Administration of adenoviral vectors induces gangrene in acutely ischemic rat hindlimbs: Role of capsid protein-induced inflammation

Purpose. The initial purpose of this study was to determine the effects of intravascular adenoviral vector-mediated gene transfer of endothelial nitri c oxide synthase (AdeNOS) on experimental hindlimb ischemia in the rat. Une xpectedly, administration of AdeNOS immediately after induction of acute li mb ischemia led to limb gangrene. We subsequently sought to define the mole cular mechanisms responsible for this unusual effect and to devise adenovir al gene transfer strategies to prevent the development of gangrene in acute ly ischemic limbs. Methods: phosphate-buffered saline or adenoviral vectors containing the bov ine endothelial nitric oxide synthase gene (AdeNOS) or no transgene (Ad-EI) were injected intra-arterially into the hindlimb of a rat under vascular i solation immediately after surgical induction of severe ischemia. Hematoxyl in and eosin staining was performed on muscle sections to evaluate inflamma tion. A separate group of animals was injected with an adenovirus containin g a nontranscribable genome, treated with cyclosporine, or received delayed administration of the adenoviral vector. Gene expression after delayed ade noviral gene transfer was assessed with immunohistochemistry, Western blott ing, and nitric oxide synthase (NOS) activity assay. Results. Both AdeNOS and Ad-E1 caused gangrene of the entire hindlimb withi n 12 days in a dose-dependent manner, at a threshold concentration of 1 x 1 0(9) plaque-forming unit/mL. Adenoviral delivery was associated with more i nflammation and edema compared with phosphate-buffered saline histologicall y. Inactivation of adenoviral DNA transcription did not affect induction of gangrene. However, gangrene was prevented by concurrent immunosuppression with cyclosporine or delayed administration of the vector. Delayed administ ration allowed adenoviral gene expression as determined by immunohistochemi stry, NOS protein levels, and an assay of NOS enzyme activity. Conclusion: Intra-arterial administration of adenoviral vectors, under vasc ular isolation, immediately after induction of acute ischemia causes inflam mation and subsequent limb gangrene. The inflammatory response is unrelated to the expression of the recombinant transgene or the adenoviral genome an d is likely due to the adenoviral capsid proteins. However, administration of cyclosporine or delayed injection of die adenoviral vector is a method t hat can be used for adenoviral mediated gene transfer in limb ischemia.