DUAL ULTRASOUND-MEDIATED DELIVERY OF MIR-126 AND VEGF PLASMID DNA FOR PRO-ANGIOGENIC GENE THERAPY IN CHRONIC DIABETIC HINDLIMB ISCHEMIA

Abstract

MicroRNA (miR)-126 is an endothelial cell specific microRNA that regulates angiogenesis by blocking endogenous inhibitors of VEGF, Sprouty-related protein (SPRED1) and phosphoinositol-3 kinase (PI3K). In diabetes, the circulating level of miR-126 is decreased and may contribute to defective angiogenesis. We hypothesized that dual ultrasound-mediated delivery of miR-126 and VEGF plasmid DNA will improve microvascular blood flow (MBF) in chronic ischemia in the setting of diabetes. In vitro: Human umbilical vascular endothelial cells (HUVECs) were transfected with scramble miR or miR-126 with non-transfected cells as controls. 1x104 cells were plated in growth factor enriched matrigel. The capillary-like network formation was captured in photomicrographs for quantification. In vivo: Unilateral hindlimb ischemia was created by left femoral artery ligation in Zucker Diabetic fatty (ZDF) rats. Blood samples were analyzed for circulating levels of miR-126 and blood glucose levels. At day 14 post-ligation, MBF was assessed by contrast-enhanced ultrasound (CEU) followed by ultrasound-mediated gene delivery (UMGD) of either 1) miR-126 (2.5 mcg), 2) VEGF plasmid DNA (500 mcg) or 3) miR-126 + VEGF plasmid DNA, with 4) control animals receiving no treatment. CEU-derived MBF was re-assessed at day 28. In vitro: HUVECs transfected with miR-126 showed greatest ability to form tubes. The number of nodes formed by the HUVECs transfected with miR-126 were higher than both control and scramble groups (p<0.05; miR-126 vs scramble and control). In vivo: ZDF animals had high blood glucose levels (>30 mmol/L) and ∼40% reduction of circulating miR-126 levels compared to non-diabetic Zucker lean animals. Ischemia induced increase circulating miR-126 levels in lean animals while it remained unchanged for ZDF rats. At 14 days post-ligation surgery, MBF of the ischemic leg was reduced by ∼50% compared to the contralateral leg. At day 28 (14 days post delivery), the MBF of miR-126 delivered animals was improved with greatest improvement observed in the miR-126 and VEGF dual delivered animals (∼80% MBF of non-ischemic contralateral leg). MBF of control animals remained at baseline levels of 50% at day 28. Dual UMGD of miR-126 and VEGF plasmid DNA increased MBF in chronic hindlimb ischemia in ZDF rats, and may be an effective technique for therapeutic angiogenesis in the setting of diabetes.