Treatment of ischemic limbs based on local recruitment of vascular endothelial growth factor-producing inflammatory cells with ultrasonic microbubble destruction

OBJECTIVES We sought to clarify the mechanism for neovascularization by ultrasonic microbubble destruction (US/MB) and its ability to improve the function of ischemic limbs. BACKGROUND In tissue, US/MB can cause capillary rupture, leading to angiogenesis and arteriogenesis. METHODS Seven days after removal of the femoral artery (day 0) in mice, microbubble/ultrasound treatment was performed by intermittent insonation (1.6 MHz, mechanical index 1.1) to the ischemic limbs after intravenous infusion of phospholipid-stabilized microbubbles BR14 (US/MB group). Effects were compared with those in untreated mice with ischemic limbs (control group). RESULTS Immunostaining of the treated muscles revealed a greater leukocyte (CD45-positive cell) count in the US/MB group on days 3 and 7. These cells included F4/80-positive cells (macrophages) and CD3-positive cells (T-lymphocytes), both of which were immunoreactive to vascular endothelial growth factor (VEGF) antibody. Muscular VEGF content by Western blotting was elevated in the US/MB group on day 3, which declined but remained greater until day 21. The US/MB group showed a greater capillary density by alkaline phosphatase stain on day 7 without further increase at day 21. Surface vascularity of the muscles and blood flow were greater in the US/MB group on day 7, which further increased by day 21. Moreover, the US/MB group showed a two-fold longer treadmill time compared with the untreated control group on day 21. None of these favorable effects were observed in mice treated with ultrasound only or microbubbles only. CONCLUSIONS Ultrasonic destruction of microbubbles delivered to the ischemic limbs can recruit inflammatory cells producing VEGF, which is followed by neovascularization and functional improvement, and thus has a therapeutic potential.