Abstract 15325: Overexpression of MicroRNA-126 Promotes Angiogenesis in a Vascular Endothelial Growth Factor Receptor 2 (VEGFR-2) and Tyrosine Kinase with Immunoglobulin-like and EGF-like Domains 2 (Tie2) Dependent Manner in Hindlimb Chronic Ischemia
Introduction: The endothelial specific microRNA-126 (miR-126) regulates angiogenesis primarily by blocking endogenous inhibitors of VEGF, Sprouty-related protein and phosphoinositol-3 kinase regulatory subunit 2. We hypothesized that ultrasound-mediated delivery of miR-126 contributes to neovascularization through VEGFR-2 and Tie2 pathway at an early and late stage respectively in chronic ischemia.
Methods: F-344 rats (n=4) were delivered with fluorescently labeled miR by ultrasound-mediated gene delivery (UMGD) to determine the miR cellular localization. Unilateral hindlimb ischemia was created by left femoral artery ligation (n=92). At day 14 post-ligation, microvascular blood flow (MBF) was assessed by contrast-enhanced ultrasound (CEU). 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 and Tie2 and pTie2 levels were quantified by western blotting at day 28. For in vitro studies, human umbilical vascular endothelial cells (HUVECs) were transfected with miR-126 or scrambled-miR in the presence or absence of Angiopoietin (Ang-1). Tie2 and pTie2 levels were quantified by western blotting. Matrigel assay was performed to investigate mechanistic aspects of miR-126 mediated angiogenesis by using VEGFR-2 and Tie2 inhibitors.
Results: miR delivered by UMGD was primarily localized in the nucleus of endothelial cells. MBF was significantly improved at day 28 in miR-126 delivered animals (0.8±0.12 vs 0.6±0.08, p < 0.001). pTie2 and Tie2 levels were significantly elevated in miR-126 delivered animals at day 28. (1.45±0.25, 2.05±0.25 fold increase over Control) In vitro studies also indicated increased levels of Tie2 and pTie2 in HUVECs when transfected with miR-126 under Ang-1 stimulation. miR-126 transfected HUVECs exhibited a reduction in formation of capillary-like structures on Matrigel by VEGFR-2 and Tie2 inhibitors at 9 hours and 18 hours respectively.
Conclusion: UMGD of miR-126 increased MBF, expression of Tie2 and pTie2 in vivo and under Ang-1 stimulation in vitro. Ability to form capillary-like structures was affected by VEGFR-2 and Tie2 inhibitors indicating a potential interplay between miR-126 and Tie2 to facilitate late angiogenesis.
Author Disclosures: P.N. Matkar: None. W.J. Cao: None. D. Rudenko: None. H.H. Chen: None. M.A. Kuliszewski: None. H. Leong-Poi: None.
- © 2014 by American Heart Association, Inc.