Effects of in vivo adventitial expression of recombinant endothelial nitric oxide synthase gene in cerebral arteries

Abstract

Nitric oxide (NO), synthesized from Larginine by NO synthases (NOS), plays an essential role in the regulation of cerebrovascular tone. Adenoviral vectors have been widely used to transfer recombinant genes to different vascular beds. To determine whether the recombinant endothelial NOS (eNOS) gene can be delivered in vivo to the adventitia of cerebral arteries and functionally expressed, a replication-incompetent adenoviral vector encoding eNOS gene (AdCMVNOS) or ␤-galactosidase reporter gene (AdCMVLacZ) was injected into canine cerebrospinal f luid (CSF) via the cisterna magna (final viral titer in CSF, 10 9 pfu͞ml). Adventitial transgene expression was demonstrated 24 h later by ␤-galactosidase histochemistry and quantification, eNOS immunohistochemistry, and Western blot analysis of recombinant eNOS. Electron microscopy immunogold labeling indicated that recombinant eNOS protein was expressed in adventitial fibroblasts. In AdCMVNOS-transduced arteries, basal cGMP production and bradykinin-induced relaxations were significantly augmented when compared with AdCMVLacZ-transduced vessels (P < 0.05). The increased receptormediated relaxations and cGMP production were inhibited by eNOS inhibitors. In addition, the increase in cGMP production was reversed in the absence of calcium, suggesting that the increased NO production did not result from inducible NOS expression. The present study demonstrates the successful in vivo transfer and functional expression of recombinant eNOS gene in large cerebral arteries. It also suggests that perivascular eNOS gene delivery via the CSF is a feasible approach that does not require interruption of cerebral blood f low.

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.…”