Abstract 3149: Long Term Effects of Ultrasound-mediated VEGF165 Delivery on Microvascular Perfusion and Endogenous Growth Factor Expression in Chronic Ischemia
Ultrasound-mediated (UM) destruction of carrier microbubbles can provide an effective method of gene delivery for therapeutic angiogenesis, however the late effects on tissue perfusion remain unexplored. Our objective was to determine the duration of the angiogenic response to UM-VEGF165 delivery, and characterize its effects on endogenous growth factor regulation. Methods: Unilateral hindlimb ischemia was produced by iliac artery ligation in 24 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). The ischemic hindlimb was then exposed to 20 minutes of intermittent high-power ultrasound during intravenous administration of VEGF165/GFP plasmid (500 μg cDNA) coupled to 1x109 cationic microbubbles. Perfusion was re-assessed by CEU at days 17, day 28 and day 52 (n=8 for each). Fluorescent microangiography (FMA) (n=3 per group) was performed at days 28 and 52 to assess microvessel density. Real time PCR was used to quantify transgene and endogenous growth factor expression. Results: Prior to VEGF165 plasmid delivery, normalized MBF in ischemic muscle was reduced. At day 17, there were no changes in tissue perfusion. By day 28, there was a significant increase in normalized MBV (1.8 ± 0.75 vs 1.1 ± 0.35, p<0.01) and MBF (0.91 ± 0.3 vs 0.44 ± 0.15, p<0.001). By day 52, normalized MBF to the ischemic leg had reduced significantly (0.63±0.15 vs 0.91 ± 0.3, p<0.01), but still remained above baseline pre-treatment values. Changes in vascular density by FMA paralleled changes in MBF, increasing at day 28, followed by regression by day 52. VEGF165/GFP mRNA expression was greatest at day 17, becoming undetectable by day 52. Expression of endogenous VEGF and Ang-2 in ischemic muscle was elevated early (3x), returning to baseline with improved perfusion. Ang-1 mRNA levels were elevated late (6x), keeping with its role in neovessel maturation. Conclusions: Ultrasound-mediated transfection of VEGF165 results in improved tissue perfusion, which despite modest regression, persisted late post-delivery. Modulation of endogenous growth factor expression occurred consistent with a positive biologic effect of ultrasound-mediated gene delivery.