Delayed Angiogenesis and VEGF Production in CCR2 -/- Mice during Impaired Skeletal Muscle Regeneration Running head: Altered Regeneration and Angiogenesis in CCR2-/- Mice

The regulation of vascular endothelial growth factor (VEGF) levels and angiogenic events during skeletal muscle regeneration remains largely unknown. This study examined angiogenesis, VEGF levels, and muscle regeneration after cardiotoxin (CT)-induced injury in mice lacking the CC chemokine receptor 2 (CCR2). Muscle regeneration was significantly decreased in CCR2 -/mice as was the early accumulation of macrophages after injury. In both mouse strains, tissue VEGF was similar at baseline (no injections) and significantly decreased at day 3 post-CT. Tissue VEGF in WT mice was restored within 7 days post-injury but remained significantly reduced in CCR2 -/mice until day 21. Capillary density (capillaries/mm) within regenerating muscle was maximal in WT mice at day 7 and double that of baseline muscle. In comparison, maximal capillary density in CCR2 -/mice occurred at 21 days post-injury. Maximal capillary density developed concurrent with the restoration of tissue VEGF in both strains. A highly significant, inverse relationship existed between the size of regenerated muscle fibers and capillaries/mm. While this relationship was comparable in WT and CCR2 -/animals, there was a significant decrease in the magnitude of this response in the absence of CCR2, reflecting the observation that regenerated muscle fiber size in CCR2 -/mice was only 50% of baseline at 42 days post-injury while WT mice had attained baseline fiber size by day 21. Thus, CCR2-dependent events in injured skeletal muscle, including impaired macrophage recruitment, contribute to restoration of tissue VEGF levels and the dynamic processes of capillary formation and muscle regeneration.