Abstract 19114: Ultrasound-Targeted Delivery of microRNA-126 for Therapeutic Angiogenesis

Abstract

Introduction: MicroRNA-126 (miR-126) is an endothelial-specific microRNA that regulates angiogenesis by blocking endogenous inhibitors of vascular endothelial growth factor (VEGF), Sprouty-related protein (SPRED1) and phosphoinositol-3 kinase (PI3K). We hypothesized that ultrasound-mediated delivery of miR-126 would result in improved angiogenesis in the setting of chronic ischemia.

Methods: Naked mature miR-126 was synthesized for transfection of rat specific miR-126 (22bp). Unilateral hindlimb ischemia was created by left femoral artery ligation in F-344 rats (n=45). At day 14 post-ligation, microvascular blood flow (MBF) in the distal hindlimb muscles were assessed by contrast-enhanced ultrasound (CEU). Ultrasound-mediated gene delivery (UMGD) of miR-126 (1x109 cationic microbubbles + 2 μ g of miR-126) (n=12) or scrambled miR (miR-SCR) (n=12) was performed, with control animals (n=11) receiving no treatment. An additional group (n=10) received UMGD of miR-126 (2 ug) three times every 48hours (day14, day 16 and day18). CEU perfusion was re-assessed at day 28, and transfection was assessed by real time PCR.

Results: At day 14, prior to miR-126 delivery, normalized MBF for the ischemic muscle was similarly reduced in all treatment groups (55-60% of normal). At day 28, MBF remained unchanged in control and miR-SCR treated groups (0.61±0.08 vs 0.62±0.09, and 0.59±0.06 vs 0.63±0.07, respectively. In miR-126 treated animals, MBF improved (0.59±0.07 vs 0.78±0.06, p < 0.001 versus control and miR-SCM). After triple delivery of miR-126, further improvement in perfusion was noted (0.58±0.04 vs 0.88±0.03, p < 0.001 versus control and miR-SCM, and p <0.01 versus single miR-126). Real time PCR showed a 14.3±3.4 fold increase over control at 3h post transfection of miR-126 that persisted to day 3 post-UMGD, and normalized by day7 (n=3 per time point).

Conclusions: UMGD of miR-126 results in improved perfusion in the setting of chronic hindlimb ischemia. Multi-gene delivery of miR-126 showed an incremental angiogenic response as compared to single gene delivery.