Rac1 and Tumor Angiogenesis Figure 3. Endothelial-specific Rac1-depletion does not impair tumor growth wild-type mice but does in b3-null mice. A. Murine B16F0 cells (106) were injected subcutaneously into the flanks of wild-type/Tie1-Cre+ or b3-null/Tie1-Cre+ mice. Lentiviral vector suspensions (106 i.u/ml) of pSico-Con, pSico-Rac1 and pSico-Flk-1 were injected intratumorally on days 5 and 10 after tumor cell injection. Representative macroscopic appearance of 14-day-old B16F0 pSico-Con-, pSico-Rac1- and pSico-Flk-1- treated melanomas in both genotypes. Scale bar: 5 mm. Bar graph shows mean tumor volume per mm3 (+ s.e.m.). Tumor size was reduced significantly in pSico-Flk-1-treated mice of both genotypes (*P,0.05) and in pSico- Rac1 treated b3-null/Tie1-Cre+ but not in pSico-Rac1-treated wild-type/Tie1-Cre+ mice (n.s.d, no significant differences). N = 4–6 animals per condition. B. Representative merged images of PECAM-1 (red) and GFP (green) -immunostained sections from pSico-Rac1-treated B16F0 tumors grown in wild- type, wild-type/Tie1-Cre+, b3-null and b3-null/Tie1-Cre+ mice. PECAM-1-positive staining identified endothelium. GFP-positive staining was observed in tumor cells (concave arrowheads) and in PECAM+ endothelium (arrows) of blood vessels in B16F0 tumors from wild-type and b3-null control mice, indicating successful pSico-Rac1 lentivirus infection in vivo. Loss of GFP detection in most PECAM-1-positive microvessels (small arrowheads), but not in B16F0 tumor cells, was observed in pSico-treated tumors grown in wild-type/Tie1-Cre+ and b3-null/Tie1-Cre+ mice, indicating successful endothelial-specific Cre recombination in vivo. Scale bar: 10 mm. Bar graph shows mean numbers of PECAM-1+/GFP2 vessels per unit area of tumor section per mm2 (+ s.e.m). Blood vessel density was reduced significantly in pSico-Flk-1-treated mice of both wild-type/Tie1-Cre+ and b3-null/Tie1- Cre+ mice (*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 = 4 animals per condition. doi:10.1371/journal.pone.0009766.g003 lentiviruses. Immunostaining for PECAM-1 identified blood vessels under both conditions. In wild-type/Tie1-Cre2 mice, GFP expression was detected in both the tumor cells and the majority of endothelial cells lining blood vessels, indicating a high efficiency of lentiviral infection in vivo. In contrast, analysis of tumors grown in wild-type/Tie1-Cre+ mice showed a loss of GFP expression in endothelial cells but not tumor cells, indicating that endothelial-specific Cre-recombination was efficient (Figure 3B). Taken together, these data indicate that, while no biological effect of Rac-1 knockdown was observed in wild-type/Tie1-Cre+, the lentivirus treatment efficiently reduced expression of Rac1 in tumor blood vessels from these animals. To examine the effect of endothelial Rac1-depletion on tumor angiogenesis, blood vessel density was quantified by counting the numbers of PECAM-1+/GFP2 blood vessels per unit area across entire midline tumor sections from size-matched tumors of wild- type/Tie1-Cre+ and b3-null/Tie1-Cre+ mice treated with either pSico-Con, pSico-Rac1 or pSico-Flk-1 lentiviruses. Once again, concurrent with our previous work [12,27], blood vessel density in pSico-Con treated b3-null/Tie1-Cre+ mice was significantly greater than in pSico-Con treated wild-type/Tie1-Cre+ mice. However, although pSico-Flk-1 treatment inhibited significantly tumor angiogenesis in both wild-type/Tie1-Cre+ and b3-null/ Tie1-Cre+ mice (P,0.05), pSico-Rac1-treatment did not affect tumor angiogenesis in wild-type/Tie1-Cre+ but did inhibit tumor angiogenesis significantly in b3-null/Tie1-Cre+ mice (Figure 3B). These results indicate that Rac1-depletion in tumor endothelial cells does not affect tumor growth or tumor angiogenesis in wild- type/Tie1-Cre+ mice but does inhibit both these processes in b3- null/Tie1-Cre+ mice. Further confirmation of the role of endothelial Rac1 in tumor growth and angiogenesis was accomplished by using a genetic ablation approach that allows conditional Rac1-deletion in an inducible-manner. Rac1-floxed mice [28] were crossed with PDGFB-iCreER transgenic mice (23) (where Cre expression, in adult animals, is restricted primarily to angiogenic/proliferating endothelial cells [29]) to generate tamoxifen-inducible endothelial- specific Rac1-deficient mice (Rac1 flox/flox PDGFB-iCreER). To verify that tamoxifen treatment of Rac1 flox/flox PDGFB-iCreER endothelial cells induced Rac1-deletion, endothelial cells and non- endothelial cells were isolated from Rac1 flox/flox PDGFB- iCreER mice and treated with tamoxifen (OHT) or ethanol (vehicle) in culture. Rac1 protein levels were barely detected in Rac1 flox/flox PDGFB-iCreER tamoxifen-treated endothelial cells when compared with vehicle-alone treated Rac1 flox/flox PDGFB-iCreER or tamoxifen-treated Rac1 flox/flox (PDGFB- iCreER negative) controls. Importantly, tamoxifen treatment of non-endothelial cells isolated from Rac1 flox/flox PDGFB- iCreER mice had no effect on Rac1 expression (Figure 4A). Rac1 flox/flox PDGFB-iCreER mice were injected subcutane- ously with B16F0 cells and, one day later, treated with tamoxifen (OHT) or placebo administered by slow-release pellets implanted subcutaneously to induce Cre-activity in PDGFB-positive cells. Deletion of Rac1 in the vascular endothelium did not affect tumor size or tumor angiogenesis when compared with controls (Figure 4B). As yet an additional test of the roles played by Rac1 and b3- integrin in angiogenesis in vivo, Rac1 flox/flox PDGFB-iCreER animals received tamoxifen pellet implants. These animals then were injected subcutaneously with B16F0 cells and subsequently received intra-tumoral injections of pSico-Con (as described above) or a pSico lentivirus directed against b3-integrin (pSico- b3). Tumors treated with pSico-Con grew to an expected size of approximately 800 mm3. pSico-b3 treated tumors, however, were significantly smaller (P,0.05, Figure 4C). Blood vessel counts of endomucin stained tumor sections showed a significant reduction in angiogenesis after treatment with pSico2b3 (P,0.001, Figure 4C). Taken together, data from Rac1 flox/flox PDGFB- iCreER animals provided a second line of evidence that tumor growth and angiogenesis in vivo does not normally depend on Rac1. Already published studies suggest that Rac1 plays a role in vascular tube formation, at least in vitro [21,22]. As such, the data describe here were somewhat surprising. To further test the effect of Rac1 deletion in another neovascularization assay, the influence of Rac1 deletion was analyzed in the post-natal developing retina [29]. Wild-type PDGFB-iCreER and Rac1 flox/flox PDGFB- iCreER newborn pups were treated daily with tamoxifen by oral gavage and their retinas were examined at P12. No significant differences in central primary plexus, or peripheral primary plexus branch points were observed between the two genotypes (Figure 5). Therefore, deficiency of Rac1 in endothelial cells is not sufficient to alter neovasculatization in vivo, at least when b3- integrin is present. Using multiple in vivo approaches, we demonstrated that endothelial Rac1 is not essential for tumor growth or tumor angiogenesis in adult wild-type mice, but that these processes become Rac1-dependent in the absence of b3-integrin. Rac1 is not required for VEGF-mediated angiogenesis in wild-type mice but is in b3-null mice Given that VEGF is a key positive regulator of pathological angiogenesis and that VEGF/VEGFR2 signalling is known to activate Rac1 in vitro, we tested the effect of endothelial-specific Rac1-depletion on VEGF-mediated neovascularization in vivo. Synthetic sponges were implanted subcutaneously into wild-type/ Tie1-Cre+ and b3-null/Tie1-Cre+ mice and injected in situ with PLoS ONE | www.plosone.org 5 March 2010 | Volume 5 | Issue 3 | e9766