Therapeutic Angiogenesis by Ultrasound-Mediated MicroRNA-126-3p Delivery

Abstract

Objective— MicroRNAs are involved in many critical functions, including angiogenesis. Ultrasound-targeted microbubble destruction (UTMD) is a noninvasive technique for targeted vascular transfection of plasmid DNA and may be well suited for proangiogenic microRNA delivery. We aimed to investigate UTMD of miR-126-3p for therapeutic angiogenesis in chronic ischemia. Approach and Results— The angiogenic potential of miR-126-3p was tested in human umbilical vein endothelial cells in vitro. UTMD of miR-126-3p was tested in vivo in Fischer-344 rats before and after chronic left femoral artery ligation, evaluating target knockdown, miR-126-3p and miR-126-5p expression, phosphorylated Tie2 levels, microvascular perfusion, and vessel density. In vitro, miR-126-3p–transfected human umbilical vein endothelial cells showed repression of sprouty-related protein-1 and phosphatidylinositol-3-kinase regulatory subunit 2, negative regulators of vascular endothelial growth factor and angiopoietin-1 signaling, increased phosphorylated Tie2 mediated by knockdown of phosphatidylinositol-3-kinase regulatory subunit 2 and greater angiogenic potential mediated by both vascular endothelial growth factor/vascular endothelial growth factor R2 and angiopoietin-1 /Tie2 effects. UTMD of miR-126-3p resulted in targeted vascular transfection, peaking early after delivery and lasting for >3 days, and resulting in inhibition of sprouty-related protein-1 and phosphatidylinositol-3-kinase regulatory subunit 2, with minimal uptake in remote organs. Finally, UTMD of miR-126-3p to chronic ischemic hindlimb muscle resulted in improved perfusion, vessel density, enhanced arteriolar formation, pericyte coverage, and phosphorylated Tie2 levels, without affecting miR-126-5p or delta-like 1 homolog levels. Conclusions— UTMD of miR-126 results in improved tissue perfusion and vascular density in the setting of chronic ischemia by repressing sprouty-related protein-1 and phosphatidylinositol-3-kinase regulatory subunit 2 and enhancing vascular endothelial growth factor and angiopoietin-1 signaling, with no effect on miR-126-5p. UTMD is a promising platform for microRNA delivery, with applications for therapeutic angiogenesis.

Endothelium

“…23,24 In this regard, microRNA seem as an interesting tool for improving angiogenesis. 24 Cao et al 25 investigated the therapeutic angiogenic effect of miR126-3p using ultrasound targeted microbubble destruction, a noninvasive technique for targeted vascular transfection of plasmid DNA, including microRNAs. In their study, transfection of miR-126-3p before and after rat left femoral artery ligation improved perfusion, vessel density, enhanced arteriolar formation, pericyte coverage, and phosphorylated Tie2 levels with no effect on miR-126-5p.…”

Neovascularization Driven by MicroRNA Delivery to the Endothelium

“…This will allow for the determination of whether ischemic regions of the infarcted heart might be targeted by angiogenic miRs, while avoiding delivery to healthy regions of the heart. Although the results of Cao et al 16 have provided optimism that UTMD approaches might be beneficial for human ischemic disease, there are several limitations of this study that should be noted. Many experimental therapies that have shown promise in preclinical models do not have similar efficacy in human clinical trials.…”

“…In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology, Cao et al 16 have provided further mechanistic insight into therapeutic modulation of angiogenesis via UTMDmediated delivery of miR-126. They demonstrate specific delivery of miR-126 to the ECs and pericytes of vessels in ischemic rat hindlimb muscle exposed to ultrasound and downregulation of known miR-126 targets, such as SPRED1 and PIK3R2.…”

“…18 To apply UTMD miR delivery as a therapy, it is critical to understand the duration of miR delivery and action. Cao et al 16 have observed that miR-126 transfection occurs from 3 hours to 3 days after ultrasound, with target gene knockdown lasting 3 days. Although target genes return to normal levels by 14…”

“…It is not clear how these comorbidities might influence the effectiveness of miR-126 delivery in humans. The hindlimb ischemia model used by Cao et al 16 is considered a chronic ischemia model because 2 weeks elapse after ligation before therapy is initiated. However, in many human patients, ischemia may be present for many months or years.…”