Modulation of NO and ROS production by AdiNOS transduced vascular cells through supplementation with L-Arg and BH4: Implications for gene therapy of restenosis

Objective: Gene therapy with viral vectors encoding for NOS enzymes has been recognized as a potential therapeutic approach for the prevention of restenosis. Optimal activity of iNOS is dependent on the intracellular availability of L-Arg and BH4 via prevention of NOS decoupling and subsequent ROS formation. Herein, we investigated the effects of separate and combined L-Arg and BH4 supplementation on the production of NO and ROS in cultured rat arterial smooth muscle and endothelial cells transduced with Ad(iNOS), and their impact on the antirestenotic effectiveness of Ad(iNOS) delivery to balloon-injured rat carotid arteries. Methods and results: Supplementation of Ad(iNOS) transduced endothelial and vascular smooth muscle cells with L-Arg (3.0 mM), BH4 (10 mM) and especially their combination resulted in a significant increase in NO production as measured by nitrite formation in media. Formation of ROS was dose-dependently increased following transduction with increasing MOIs of Ad(iNOS). Exposure of RASMC to Ad(iNOS) tethered to meshes via a hydrolyzable cross-linker, modeling viral delivery from stents, resulted in increased ROS production, which was decreased by supplementation with BH4 but not L-Arg or L-Arg/BH4. Enhanced cell death, caused by Ad(iNOS) transduction, was also preventable with BH4 supplementation. In the rat carotid model of balloon injury, intraluminal delivery of Ad(iNOS) in BH4-, L-Arg-, and especially in BH4 and L-Arg supplemented animals was found to significantly enhance the antirestenotic effects of Ad(iNOS)-mediated gene therapy. Conclusions: Fine-tuning of iNOS function by L-Arg and BH4 supplementation in the transduced vasculature augments the therapeutic potential of gene therapy with iNOS for the prevention of restenosis. (C) 2013 Elsevier Ireland Ltd. All rights reserved.