Division of Cardiology, The Children's Hospital of Philadelphia, Abramson Research Center, PA, USA.
Atherosclerosis. 2013 Sep;230(1):23-32. doi: 10.1016/j.atherosclerosis.2013.06.002. Epub 2013 Jun 29.
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.
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.
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.
用编码 NOS 酶的病毒载体进行基因治疗已被认为是预防再狭窄的一种潜在治疗方法。iNOS 的最佳活性取决于细胞内 L-Arg 和 BH4 的可用性，这可通过防止 NOS 解偶联和随后的 ROS 形成来实现。在此，我们研究了分别和联合补充 L-Arg 和 BH4 对转染 AdiNOS 的培养大鼠动脉平滑肌和内皮细胞中 NO 和 ROS 产生的影响，及其对 AdiNOS 递送至球囊损伤大鼠颈动脉的抗再狭窄效果的影响。
用 L-Arg（3.0mM）、BH4（10μM）和特别是它们的组合补充转染了 AdiNOS 的内皮和血管平滑肌细胞，导致培养基中硝酸盐形成所测量的 NO 产量显著增加。随着转染 AdiNOS 的 MOI 增加，ROS 的形成呈剂量依赖性增加。通过使用可水解交联剂将 AdiNOS 固定在网格上，模拟病毒从支架递送至血管壁，从而使 RASMC 暴露于 AdiNOS 中，导致 ROS 产生增加，而 BH4 但不是 L-Arg 或 L-Arg/BH4 补充可降低 ROS 产生。AdiNOS 转导引起的细胞死亡也可以通过 BH4 补充来预防。在球囊损伤的大鼠颈动脉模型中，在 BH4-、L-Arg-、特别是 BH4 和 L-Arg 补充的动物中，经腔内递送 AdiNOS 被发现可显著增强 AdiNOS 介导的基因治疗预防再狭窄的效果。
在转导的血管中通过 L-Arg 和 BH4 补充来微调 iNOS 功能，可增强 iNOS 基因治疗预防再狭窄的治疗潜力。
