Abstract 14030: Therapeutic Angiogenesis by Ultrasound-Mediated miRNA-126 Delivery
Introduction: The endothelial specific MicroRNA-126 (miR-126) regulates angiogenesis by blocking endogenous inhibitors of VEGF, Sprouty-related protein (SPRED1) and phosphoinositol-3 kinase (PI3K). We hypothesized that ultrasound-mediated delivery of miR-126 would lead to improved microvascular blood flow (MBF) in chronic ischemia.
Methods: Unilateral hindlimb ischemia was created by left femoral artery ligation in F-344 rats (n=92). At day 14 post-ligation, MBF was assessed by contrast-enhanced ultrasound (CEU). Ultrasound-mediated gene delivery (UMGD) of miR-126 (n=35) or scrambled miR (n=25) was performed, with control animals (n=32) receiving no treatment. Perfusion was re-assessed at day 28 and fluorescent microangiography (FMA) was performed for vessel density quantification. Transfection of miR-126 was assessed by real time PCR. SPRED1 and PIK3R2 knockdown were quantified by western blots. For in-vitro studies, human umbilical vascular endothelial cells (HUVECs) were transfected with miRNA-126 or scrambled-miRNA in the presence or absence of Angiopoeitin (Ang)-1. Tie2 and pTie2 levels were quantified by western blots.
Results: The miR-126 delivered groups showed 2.1±0.2 fold increased miR-126 levels over control at day 1 post delivery, 1.7±0.3 fold increase at day 3 post delivery, which normalized at day 28. Western blots showed significant knockdown of SPRED1 and PIK3R2 protein levels at day 1 post delivery. At day 14, prior to delivery, normalized MBF for the ischemic muscle were reduced in all treatment groups (~60%). In control and scramble groups, MBF remained unchanged. In miR-126 treated animals, MBF was significantly improved at day28 (0.8±0.12 vs 0.6±0.08, p < 0.001). FMA analysis showed 1.5±0.2 fold increase (p<0.01 versus Control and Scramble) in vascular density for miR-126 treated animals. In-vitro studies also showed increased levels of Tie2 and pTie2 in HUVECs only when transfected with miR-126 under Ang-1 stimulation.
Conclusion: UMGD of miR-126 increased MBF and vessel density in chronic hindlimb ischemia, with knock down of SPRED1 and PIK3R2. miR-126 increases expression of Tie2 and pTie2 under Ang-1 stimulation, indicating potential interplay between miR-126 and Ang-1 in the activation of Tie2.
- © 2013 by American Heart Association, Inc.