AAV-PDGF-D induces macrophage accumulation in skeletal muscle. (A) Schematic representation of the AAV constructs. Human PDGF-DFL, PDGF-DΔN, VEGF-E, and HSA cDNAs were driven by the CMV promoter and early enhancer and were followed by the Woodchuck posttranscriptional enhancer-element (WPRE) and the SV40 polyadenylation signal (pA). (B) In vitro expression analysis of the AAV vectors in HeLa cell cultures. PDGFs were precipitated from the media of the AAV-transduced cells with PDGFR-β immunoglobulin, and VEGF-E was precipitated with VEGFR-2 immunoglobulin. Fluorescence photomicrographs from the ears of mice that received transplanted GFP-marked bone marrow cells from a donor of the same mouse strain. Ears were injected with (C) AAV-PDGF-DFL, (D) AAV-PDGF-DΔN, (E) AAV-PDGF-B, or (F) PBS. Note the strong accumulation of GFP-positive cells in the ears expressing PDGF-D (original magnification, × 50). Staining of the macrophage antigen F4/F80 in mice injected with (G) AAV-PDGF-DFL and (H) AAV-PDGF-DΔN compared with (I-J) AAV-PDGF-B or PBS control (original magnification, × 200). (I, inset) Human PDGF-B (lane 1) and β-actin (lane 2) RT-PCR from RNA extracted from (B) AAV-PDGF-B– and (H) AAV-HSA–injected muscle. Bars represent 230 μm (F) and 20 μm (J).