Rac1 and Tumor Angiogenesis Figure 9. Endothelial Rac1-depletion does not affect the expression of Flk-1. A. Western blot analysis of Flk-1 expression levels in primary wild-type and b3-null endothelial cells transfected with scrambled (Con siRNA) or Rac1 (Rac1 siRNA) siRNAs. Although Flk-1 expression was increased significantly in b3-null endothelial cells when compared with wild-types (*P,0.01), Rac1-depletion did not affect Flk-1 levels (n.s.d) in either genotype. Bar graph represents mean (+ s.e.m.) of Flk-1 expression relative to Hsc-70 loading control (N = 3–4 independent experiments). B. HUVEC were transfected with vector only, a wild-type Rac1 construct (Rac1 WT), or a constitutively active Rac1 construct (Rac1 QL) and seeded on confluent fibroblasts. Tubules were visualized by PECAM1 staining 5 days after seeding. The mean number of branch points (+SEM) is shown in the accompanying bar chart. (n = 12 microscopic fields per condition). Scale bar: 100 mm. doi:10.1371/journal.pone.0009766.g009 endothelial b3-integrin expression levels decrease as malignant transformation progresses [16]. In addition, vascular b3-integrin levels were significantly lower in lung metastases from colorectal carcinomas when compared with those seen in primary colorectal tumors [17,18]. Thus, targeting b3-integrin might not be a useful anti-angiogenic strategy for this particular subset of patients. One question that remains unanswered is: ‘‘when is b3-integrin actually bound to ligand during the angiogenic process?’’ Wow-1 is an antibody that recognizes ligated b3 integrin in cells in culture [58] but, unfortunately, has yet to be proven to work in whole tissue sections or in vivo. Thus testing b3-integrin ligation in vivo is presently impossible without the use of inhibitors. Importantly, since inhibition of b3-ligation does not phenocopy the deficiency of b3-integrin, where such angiogenic responses are enhanced [12], it is also not possible to test whether b3-integrin ligation is truly relevant to our in vitro findings. This is because inhibition of b3-ligation, using RGD mimetics or antibodies, can inhibit tubule formation, for many reasons including an increase in cell death [59]. Thus inhibiting b3-integrin function not only inhibits b3- ligation but also simultaneously alters downstream signaling. Furthermore, we have recently shown that low doses of such agents can actually enhance angiogenesis, both in vitro and in vivo, but that these doses do not affect ligand binding [60]. Thus in conclusion, although such experiments would be of great interest we await the technology to enable us to test the effect of b3- ligation effectively in vivo without the use of inhibitors or knockout mice. Our findings suggest that inhibition of Rac1 might be an effective anti-angiogenic therapeutic strategy in tumor blood vessels that display low levels of b3-integrin. Furthermore, combination therapy of anti-Rac1 and anti-b3-integrin targeted to endothelial cells may be an effective general anti-angiogenic strategy. PLoS ONE | www.plosone.org 13 March 2010 | Volume 5 | Issue 3 | e9766