Mononuclear cells infiltrate the foci of VEGF-induced neovascularization. (A) Immunohistochemical time course analysis, by the visualization of small arteries using an antibody against smooth muscle actin (α-SMA), of the angiogenic response induced by AAV-mediated VEGF expression in normal skeletal muscle at different times after vector injection. Abundant mononuclear cell infiltrates are detectable in the perivascular spaces of the injected muscles (enlargements i-iii in panel B). Images were obtained using an Olympus CX40 microscope and 10 ×/0.30 NA and 40 × 0.75 UPlan FL objective lenses (Olympus, Tokyo, Japan). Images were captured using Olympus Camedia C-3030 digital camera and processed with Adobe Photoshop 7.0 software. (C) Quantification of the number of α-SMA–positive vessels over time. Shown are means and standard deviations of counts, expressed as number of α-SMA vessels per muscle fiber. The quantifications were carried out by 3 independent investigators who observed 10 different sections from 3 animals per time point. d indicates days; mo, months. (D) Quantification of the number of the infiltrating cells in the AAV-VEGF–treated muscles. Shown are means and standard deviations of counts, expressed as number of nuclei per muscle fiber. The quantifications were carried out by 3 independent investigators who observed 10 different sections from 3 animals per time point.