Abstract 2894: Modulation of the Pulsing Interval During Ultrasound-mediated VEGF Plasmid Delivery Can Selectively Target Transfection to Different Vascular Compartments
Ultrasound-mediated (UM) destruction of plasmid-bearing microbubbles targets gene delivery to the vascular endothelium, promoting therapeutic angiogenesis. We hypothesized that modulation of the ultrasound pulsing intervals (PI) would allow targeting of gene delivery to selective vascular compartments based upon blood flow velocities, allowing differential targeting of faster filling vessels (arterioles) versus slower vessels (capillaries).
Methods: Unilateral hindlimb ischemia was produced by iliac artery ligation in 60 rats. At day 14 post-ligation, microvascular blood volume (MBV) and blood flow (MBF) in the proximal hindlimb muscles were assessed by contrast-enhanced ultrasound (CEU). UM-delivery of VEGF165/GFP plasmid (500 μg cDNA)-bearing microbubbles (1x109), was performed, at PIs of 2, 5 and 10 s (n=20 for each). Perfusion was re-assessed by CEU at days 17 and 28 (n=10 for each). Fluorescent microangiography (FMA) was performed at day 28 to assess vessel density. Transfection was localized using immunohistochemistry. PCR was used to quantify transgene expression.
Results: VEGF165/GFP mRNA expression was greatest at day 17, and was similar for PI 5 and 10 s, however reduced for PI 2 s. Fluorescent microscopy demonstrated GFP transfection predominantly within arterioles for shorter PIs (PI 5 s >> PI 2 s), and more capillary expression with PI 10 s. Prior to delivery, normalized MBV and MBF in ischemic muscle were similarly reduced in all groups. By day 28, for PI 5 and 10 s, there was a significant increase in normalized MBV (1.4±0.2 vs 0.9±0.2, p<0.001, and 1.3±0.3 vs 0.9±0.2, p<0.001 respectively) and MBF (0.9±0.3 vs 0.3±0.1, p<0.001, and 0.5±0.2 vs 0.3±0.1, p<0.001 respectively), with increases in MBF significantly (p<0.001) higher for PI 5 s. There were no significant changes in MBV or MBF with delivery at PI 2 s. Changes in vascular density by FMA paralleled changes in MBV, increasing at day 28 for both PI 5 and 10 s, and remaining unchanged for PI 2 s.
Conclusions: Altering the PI during UM-gene delivery can result in targeted delivery to selective vascular compartments. For the purposes of gene therapy for angiogenesis in chronic ischemia, a strategy that selectively targets arterioles is more effective than one that targets capillaries.