Administration of adenoviral vectors induces gangrene in acutely ischemic rat hindlimbs: role of capsid protein-induced inflammation.
OBJECTIVE
The initial purpose of this study was to determine the effects of intravascular adenoviral vector-mediated gene transfer of endothelial nitric oxide synthase (AdeNOS) on experimental hindlimb ischemia in the rat. Unexpectedly, administration of AdeNOS immediately after induction of acute limb ischemia led to limb gangrene. We subsequently sought to define the molecular mechanisms responsible for this unusual effect and to devise adenoviral gene transfer strategies to prevent the development of gangrene in acutely ischemic limbs.


METHODS
Phosphate-buffered saline or adenoviral vectors containing the bovine endothelial nitric oxide synthase gene (AdeNOS) or no transgene (Ad-E1) were injected intra-arterially into the hindlimb of a rat under vascular isolation immediately after surgical induction of severe ischemia. Hematoxylin and eosin staining was performed on muscle sections to evaluate inflammation. A separate group of animals was injected with an adenovirus containing a nontranscribable genome, treated with cyclosporine, or received delayed administration of the adenoviral vector. Gene expression after delayed adenoviral gene transfer was assessed with immunohistochemistry, Western blotting, and nitric oxide synthase (NOS) activity assay.


RESULTS
Both AdeNOS and Ad-E1 caused gangrene of the entire hindlimb within 12 days in a dose-dependent manner, at a threshold concentration of 1 x 10(9) plaque-forming unit/mL. Adenoviral delivery was associated with more inflammation and edema compared with phosphate-buffered saline histologically. 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 administration allowed adenoviral gene expression as determined by immunohistochemistry, NOS protein levels, and an assay of NOS enzyme activity.


CONCLUSIONS
Intra-arterial administration of adenoviral vectors, under vascular isolation, immediately after induction of acute ischemia causes inflammation and subsequent limb gangrene. The inflammatory response is unrelated to the expression of the recombinant transgene or the adenoviral genome and is likely due to the adenoviral capsid proteins. However, administration of cyclosporine or delayed injection of the adenoviral vector is a method that can be used for adenoviral mediated gene transfer in limb ischemia.
Brevetti
L S
LS
Department of Surgery, Division of Vascular Surgery, University of California, San Francisco 94143-0222, USA.
Sarkar
R
R
Chang
D S
DS
Ma
M
M
Paek
R
R
Messina
L M
LM
D000208:Acute Disease; D000256:Adenoviridae; D000818:Animals; D002213:Capsid / Q000276:immunology*; D005734:Gangrene; D005822:Genetic Vectors; D006614:Hindlimb / Q000098:blood supply* / Q000473:pathology*; D007249:Inflammation / Q000821:virology*; D007511:Ischemia / Q000150:complications*; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley