Rac1 and Tumor Angiogenesis Figure 8. Effect of Rac1-depletion in wild-type and b3-null cells in 2D versus 3D. A. Endothelial cell growth over 3 days was assessed by counting the total number of Mock, Con- or Rac1-siRNA transfected wild-type and b3-null endothelial cells. Although b3-null cells grew faster than wild-type, Rac1-depletion did not affect cell growth in either genotype. Symbols (N, Mock; &, Con-; and m, Rac1-siRNA) values given represent mean total cell numbers (+ s.e.m) from 3 independent experiments. B. Trypan blue exclusion, used to assess cell viability, shows that Rac1 knockdown has no effect on endothelial cell viability in either genotype. Bar graph represents mean percentage of viable cells (+ s.e.m) from 3 independent experiments. C. Wild-type and b3-null endothelial cell migration in response to VEGF was assessed in scratch wound healing assays. Representative photomicrographs (20 x magnification) of scratches at 0 hr and after 8 hr of migration after VEGF (25 ng/ml) stimulation. Bar graphs represent the mean percentage migration relative to 0 hours (+ s.e.m.), from 3 independent experiments, displayed by Mock (white), Con- (grey) and Rac1-siRNA (black) transfected wild-type and b3-null endothelial cells. Under 2D conditions, Rac1-depletion inhibited VEGF-induced migration of both wild-type and b3-null endothelial cell cells to baseline levels. D. VEGF-stimulated endothelial cell tube formation was assessed in 2D Matrigel cultures. Representative phase contrast micrographs (20 x magnification) showing cord structures (arrows) that formed 8 hr after seeding of Con- or Rac1- siRNA transfected cells on 2D Matrigel. Bar graphs represent mean numbers of branch points per field (+ s.e.m.) from 3 independent experiments. Rac1-depletion inhibited significantly VEGF-mediated tube formation by both wild-type and b3-null endothelial cells (*P,0.0001). Trypan blue exclusion assays demonstrated that the percentage of cell death of cells isolated from the cord-formation experiment did not change. Bar graph represents mean percentage of cell death (+ s.e.m.) from 3 independent experiments. E. In vitro VEGF-mediated endothelial cell migration was assessed in a modified 3D Boyden chamber assay. Quantification of VEGF-mediated migration of Mock, Con- or Rac1-siRNA transfected wild-type (left) and b3-null (right) endothelial cells in the presence or the absence of DC101. Bar graphs represent mean percentage migration relative to Mock treated cells in the absence of VEGF (+ s.d). VEGF-mediated migration was significantly reduced in Con siRNA DC101 treated endothelial cells, confirming the requirement of Flk-1 in VEGF-induced migration and provides a control (*P,0.05) siRNA targeting of Rac1 did not affect wild-type (n.s.d), but did inhibit b3-null, endothelial cell migration in 3D conditions (*P,0.01). N = 3–5 independent experiments. doi:10.1371/journal.pone.0009766.g008 chamber assays), Rac1-depleted cells were less motile than controls in 2D; whereas they migrated as well as controls in 3D. Several studies have shown that VEGF-mediated endothelial cell migra- tion in 2D involves activation of Rac1 [38,39,40] and our data are in line with these observations. Importantly, however, our findings demonstrate that the involvement of Rac1 in 3D endothelial cell migration is not as critical as originally thought and this is likely to reflect the reason for the normal tumor angiogenesis in Rac1- depleted mice in vivo. The reduced VEGF-induced 2D tube formation, after Rac1- depletion in endothelial cells, that we observed is also in line with several reports [41,42,43,44,45,46,47]. In contrast, using a novel 3D model involving co-culture with VEGF-A-overexpresing fibroblasts, others have shown that dominant-negative N17Rac1 has no effect on VEGF-induced capillary-like network formation of endothelial cells [48]. These investigators showed that a dominant negative mutant of N-Ras (a small GTPase from a different family to that of Rac1) suppressed capillary-network formation of endothelial cells induced by VEGF-A-producing fibroblasts whereas the mutants of Rac1, RhoA or Cdc42 did not. Our observations also support the notion that, in 3D environ- ments, Rac1 is dispensable for VEGF-mediated capillary- formation in wild-type but not b3-null aortic rings and angiogenesis in vivo. Previous studies have suggested that b3-integrin activation induces Rac1 activity. For example, inhibition of endothelial cell cyclooxygenase-2, by nonsteroidal anti-inflammatory drugs, suppresses avb3-dependent activation of Rac1 [49,50]. Given such crosstalk between integrins and Rac1, it is surprising that Rac1-activity is enhanced in b3-null endothelial cells. The increased active levels of Rac1 may reflect a compensatory response due to the deletion of b3-integrin, since rescuing the expression of b3-integrin in b3-null endothelial cells diminished the activity of Rac1 to levels similar as those seen in wild-type. Importantly, however, the expression and activity levels of other tested Rho-related GTPases (e.g. Rac2, Rac3, Cdc42 and RhoA) were equal in wild-type and b3-null endothelial cells suggesting that these Rho GTPases are unlikely to compensate for the loss of b3-integrin. Although recent work has shown that molecules such as RhoG can compensate for Rac-1 [51] there is also evidence that this depends on specific signaling cascades and is not always the case [52]. Our data suggest that this is unlikely in b3-null endothelial cells, since we have shown, that by inhibiting Rac1 expression alone, we can reduce the enhanced angiogenic responses of b3- mice back to levels observed in wild-type mice either with or without depletion of Rac1. Although it is tempting to speculate that RhoG activity may compensate for Rac1 in wild-type endothelial cells, thus explaining the reduced requirement for Rac1 in wild-type angiogenic events in vivo, this is also not likely because Rac1-deletion in both b3-null and wildtypes brings tumor growth and blood vessel density to the same level in both genotypes. Thus the most logical assumption is simply that Rac1 is not involved in tumor angioegenesis in vivo unless b3-integrin is absent. Our data indicate that the importance of Rac1 in angiogenic processes is revealed only after b3-integrin is absent. We show that the enhanced Rac1 activity in b3-null endothelial cells is responsible, at least in part, for the elevated tumor angiogenesis and VEGF-mediated angiogenic responses in b3-null mice. Despite the fact that b3-null endothelial cells have elevated VEGFR2 (Flk-1) levels [12,27,53], even after Rac1-depletion, indicates that elevated VEGFR2 levels alone are not sufficient to enhance angiogenesis. We show that VEGF/VEGFR2-mediated responses are Rac1-dependent in the absence, but not in the presence of b3-integrin, raising the possibility that downstream signalling of VEGFR2 can either involve Rac1 or not, but that this depends on b3-integrin. In b3-null mice the increased Rac1-1 activity likely results in changes in downstream signaling that confer the enhanced angiogenic responses. For example, p38 MAP kinase signaling is known to be downstream of Rac1 and b3-integrin and plays a role in angiogenic processes such as endothelial cell migration and regulation of cell-cell adhesion [54,55,56]. Thus, in future experiments it would be of interest to examine the role of p38 MAP-kinase signaling in b3-null endothelial cells. Several studies have shown that the blockade of avb3 using monoclonal antibodies or antagonist peptides can inhibit tumor growth and tumor angiogenesis in mouse xenografts models and other preclinical tests [57] and some of these agents, such as the humanized monoclonal antibody Vitaxin [5] and the cyclic RGD peptide Cilengitide [3,4,6,7] are in clinical trials for the anti-angiogenic treatment of cancer. However, to date, there is very little evidence showing these inhibitors can in fact suppress tumor angiogenesis in humans [3,4,6,9,10,11]. This may simply reflect that tumor angiogenesis is not actually dependent on the activity of avb3-integrin in endothelial cells in several tumor types. Indeed, in some human tumors, such as angiosarcomas, PLoS ONE | www.plosone.org 12 March 2010 | Volume 5 | Issue 3 | e9766