Protective Vasomotor Effects of In Vivo Recombinant Endothelial Nitric Oxide Synthase Gene Expression in a Canine Model of Cerebral Vasospasm

Abstract

Background and Purpose-Post-subarachnoid hemorrhage (SAH) cerebral vasospasm is a potentially devastating condition whose pathogenesis involves impaired nitric oxide (NO) bioavailability. We aimed to determine whether recombinant endothelial NO synthase (eNOS) gene expression may protect vasomotor function and prevent vasospasm in a canine experimental SAH model. Methods-Recombinant adenoviral vectors (5ϫ10 9 plaque-forming units/animal) encoding genes for eNOS (AdeNOS) and ␤-galactosidase (AdLacZ) or vehicle were injected into the cerebrospinal fluid (CSF) of dogs on day Ϫ1 (ie, 24 hours before the first intra-CSF injection of blood on day 0). Cerebral angiography was performed at day 0 (baseline) and day 7 (immediately before death), and tissues were harvested for additional studies. Results-Western analysis and immunohistochemistry detected recombinant eNOS exclusively in cerebral arteries isolated from AdeNOS-transduced dogs, and in this group of animals CSF NO concentrations were significantly elevated by day 2. Analysis of day 7 versus day 0 cerebral angiograms for each group revealed significant spasm at the basilar artery midpoint in AdLacZ-transduced and nontransduced dogs but not in AdeNOS-transduced dogs. Isometric force recording of basilar arteries isolated from AdeNOS-transduced dogs showed significantly augmented relaxations to bradykinin and reduced contractions to endothelin-1. Conclusions-Our

A direct mechanical method for accurate and efficient adenoviral vector delivery to tissues

“…An animal model involving intact carotid artery was chosen as the focus for this study in the context of our ability to determine the effects of mechanical vector delivery in this tissue preparation morphologically, biochemically, and functionally, and because of our previous experience with vascular gene transfer in dogs 3,4,13,22,31 and rabbits. 19,20 Ex vivo experiments were carried out using arteries harvested from mongrel dogs.…”

“…Experimental studies have demonstrated successful transfer of a variety of recombinant genes in the cardiovascular system both ex vivo and in vivo in animals and humans [1][2][3][4][5][6][7][8][9][10][11][12][13] suggesting that gene transfer technology may, in future, offer an effective therapeutic option for the spectrum of human cardiovascular diseases. [14][15][16][17][18] Most vascular gene transfer studies, to date, have utilized replication-incompetent adenovirus as a delivery vehicle for recombinant cDNA.…”

“…Ex vivo delivery of this vector has been carried out by immersion of arterial tissue in a chamber containing vector-solution, 3,5,11 while in vivo delivery has been carried out through the lumen of an artery, that is, by direct injection, or via its outer ''adventitial'' layer, that is, perivascularly. 2,4,7,8,10,13,[19][20][21] A number of groups have demonstrated the ability of perivascular (adventitial) gene delivery to favorably and profoundly modify vasomotor function in a variety of cardiovascular models [3][4][5][11][12][13]19,20,22 and this approach, which has been reported to minimize the proinflammatory effects of adenoviral vectors, 10 can be used as an effective adjunct to the deployment of intravascular gene delivery stents and catheters. In vivo, current vascular gene delivery techniques have limited ability to produce specific targeting and selective localization of vector in a desired arterial territory or individual vessel.…”

“…While this method was shown to be helpful in similar in vivo gene transfer studies carried out in a largeanimal model, 4,13 vector delivery was still relatively indirect and nonfocal. In a more direct method of mechanical delivery to the carotid artery described by Kullo et al, 19 sequestration of vector around the vascular adventitia was carried out to facilitate more focal gene transfer.…”

“…10,19 Several groups have used viral titers of 10 9 -10 10 plaque forming units (PFU) per ''target'' (ie, vessel segment of vascular territory within a living host) to facilitate effective expression of recombinant proteins in the vessel wall. [2][3][4][5][9][10][11][12][13]19,20,23,32 To explore the possibility of effective mechanical delivery of adenoviral vector to tissues, we postulated that a small paintbrush could be used to rapidly, efficiently, and accurately apply an adenoviral vector directly to a desired tissue target in vivo. This work suggests that our mechanical vector delivery method facilitates enhanced concentration and localization of vector at the tissue surface to which it was directly applied, enabling reduction of vector incubation time and titer in vivo.…”