Role of Rap1a for neovascularization capacity in vivo. (A,B) Rap1a^+/+ (n = 11), Rap1a^+/− (n = 11), and Rap1a^−/− (n = 4) mice were subjected to hind limb ischemia as described in “Methods.” (A) Capillary density (ratio of the number of capillaries to the number of myocytes) was determined in 8-μm frozen sections of ischemic muscles. Representative images of ischemic muscles are shown on the left panel (CD31, red fluorescence; laminin, green fluorescence). A quantitative analysis of capillary density is shown. Data are presented as mean plus or minus SEM (*P < .05 vs Rap1a^+/+). Bar represents 20 μm. Micrographs were acquired with an LSM 510 confocal microscope fitted with a Plan-Neofluar 40×/1.3 NA oil objective and LSM5 image acquisition software (all from Carl Zeiss). (B) Conductance vessels in the adductor muscles were identified by size and smooth muscle actin (SMA) staining using a Cy3-labeled mouse monoclonal antibody for SMA. The number of small (< 50 μm), medium (50-100 μm), and large vessels was determined separately. Data are presented as mean plus or minus SEM (*P < .05 vs Rap1a^+/+, < 50 μm). Evaluation was performed in a blinded fashion. (C) The perfusion of ischemic limbs was assessed by high frequency ultrasound in Rap1a^−/− and Rap1a^+/+ (wild-type) mice. (D) Statistical summary of blood vessel infiltration in Matrigel sections stained with an anti-SMA antibody in wild-type and Rap1a^+/− mice. Quantitative results are presented as mean plus or minus SEM; n = 4 (Rap1a^+/+), n = 4 (Rap1a^+/−). Evaluation was performed in a blinded fashion. Sections of Matrigel plugs were stained with hematoxylin and eosin. Bar represents 20 μm (right panel).