A selective microRNA-based strategy inhibits restenosis while preserving endothelial function
Gaetano Santulli, … , Andrew R. Marks, Hana Totary-Jain
Gaetano Santulli, … , Andrew R. Marks, Hana Totary-Jain
Published August 18, 2014
Citation Information: J Clin Invest. 2014;124(9):4102-4114. https://doi.org/10.1172/JCI76069.
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Research Article

A selective microRNA-based strategy inhibits restenosis while preserving endothelial function

Abstract

Drugs currently approved to coat stents used in percutaneous coronary interventions do not discriminate between proliferating vascular smooth muscle cells (VSMCs) and endothelial cells (ECs). This lack of discrimination delays reendothelialization and vascular healing, increasing the risk of late thrombosis following angioplasty. We developed a microRNA-based (miRNA-based) approach to inhibit proliferative VSMCs, thus preventing restenosis, while selectively promoting reendothelialization and preserving EC function. We used an adenoviral (Ad) vector that encodes cyclin-dependent kinase inhibitor p27Kip1 (p27) with target sequences for EC-specific miR-126-3p at the 3′ end (Ad-p27-126TS). Exogenous p27 overexpression was evaluated in vitro and in a rat arterial balloon injury model following transduction with Ad-p27-126TS, Ad-p27 (without miR-126 target sequences), or Ad-GFP (control). In vitro, Ad-p27-126TS protected the ability of ECs to proliferate, migrate, and form networks. At 2 and 4 weeks after injury, Ad-p27-126TS–treated animals exhibited reduced restenosis, complete reendothelialization, reduced hypercoagulability, and restoration of the vasodilatory response to acetylcholine to levels comparable to those in uninjured vessels. By incorporating miR-126-3p target sequences to leverage endogenous EC-specific miR-126, we overexpressed exogenous p27 in VSMCs, while selectively inhibiting p27 overexpression in ECs. Our proof-of-principle study demonstrates the potential of using a miRNA-based strategy as a therapeutic approach to specifically inhibit vascular restenosis while preserving EC function.

Authors

Gaetano Santulli, Anetta Wronska, Kunihiro Uryu, Thomas G. Diacovo, Melanie Gao, Steven O. Marx, Jan Kitajewski, Jamie M. Chilton, Kemal Marc Akat, Thomas Tuschl, Andrew R. Marks, Hana Totary-Jain

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Figure 2

Ad-p27-126TS inhibits VSMC migration while preserving EC properties in vitro.

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Ad-p27-126TS inhibits VSMC migration while preserving EC properties in v...
Migration assays of VSMCs (A) and ECs (B) transduced with the indicated Ad vector. Representative images at the indicated time points are shown. Wound areas were measured using ImageJ64 software. (C) Formation of network-like structures in ECs was quantified by counting branch points. Scale bars: 100 μm (A–C). Data represent the means ± SEM of at least 3 experiments performed in quadruplicate. (D) Representative photomicrographs of platelet adhesion to ECs transduced with the indicated Ad vector in a parallel-plate flow chamber apparatus before (upper panels, t = 0) and after perfusion of PPACK-treated blood and washing (lower panels, t = 3 minutes). Yellow arrows represent human platelets. Scale bars: 30 μm. Graph on the right represents a quantification of the platelet adhesion test performed using Fiji software. Data represent the means ± SEM of at least 3 experiments performed in triplicate. Data comparisons were made using 1-way ANOVA with Tukey-Kramer’s post hoc test. *P < 0.01 versus Ad-GFP.