526 Targeted Delivery of MicroRNA-126 Improves Perfusion in Chronic Hindlimb Ischemia

Abstract

MicroRNA-126 (miR-126) is an endothelial-specific microRNA that regulates angiogenesis, acting as an inhibitor of endogenous inhibitors of vascular endothelial growth factor (VEGF), namely Sprouty-related protein (SPRED1) and phosphoinositol-3 kinase (PI3K). In this study we examined the in vivo angiogenic effect of targeted delivery of miR-126 in chronic hindlimb ischemia in rats. 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=35). 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 ìg of miR-126) (n=7) or scrambled miR (miR-SCR) (n=6) was performed, with control animals (n=3) 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. At day 14, prior to miR-126 delivery, normalized MBF for the ischemic muscle was similarly reduced in all treatment groups (∼55% of normal). At day 28, MBF remained unchanged in control and miR-SCR treated groups (0.59±0.06 vs 0.56±0.04, and 0.59±0.07 vs 0.58±0.05, respectively. In miR-126 treated animals, MBF improved (0.60±0.08 vs 0.78±0.06, p < 0.01 versus control and miR-SCM). After triple delivery of miR-126, further improvement in perfusion was noted (0.57±0.04 vs 0.88±0.03, p < 0.01 versus control and miR-SCM, and p <0.05 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). 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.