessary in adult neovascularization in vivo and that the role for this Rho GTPase in vivo may well be different to its functions in vitro. Results Tumor growth and tumor angiogenesis in b3-null, but not wild-type mice, are dependent on adult endothelial Rac1 expression Endothelial cell migration is a critical feature of tumor angiogenesis and is thought to involve both changes in integrin expression and the activity of Rho GTPases such as Rac1 [26]. However, a precise role for Rac1 in tumor angiogenesis has never been addressed. We provide evidence that Rac1 activity (Rac1- GTP) is elevated in b3-integrin knockout endothelial cells without affecting total Rac1 levels, and that transduction of human b3- integrin into b3-null endothelial cells is sufficient to reduce Rac1- GTP levels back to wild-type levels (Figure 1A, B). Given the enhanced pathological angiogenic responses in b3-null mice [12] we asked whether endothelial Rac1 was differentially required in tumor angiogenesis in adult wild-type or b3-null mice. To investigate whether Rac1-deficiency in endothelial cells affected tumor growth and tumor-associated angiogenesis in adult Figure 1. Increased active levels of Rac1 in b3-integrin-deficient endothelial cells. A. Active levels of Rac1 were examined by GST-PAK pull- downs. Western blot analysis of active Rac1 bound to GST-PAK (Rac1-GTP) and total Rac1 from wild-type, b3-integrin null, and b3-null primary lung endothelial cells transduced with human b3-integrin (rescue). Immunoblots were quantified by densitometry, and the levels of GTP-bound Rac1 normalised to total Rac1 levels. Active levels of Rac1 were increased approximately 3 fold in b3-null endothelial cells when compared with wild-type controls (*P,0.01), however, total levels of Rac1 were expressed equally in both genotypes. Furthermore, active levels of Rac1 were reduced to wild- type levels in rescue cells (*P,0.01). Results shown are the means + s.e.m of 3-4 independent experiments. B. Flow-cytometric analysis shows that surface levels of b3-integrin were not detectable in b3-null (dashed line) cells when compared with wild-types (bold line) endothelial cells (left panel). Rescue cells expressed b3-integrin (bold line, right panel). Grey peaks represent isotope IgG controls. Bar graph shows means + s.e.m of relative surface b3-integrin expression in wild-type (white), b3-null (black) and rescue (grey) endothelial cells compared with negative control (*P,0.001, N = 3 independent experiments). doi:10.1371/journal.pone.0009766.g001 PLoS ONE | www.plosone.org 2 March 2010 | Volume 5 | Issue 3 | e9766