title: Modulation of NO and ROS production by AdiNOS transduced vascular cells through supplementation with L-Arg and BH4: implications for gene therapy of restenosis.
abstract: 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 AdiNOS, and their impact on the antirestenotic effectiveness of AdiNOS delivery to balloon-injured rat carotid arteries.
  
  
  RESULTS
  Supplementation of AdiNOS transduced endothelial and vascular smooth muscle cells with L-Arg (3.0 mM), BH4 (10 μM) 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 AdiNOS. Exposure of RASMC to AdiNOS 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 AdiNOS transduction, was also preventable with BH4 supplementation. In the rat carotid model of balloon injury, intraluminal delivery of AdiNOS in BH4-, L-Arg-, and especially in BH4 and L-Arg supplemented animals was found to significantly enhance the antirestenotic effects of AdiNOS-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.
authors: Division of Cardiology, The Children's Hospital of Philadelphia, Abramson Research Center, PA, USA.
authors: Scott P
authors: SP
authors: Forbes
authors: Ivan S
authors: IS
authors: Alferiev
authors: Michael
authors: M
authors: Chorny
authors: Richard F
authors: RF
authors: Adamo
authors: Robert J
authors: RJ
authors: Levy
authors: Ilia
authors: I
authors: Fishbein
mesh_terms: D000256:Adenoviridae / Q000378:metabolism; D000818:Animals; D001011:Aorta / Q000378:metabolism; D001120:Arginine / Q000737:chemistry; D001708:Biopterins / Q000031:analogs & derivatives / Q000737:chemistry; D002339:Carotid Arteries / Q000378:metabolism; D049109:Cell Proliferation; D002470:Cell Survival; D023903:Coronary Restenosis / Q000628:therapy*; D003432:Cross-Linking Reagents / Q000737:chemistry; D004195:Disease Models, Animal; D015316:Genetic Therapy; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D032389:Myocytes, Smooth Muscle / Q000166:cytology; D019331:NG-Nitroarginine Methyl Ester / Q000737:chemistry; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism*
chemical_list: D003432:Cross-Linking Reagents; D017382:Reactive Oxygen Species; D001708:Biopterins; D009569:Nitric Oxide; D001120:Arginine; D052247:Nitric Oxide Synthase Type II; C003402:sapropterin; D019331:NG-Nitroarginine Methyl Ester
keywords: Gene therapy; Inducible nitric oxide synthase; L-Arg; Nitric oxide; Restenosis; Smooth muscle cells; Tetrahydrobiopterin
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