Optimization of ex vivo inducible nitric oxide synthase gene transfer to vein grafts

Background. Vein graft failure as the result of intimal hyperplasia (IH) re mains a significant clinical problem. Ex vivo modification of vein grafts u sing gene therapy is all attractive approach to attenuate IH. Gene transfer of the inducible nitric oxide synthase (iNOS) gene effectively reduces IH. However, iNOS activity after gene transfer may be impaired by the availabi lity of cofactor, such as tetrahydrobiopterin (BH4). The purpose of this st udy is to determine the optimal conditions for ex vivo adenoviral-mediated iNOS gene transfer into arterial and venous vessels. Methods. Porcine internal jugular veins and carotid arteries were infected ex vivo with the adenoviral iNOS vector (AdiNOS) and with an adenovirus car rying the cDNA encoding guanosine triphosphate cyclohydrolase I (AdGTPCH), the rate-limiting enzyme for BH4 synthesis. The production of nitrite, cycl ic guanosine monophosphate (cGMP), and biopterin were assessed daily. Results. Nitric oxide (NO) production after iNOS gene transfer was maximal when vessels were cotransduced with AdGTPCH. NO production in these vessels persisted for more than 10 days. Vein segments generated approximately 2-f old more nitrite, cGMP, and biopterin than arterial segments infected with AdiNOS/AdGTPCH. Submerging vein segments into adenoviral solution resulted in improved gene transfer with greater nitrite and cGMP release compared wi th infections carried out under pressure intraluminally. Similarly, injury to the vein segments before infection with AdiNOS resulted in less nitrite production. Conclusions. These data demonstrate that AdiNOS can efficiently transduce v ein segments ex vivo and that the cotransfer of GTPCH can optimize iNOS enz ymatic activity. This cotransfer technique may be used to engineer vein gra fts before coronary artery bypass to prevent IH.