Active Rac1 improves neovessel architecture and lumen formation in vivo. (A) VEGF₁₆₅ transfectants were mixed with packaging cells expressing retrovirus encoding active L61Rac1, dominant-negative N17Rac1, or empty vector (control) and injected together with Matrigel subdermally. Animals were harvested after 7 days. Gross: gross images of dermis overlying the Matrigel implants show that active L61Rac1 improved formation of new blood vessels relative to control, whereas DN Rac1was inhibitory. Scale bar = 375 μm. CD31 Stain: ECs in cross section stained with CD31 antibody (brown color), illustrating that active Rac1 improved lumen formation (arrows) relative to empty vector control, whereas DN Rac1 abolished lumen formation. Scale bar = 15 μm. (B) Tx-Red dextran: perfusion of vessels with 70 kDa, lysine-fixable, Texas-Red dextran (10 minutes), viewed with confocal microscopy (scale bar = 300 μm) confirming that active Rac1 promoted formation of larger, well-perfused, architecturally improved blood vessels. Microfil Vascular Cast: the entire vasculature was perfused with Microfil, illustrating improvement in neovessel diameter and architecture mediated by active Rac1 (scale bar = 250 μm). (C) Quantification of vascular parameters; n > 20 for all groups. From gross images: relative neovascularization (ie, percentage of relative area occupied by neovessels in flat mount; P < .001). From CD31-stained cross-sections: quantification of ECs per 0.015 mm², relative total lumen area (P < .001), numbers of neovessels per 0.015 mm² (P < .01), and average internal neovessel diameter (P < .01).