Abstract-Acute injury after adenoviral vascular gene transfer remains incompletely characterized. Here, we describe the early response (Յdays) in 52 New Zealand White rabbits undergoing gene transfer (␤-galactosidase or empty vector) or sham procedures to both carotid arteries. After gene transfer, arteries were either left in vivo for 1 hour to 3 days (in vivo arteries) or were excised immediately after gene transfer and cultured (ex vivo arteries). Within 1 hour, in vivo arteries receiving infectious titers of Ն4ϫ10 9 plaque-forming units (pfu)/mL showed endothelial activation, with an acute inflammatory infiltrate developing by 6 hours. Ex vivo arteries showed endothelial activation but no inflammatory infiltrate. There were also significant differences in transgene expression between in vivo and ex vivo arteries. Ex vivo arteries showed titer-dependent increases in ␤-galactosidase expression through 2ϫ10 10 pfu/mL, whereas in in vivo arteries, titers above 4ϫ10 9 pfu/mL merely increased acute inflammatory response, without increasing transgene expression. In vivo arteries showed significant time-and titer-dependent impairment in endothelium-dependent relaxation, with no effect on contraction or nitroprusside-induced relaxation. Interestingly, however, if rabbits were made neutropenic with vinblastine, their arteries maintained full endothelium-dependent relaxation, even after very high titer vascular infection (up to 1ϫ10 11 pfu/mL). These findings show that recombinant adenovirus triggers an early inflammatory response, and it is the inflammatory response that in turn causes functional endothelial injury. This occurs at much lower titers than previously appreciated (though the precise threshold will undoubtedly vary between laboratories). However, titers below the inflammatory threshold produce excellent transgene expression without inflammation or vascular injury. (Circ Res. 1998;82:1253-1262.)

Background-Hypercholesterolemia reduces nitric oxide bioavailability, manifested by reduced endothelium-dependent vascular relaxation, and also induces vascular adhesion molecule expression and inflammatory cell infiltration. We have previously shown that gene therapy with NO synthase in hypercholesterolemic rabbits substantially reverses the deficit in vascular relaxation. In the present study, we show that NO synthase gene therapy rapidly and substantially reduces vascular adhesion molecule expression, lipid deposition, and inflammatory cell infiltration. Methods and Results-Thirty male New Zealand White rabbits were maintained on a 1% cholesterol diet for 11 to 13 weeks, then underwent carotid artery gene transfer with Ad.nNOS or Ad.␤Gal (recombinant adenoviruses expressing neuronal NO synthase or ␤-galactosidase, respectively), or received medium alone in a sham procedure. Arteries were harvested at 1 and 3 days after gene transfer, and the following parameters were determined by immunohistochemical and image-analysis techniques: intercellular adhesion molecule-1, vascular cell adhesion molecule-1, lipid deposition by oil red O staining, lymphocyte infiltration (CD43-positive cells), and monocyte infiltration (RAM-11-positive cells). In Ad.nNOS-treated arteries, all markers were significantly decreased relative to Ad.␤Gal or sham-treated arteries within 3 days after gene transfer. Ad.nNOS had a particularly striking impact on monocyte infiltration; as early as 24 hours after gene transfer, Ad.nNOS-treated arteries had Ͼ3-fold fewer monocytes than Ad.␤Gal-or sham-treated arteries. Conclusions-NO synthase gene therapy rapidly ameliorates several markers of atherosclerosis in the cholesterol-fed rabbit. (Circulation. 1999;99:2979-2982.)

Abstract-Gene therapy aims to intervene in a disease process by transfer and expression of specific genes in a target tissue or organ. Cardiovascular gene therapy in humans remains in its infancy, but in the last decade, experimental gene transfer has emerged as a powerful biological tool to investigate the function of specific genes in vascular disease pathobiology. Nitric oxide synthases, the enzymes that produce nitric oxide, have received considerable attention as potential candidates for vascular gene therapy because nitric oxide has pleiotropic antiatherogenic actions in the vessel wall, and abnormalities in nitric oxide biology are apparent very early in the atherogenic process. In this article, we review the use of nitric oxide synthases in experimental vascular gene therapy and assess the utility of these approaches for investigating the role of nitric oxide in atherosclerosis and their potential for human gene therapy. NO and Atherogenesis NO SynthasesNitric oxide (NO) is produced by NO synthases (NOs), which oxidize L-arginine to L-citrulline (reviewed in References 1 and 2). All 3 NOS isoforms have a similar molecular structure and require multiple cofactors, including flavins, NADPH, and tetrahydrobiopterin, that are required to maintain dimerization and NO production 1,2 (the Figure). Neuronal (nNOS, or NOS I) and endothelial (eNOS, or NOS III) isoforms are constitutively expressed and are activated by calcium-calmodulin. The inducible isoforms (iNOS, or NOS II) are regulated primarily at the transcriptional level, independent of agonist stimulation and intracellular calcium levels. 3,4 eNOS, expressed in endothelial cells, is the predominant NOS isoform in the vessel wall. Under basal conditions, eNOS is inactive and remains membrane bound by virtue of myristoylation, palmitoylation, and an inhibitory interaction with caveolin, the principal structural protein in caveolae. Receptor-mediated agonist stimulation (eg, bradykinin, substance P) leads to rapid enzyme activation by depalmitoylation, binding of calcium-calmodulin, displacement of caveolin, and release from the plasma membrane. 5,6 Shear stress is an important physiological stimulator of eNOS activity, causing rapid membrane release and activation by Aktdependent serine phosphorylation 7,8 and upregulating eNOS gene expression by transcriptional activation of the eNOS promoter. 9 After vessel injury or in disease states, iNOS expression may be induced in the media, 10 atherosclerotic plaque, 11 or neointima. 4,12 In normal blood vessels, nNOS is present in neurons in the adventitia and may be expressed by medial smooth muscle cells (SMCs) under pathophysiological conditions. Recent data also suggest that nNOS and its splice variant NOS may be expressed at low levels in the media and adventitia. 13 Functions of Vascular NONO produced in the endothelium rapidly diffuses to interact with molecular targets in cells in the vascular wall and lumen. 14 NO interacts with thiol groups and with metal centers in diverse protein targets, including me...

Background-The vascular endothelium is anatomically intact but functionally abnormal in preatherosclerotic states, and an early deficit in the bioavailability of nitric oxide (NO) or related molecules has been described in both humans and animal models. We hypothesized that the targeted gene transfer of NO synthase (NOS) isoforms might ameliorate or reverse the deficit. Methods and Results-We constructed a recombinant adenovirus, Ad.nNOS, that expresses the neuronal isoform of NOS (nNOS) and used it for in vivo endovascular gene transfer to carotid arteries (CA) from normal and cholesterol-fed rabbits. Vessels were harvested 3 days after gene transfer. In CA from normal rabbits, Ad.nNOS generated high levels of functional nNOS protein predominantly in endothelial cells and increased vascular NOS activity by 3.4-fold relative to sham-infected control CA. Ad.nNOS gene transfer also significantly enhanced endothelium-dependent vascular relaxation to acetylcholine; at 3 mol/L acetylcholine, Ad.nNOS-treated arteries showed an 86Ϯ4% reduction in precontracted tension, whereas control CA showed a 47Ϯ6% reduction in tension. Contraction in response to phenylephrine and relaxation in response to nitroprusside were unaffected in both control and Ad.nNOS-treated CA. To determine the effect of Ad.nNOS in atherosclerotic arteries, 10 male New Zealand White rabbits maintained on a 1% cholesterol diet for 10 to 12 weeks underwent gene transfer according to the same protocol used in normal rabbits. Ad.nNOS-treated arteries showed a 2-fold increase in NADPH-diaphorase staining intensity relative to sham-infected and Ad.␤Gal-treated arteries. The CA from cholesterol-fed rabbits showed impaired acetylcholine-induced relaxation, but this abnormality was almost entirely corrected by Ad.nNOS gene transfer. Conclusions-In vivo adenovirus-mediated endovascular delivery of nNOS markedly enhances vascular NOS activity and can favorably influence endothelial physiology in the intact and atherosclerotic vessel wall.