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Cardiovascular Research 1997 35(3):505-513; doi:10.1016/S0008-6363(97)00098-9
© 1997 by European Society of Cardiology
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Copyright © 1997, European Society of Cardiology

Efficient adenoviral gene transfer to early venous bypass grafts: comparison with native vessels

Keith M Channona, Gregory J Fultonb, John L Grayb, Brian H Annexa, Geetha A Shettya, Michael A Blazinga, Kevin G Petersa, Per-Otto Hagenb and Samuel E Georgea,*

aDepartment of Medicine, Division of Cardiology, Duke University Medical Center, Box 3060, Durham, NC 27710 USA
bDepartment of Surgery, Duke University Medical Center, Durham, NC 27710 USA

* Corresponding author. Tel: +1 (919) 681 8446; fax: +1 (919) 684 8591; e-mail: samuel.george@duke.edu

Received 7 June 1996; accepted 20 January 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
 References
 
Objectives: Gene therapy may provide new approaches to reduce vein graft failure following coronary or peripheral bypass surgery. The aim of this study was to investigate the relative efficacy of intraoperative adenoviral gene transfer to vein grafts, comparing transgene expression in vein grafts with that in matched native vessels in the same animal. In addition, we assessed the impact of bypass grafting on the cellular targets of gene transfer. Methods: New Zealand White rabbits underwent interposition bypass grafting of the carotid artery, using the ipsilateral external jugular vein, which was infected with an adenovirus expressing β-galactosidase immediately prior to bypass grafting (n = 16). The contralateral native jugular vein (n = 16) and carotid artery (n = 8) were infected concurrently with the same adenoviral preparation. After 3, 7 or 14 days, β-galactosidase protein expression was quantified by ELISA, and specific cell types expressing β-galactosidase were identified by X-Gal staining and by immunohistochemistry. Results: After 3 days, endothelial cells were efficiently transduced in all vessels; medial smooth muscle cells were transduced infrequently. In contrast to jugular veins after gene transfer, endothelium in vein grafts showed expression of VCAM-1 and ICAM-1, and intense inflammation with CD18+ leukocytes. Transgene expression in vein grafts at day 3 was maintained at levels approximately 50% of that in ungrafted jugular veins, but continued to decrease through day 7. Conclusions: Although vascular injury in early venous bypass grafts reduces gene transfer efficacy, significant transgene expression is maintained for at least 7 days. These findings have important implications for intraoperative gene transfer strategies in vein grafts.

KEYWORDS Gene transfer; Adenovirus; Bypass grafts; Endothelium; Smooth muscle; Rabbit, endothelial cells


   1 Introduction
Top
 Abstract
 1 Introduction
2 Materials and methods
 3 Results
 4 Discussion
 References
 
Vein grafts remain the most frequently used conduit for peripheral and aorotocoronary bypass grafting (CABG). Although CABG improves both survival and anginal symptoms in patients with severe coronary artery disease, the problem of vein graft failure continues to limit its long-term success. Vein grafts fail due to early thrombosis, or due to intimal hyperplasia, which develops as a consequence of early vein graft injury [1–3]. More than 50% of coronary venous bypass grafts fail within 10 years of implantation, making vein graft failure the leading indication for repeat CABG [4, 5]. Medical therapies such as antiplatelet agents and aggressive risk factor modification have had limited impact in reducing vein graft failure, so new therapeutic approaches remain of great clinical and economic importance. Intraoperative gene therapy may provide novel strategies to limit vein graft failure, by inhibiting the early molecular events in these pathological processes.

Gene transfer approaches using adenovirus have shown great promise for gene therapy of vascular disease [6–8]. Adenoviral gene transfer of marker genes has been extensively evaluated in normal and injured arteries, in atherosclerotic vessels, and in the setting of balloon angioplasty [9]. In addition, the use of therapeutic transgenes has been effective in inhibiting intimal proliferation after balloon injury [10–12]. Venous bypass grafts are readily available for ex-vivo genetic modification during bypass surgery, without the need for percutaneous delivery devices which are necessary for delivering genetic material during arterial interventions [13].

Although a number of studies address the molecular and cellular events that accompany arterial gene transfer [14–17], comparatively little is known about gene transfer to vein grafts [18]. Importantly, the characteristics of arterial gene transfer may not apply to vein grafts. First, the structural and functional differences between veins and arteries may have significant effects on gene transfer efficacy [19, 20]. Second, in arterial studies, vessel injury typically occurs before gene transfer, whereas in vein grafts profound biologic changes occur after implantation of the vein into the arterial circulation [1, 21]. Arterial pressure and flow result in endothelial damage, fibrin and platelet deposition, and infiltration by inflammatory cells. The extent to which these factors influence the efficacy of intraoperative gene transfer to vein grafts is unknown, but is an important issue in determining the practical utility of gene therapy approaches in vein grafts.

In this study, we characterize the efficacy, cellular targets and time course of adenoviral gene transfer to venous bypass grafts in a well-defined rabbit jugular vein interposition bypass graft model [21, 22]. These jugular vein grafts undergo profound changes in the first 3 days, with prominent evidence of endothelial injury and extensive transmural inflammation. This early injury subsequently results in marked intimal hyperplasia [9, 21, 22]. These and other changes are similar to those seen in human saphenous vein bypass grafts. We compare transgene expression in jugular vein grafts with transgene expression in the paired contralateral carotid artery and jugular vein. We show that transgene expression is well maintained in vein grafts relative to native vessels at 3 days, but thereafter decreases more rapidly than in native vessels as a result of the vascular injury and remodeling that occurs after bypass grafting.


   2 Materials and methods
Top
 Abstract
 1 Introduction
 2 Materials and methods
3 Results
 4 Discussion
 References
 
2.1 Animals
Male New Zealand white rabbits (2–2.5 kg) were maintained on a normal diet of rabbit chow with water ad libitum. Anesthesia for surgery was induced using ketamine (Ketastat, 60 mg·kg–1, Bristol Laboratories, Syracuse, NY) and Xylazine (Anased, 6 mg·kg–1, Lloyd Laboratories, Shenandoah, IA) subcutaneously. All animal care and procedures complied with the Principles of Laboratory Animal Care as formulated by the National Society for Medical Research and with the Guide for the Care and Use of Laboratory Animals issued by the National Institutes of Health (NIH publication 80-23, revised 1985).

2.2 Preparation of adenovirus vector
We generated an adenovirus vector, Ad.Pac β Gal, derived from the in340 mutant strain of adenovirus type 5 [23], as previously described [24]. Ad.Pac β Gal contains a nuclear localizing β-galactosidase cDNA in the E1 cloning site with a CMV early enhancer-promoter and an SV40 VP 2 polyadenylation signal. This vector directs high-level transgene expression in cultured vascular smooth muscle and endothelial cells [24]. High-titer purified stock of Ad.Pac β Gal was generated by infecting 150 mm plates of confluent 293 cells at an multiplicity of infection of 1 in Dulbecco's modified Eagle medium (DMEM) containing 2% fetal bovine serum (FBS). After observation of a cytopathic effect, cells were harvested and the virus was purified on a cesium chloride gradient using modifications of existing methods [25]. Briefly, infected 293 cells were washed from the plates, pelleted by centrifugation at 500 g and lysed by sonication in virus storage buffer (VSB; 20 mM Tris pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2). Cell debris was pelleted and the supernatant was adjusted to a density of 1.1 g·ml–1 by addition of solid cesium chloride. This solution was layered onto 1.3 g·ml–1–1.4 g·ml–1 cesium chloride step gradient and ultracentrifuged at 100 000 g for 2.5 h at 4°C. The visible band of pure virus was harvested and desalted by serial gel filtration on sepharose CL-6B spin columns (Pharmacia, Uppsala, Sweden) in VSB. Gel-filtered virus was diluted 1:1 with 80% normal rabbit serum/20% glycerol and immediately frozen in aliquots. The viral concentration was initially estimated spectrophotometrically by absorbance at 260 nm and infectious titer of all viral stocks was determined by plaque assay on 293 cells using standard techniques [25].

2.3 Venous bypass graft surgery and gene transfer
Male New Zealand white rabbits (2–2.5 kg) underwent right carotid artery interposition bypass graft surgery using the ipsilateral external jugular vein, as previously described [26]. Gene transfer was carried out, in the same animal, to: (1) the right external jugular vein, which was then implanted as a vein graft; (2) the left external jugular vein; and, (3) in some animals, the left carotid artery. This approach provided within-animal controls of gene transfer efficacy in native vessels, and assured similar conditions for each vessel. High-titer viral stock, maintained on dry ice until immediately before use, was thawed and diluted to a titer of approximately 1x109 pfu·ml–1 with serum-free DMEM. Preliminary experiments using this concentration of virus generated high-level β-galactosidase expression in both cultured rabbit vascular smooth muscle cells and in ex-vivo vessel segments. At surgery, vessels were exposed through a midline neck incision. Heparin (700 IU i.v.) was administered and gene transfer was performed by isolating the exposed vessel segment between microvascular clamps. A 20-gauge teflon cannula was inserted into the vessel either through an arteriotomy or through a transected venous side branch. The lumen of the vessel was cleared of blood by gentle flushing with DMEM. Approximately 200 µl of virus solution was instilled into the vessel lumen and gentle hydrostatic pressure was applied to distend the vessel to its physiologic dimensions. Incubation was continued for 30 min after which the arteriotomy was repaired using 10/0 nylon sutures or the venous side branch was ligated. Flow was restored in the native left carotid artery or left jugular vein. The right jugular vein was tied off, resected, reversed and used as an interposition venous bypass graft across the right carotid artery, as described [26].

2.4 Vessel harvesting and analysis
Vessels were harvested 3, 7 or 14 days after surgery. Animals were anesthetized and the native carotid artery, jugular vein and the venous bypass graft were dissected free through a midline neck incision. Animals were heparinized (700 IU i.v.) and sacrificed with an intravenous overdose of pentobarbital sodium (100 mg·kg–1, Anthony Products, Arcadia, CA). Immediately after sacrifice, vessels were rapidly harvested and washed in phosphate-buffered saline (PBS). A segment of aorta was also harvested from animals sacrificed at 3 days, to provide an uninfected control vessel. The length of all vessels was measured and wet weight was determined after carefully blotting dry of excess PBS. Segments from all vessels were immediately frozen at –80°C for protein extraction, or were processed for frozen sections and immunohistochemistry. The remaining portion of each vessel was fixed for 30 min in 2% formalin/0.2% glutaraldehyde under gentle luminal pressure. Fixed vessels were stained for β-galactosidase by overnight incubation at room temperature in X-Gal solution (20 mM Tris, pH 7.4, 150 mM NaCl, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2 containing 0.5 mg·ml–1 X-Gal [3-chloro-5-bromo-indolyl-β-galactopyranoside]). Stained vessels were either cut open longitudinally for photography, or intact rings were dehydrated through graded alcohols to xylene, and paraffin-embedded. Sections (6 µM) were counterstained with eosin. To define vessel architecture, representative sections from each vessel were also stained with hematoxylin–eosin or with Masson trichrome. Endothelial β-galactosidase gene transfer was assessed by counting luminal blue-stained cell nuclei in vessel cross-sections. To account for the increase in vessel diameter occurring in vein graft after gene transfer, we counted the absolute number of transduced endothelial cells in a complete vessel cross-section, rather than the number of cells per unit area, or the percentage of transduced cells. For counting, sections (at least 2 for each vessel segment, taken at least 100 µM apart) were photographed at 50–100x magnification and the image was projected to allow visualization of a complete vessel cross-section at high resolution.

β-Galactosidase protein concentration was quantified using an ELISA (3 Prime–5 Prime, Boulder, CO), following the manufacturer's instructions. Frozen vessel segments were thawed, blotted dry, and sonicated on ice in lysis buffer (50 mM Tris, pH 7.4, 1 mM EDTA, 0.1% Triton-X100 containing phenylmethyl-sulfonylfluoride [0.2 mg·ml–1] and leupeptin [0.5 µg·ml–1]). Tissue debris was pelletted by centrifugation at 14 000 g and total protein concentration was determined using the Bradford assay (Biorad Laboratories, Richmond, CA). There was a linear relationship between wet vessel weight and quantity of protein extracted (r = 0.92, P<0.0001), indicating quantitatively uniform protein extraction between vessel categories. β-Galactosidase protein quantity was determined as ng β-galactosidase per mm of vessel length, to standardize for the differences in vessel mass per unit length.

2.5 Immunohistochemistry
Vessel segments were equilibrated in 30% sucrose in phosphate-buffered saline (PBS) at 4°C, then embedded in OCT compound (Miles Scientific, Elkhart, IN) and frozen in liquid nitrogen. Sections (6 µm) were prepared on silane-coated glass microscope slides. Immunohistochemistry to identify smooth muscle cells, neutophils or macrophages was performed using primary antibodies directed against human smooth muscle actin (HHF 35, Dako, Carpenteria, CA), rabbit CD18 (Serotec, Oxford, UK) [27], or rabbit RAM 11 (Dako, Carpenteria, CA) [27], respectively. For staining of endothelial cells, we used a mouse monoclonal antibody raised against a recombinant extracellular domain of the endothelium-specific receptor tyrosine kinase, Tie-2/Tek [28]. Vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) were identified using monoclonal antibodies raised against rabbit VCAM-1 and ICAM-1 [29](a generous gift of Dr. M. Cybulsky, Harvard University). Briefly, frozen sections were thawed and dried at room temperature, then equilibrated in PBS. Xylene was used to remove paraffin from paraffin-embedded sections, which were then rehydrated through graded alcohols to PBS. Blocking solution (1.5% horse serum in PBS) was applied for 1 h at room temperature or overnight at 4°C. Antibodies were diluted in blocking solution at the manufacturer's recommended concentration and were applied to tissue sections for 1 h. This was followed by sequential incubation with biotinylated anti-mouse IgG and ABC reagent, according to the manufacturer's specifications (Vectastain ABC kit, Vector Laboratories, Inc.). Levamisole was added to block endogenous alkaline phosphatase activity and immune complexes were localized using the chromogenic alkaline phosphatase substrate, Vector Red (Vector Laboratories, Inc.). For vWF staining, immune complexes were visualized by fluorescence. The sections were lightly counterstained with hematoxylin, dehydrated and mounted with Permount (Fisher Scientific). In all experiments an adjacent section was incubated with an irrelevant murine IgG monoclonal antibody to serve as a negative control.


   3 Results
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
4 Discussion
 References
 
A total of 54 vessels were analyzed, from 20 rabbits (16 native jugular veins, 16 vein grafts, 8 carotid arteries, 8 uninfected control aortas and 4 uninfected control vein grafts).

3.1 Cellular targets of gene transfer in veins, arteries and vein grafts
Three days after gene transfer, both native jugular veins and vein grafts stained intensely blue with X-Gal, indicating β-galactosidase expression (Fig. 1). A high proportion of endothelial cells on the luminal surface of these vessels were transduced (Fig. 1A,B). Cells in the adventitia also expressed β-galactosidase, due to exposure of the outer surface of the vessel to virus solution during the surgical procedure. At the vein graft–carotid artery anastomosis, a clear demarcation of blue staining was apparent between the vein graft and the artery that had not been exposed to adenovirus (Fig. 1B).


Figure 1
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  		Fig. 1 Macroscopic views of jugular vein and vein graft after β-galactosidase gene transfer. Segments of jugular vein (JV; panel A) or venous bypass graft (VG; panel B) were harvested 3 days after gene transfer, fixed, and stained with X-Gal. In panel A, the luminal surface of a segment of JV is shown opened longitudinally (original magnification x10). The luminal surface is intensely blue. Vein graft segments (panel B, x10) also show high-efficiency endothelial gene transfer. The body of the vein graft (‘VG’) stains intensely blue, while only occasional cells on the luminal surface of the anastomosed carotid artery (‘CA’) are transduced. Black arrowheads in panel B mark the suture line between vein graft and artery.

 
We used immunohistochemistry to identify the specific cell types transduced by adenoviral gene transfer. In both native vessels and vein grafts, transduced cells on the luminal surface of the vessel were confirmed as endothelial cells, as they stained with an anti Tie-2/Tek monoclonal antibody (Fig. 2A,F). Interestingly, vein graft endothelium stained much more weakly for Tie-2/Tek than ungrafted jugular vein. Numerous cells throughout the adventitia also expressed β-galactosidase, but β-galactosidase expression was seen only infrequently in medial smooth muscle cells of jugular veins, vein grafts (Fig. 2B,G) or carotid arteries (data not shown). No X-Gal staining cells were seen in control vein grafts not exposed to adenovirus (Fig. 2K–O).


Figure 2
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  		Fig. 2 Immunohistochemistry of jugular vein and venous bypass graft after β-galactosidase adenoviral gene transfer. Sections of jugular vein 3 days after gene transfer (JV, panels A–E), venous bypass graft 3 days after after gene transfer(VG, panels F–J), or uninfected control vein graft (Control VG, panels K–O) were first stained with X-Gal to demonstrate transduced cells (blue nuclei), then immunostained for the following: (1) endothelial cells, using an anti Tie-2/Tek antibody (panels A, F, K); (2) smooth muscle cells (panels B, G, L); (3) VCAM-1 (panels C, H, M); (4) ICAM-1 (panels D, I, N), or (5) CD 18+ leukocytes (panels E, J, O); (original magnification x150). Numerous blue-stained cell nuclei, indicating β-galactosidase expression (outlined arrows), are present on the luminal surface and in the adventitia of both JV and VG, but not Control VG. Transduced cells on the luminal surface are endothelial cells; smooth muscle cells in the media of jugular vein or vein graft are only infrequently transduced. Endothelial expression of VCAM-1 or ICAM-1 are not seen in JV, but are clearly upregulated in both VG and Control VG (panel I; black arrows). Infrequent CD 18+ polymorphonuclear leukocytes (PMN) are present in the adventitia of JV. In contrast, the luminal surface and adventitia of VG and Control VG are heavily infiltrated with PMN (panel J, O; black arrows) which are adherent to the endothelial surface and form aggregations in the subendothelial space, with evidence of endothelial disruption (outlined arrows).

 
3.2 Impact of vascular injury on gene transfer in vein grafts
To investigate the vessel injury sustained by ungrafted veins and vein grafts after intraoperative gene transfer, we used immunohistochemistry to identify VCAM-1 and ICAM-1 expression, RAM 11+ macrophages and CD18+ leukocytes (polymorphonuclear leukocytes; PMNs). Jugular vein exposed to adenovirus at a titer of 1x109 pfu·ml–1 showed no evidence of endothelial VCAM-1 expression, little or no ICAM-1 expression, and did not show significant PMN infiltration, except for infrequent cells in the surgically exposed adventitia (Fig. 2C,D,E, respectively). In contrast, VCAM-1 and ICAM-1 immunostaining was clearly evident in the intima of 3-day vein grafts (Fig. 2H,I). Furthermore, vein grafts were heavily infiltrated by PMNs, which were adherent to transduced endothelial cells and formed aggregates in the subendothelial space (Fig. 2J). In many places infiltrating PMNs were associated with disrupted endothelial integrity and fragmention of transduced endothelial cells (Fig. 2, panel J). RAM 11+ macrophages were only infrequently identified in vein grafts, predominantly in the adventitia (data not shown). Control vein grafts not exposed to adenovirus showed a similar degree of endothelial injury and leukocyte infiltration (Fig. 2M–O), further establishing that the observed inflammation in vein grafts at this time point is due to the sequelae of vein grafting, rather than to adenoviral infection. These findings show that after intraoperative gene transfer, endothelial cells in vein grafts are subject to profound injury when the vein is implanted into the arterial cirulation.

3.3 Relative efficacy of gene transfer to vein grafts and native vessels
We hypothesized that the vascular injury that accompanies bypass grafting might have a significant effect on gene transfer efficacy in vein grafts. Accordingly, we compared β-galactosidase protein expression in vein grafts (VG) with that in jugular veins (JV) and carotid arteries (CA). Vein grafts enlarged considerably after implantation in the arterial circulation (mean vessel mass per unit length: CA, 1.67±0.15 mg·mm–1; JV, 1.71±0.37 mg·mm–1; VG, 4.22±1.22 mg·mm–1). To control for these changes, β-galactosidase protein was determined per mm of vessel length, rather than per mg of vessel weight. High levels of β-galactosidase protein were present in both carotid artery and jugular vein (Fig. 3; CA mean±s.d. [n = 8] 2.96±1.62 vs. JV 2.49±1.41 ng β-galactosidase·mm vessel length–1; P = n.s.), whereas the concentration of β-galactosidase in vein graft extracts was reduced by about half (mean±s.d. 1.42±0.83 ng·mm–1 [n = 8]: P<0.05 vs. carotid artery, P<0.05 vs. jugular vein).


Figure 3
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  		Fig. 3 Relative efficacy of β-galactosidase protein expression after adenoviral gene transfer to carotid arteries, jugular veins and venous bypass grafts. β-Galactosidase (β-Gal) protein expression was determined by ELISA in protein extracts from segments of rabbit carotid artery (CA), native jugular vein (JV), vein graft (VG), 3 days after gene transfer, or in uninfected aorta (Ao) (n = 8 for each group). β-Galactosidase levels are expressed as ng β-galactosidase protein per mm of vessel length. Solid black circles represent the values for individual vessel segments. Bars represent the means for each group of vessels. Asterisks denote statistical significance of comparison with CA or JV (*P<0.05; **P<0.01).

 
As an alternative means of evaluating gene transfer efficacy, we counted β-galactosidase positive cells in vessel cross-sections. Virtually all endothelial cells in native carotid arteries and jugular veins stained blue, indicating a very high transfection efficiency. A similar number of blue-staining cells were identified in sections of carotid artery and jugular vein (Fig. 4; means±s.d. [n = 7]: CA, 196.8±57.1; JV, 181.7±56.9 cells·section–1; P = n.s.). In vein grafts, the number of luminal blue cells was lower than in carotid arteries or jugular veins (Fig. 4; mean±s.d. [n = 7] 109.0±51.4 cells·section–1; P<0.01 vs. CA, P<0.05 vs. JV), but this reduction was less than two-fold. The number of blue-staining endothelial cells per vessel section correlated significantly with β-galactosidase protein expression per mm vessel length (r = 0.89; P<0.0001), indicating that transduction of endothelial cells was a major determinant of overall gene transfer efficacy. Taken together, these findings show that transgene expression in vein grafts at 3 days is well maintained compared with native ungrafted vessels.


Figure 4
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  		Fig. 4 Relative efficacy of endothelial cell gene transfer in carotid arteries, jugular veins and venous bypass grafts. Transduction of endothelial cells by adenoviral gene transfer of rabbit carotid artery (CA), native jugular vein (JV) or vein graft (VG) was assessed by counting the total number of X-Gal stained endothelial cell nuclei in histologic sections, 3 days after β-galactosidase gene transfer. Uninfected aorta (Ao) was used as a negative control. Solid circles represent the values for individual vessels. Bars represent the means for each group of vessels. Asterisks denote statistical significance of comparison with CA or JV (*P<0.05; **P<0.01).

 
3.4 Time course of transgene expression in native jugular vein and vein grafts
To investigate the time course of transgene expression in vein grafts, we compared β-galctosidase protein expression in vein grafts harvested at 7 or 14 days after gene transfer, with that in the paired native jugular vein (Fig. 5). The acute inflammation seen in vein grafts at day 3 was decreased by day 7, and had further reduced by day 14. Vein grafts showed early intimal hyperplasia at day 7, which increased through day 14. No excess of CD18+ inflammatory cells was seen in infected vein grafts at these time points compared with control (uninfected) grafts.


Figure 5
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  		Fig. 5 Relative time course of transgene expression in jugular veins and venous bypass grafts. β-Galactosidase (β-Gal) protein expression was determined by ELISA in protein extracts from segments of native jugular veins (JV) or paired vein grafts (VG), harvested 3 days (n = 8 pairs), 7 days (n = 4 pairs) or 14 days (n = 4 pairs) after gene transfer. β-Galactosidase levels are expressed as ng β-galactosidase protein per mm of vessel length. Solid black circles represent the values for individual vessels. Bars represent the means for each group of vessels. Asterisks denote statistical significance of the comparison between 7 day or 14 day vessels and 3 day vessels (*P<0.05; **P<0.01).

 
β-Galactosidase protein levels in jugular veins decreased modestly between day 3 and day 7 (mean±s.d.: 2.23±1.33 vs. 2.49±1.49 ng β-galactosidase·mm vessel length–1; P = n.s.). In contrast, β-galactosidase levels in 7-day vein grafts were significantly reduced compared with both 7-day jugular vein and 3-day vein grafts (mean±s.d.: 0.37±0.66 vs. 2.23±1.33 and 1.43±0.83 ng β-galactosidase·mm vessel length–1; P<0.05 and P<0.01, respectively). Thus, the mean rate of decrease of transgene expression from day 3 to day 7 after gene transfer was more than 4-fold greater in vein grafts than in jugular veins, after identical gene transfer procedures. By 14 days, β-galactosidase levels in both jugular veins and vein grafts were barely detectable.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
References
 
The widespread application of vascular gene transfer techniques to models of arterial disease [6, 7, 9]contrasts sharply with the limited reports of gene transfer in venous bypass grafts. The only previous study of adenoviral gene transfer in vein grafts established the feasibility of the procedure, but did not quantify transgene expression or determine the cellular targets of vein graft gene transfer in comparison with that in native vessels [18]. We now extend the characterization of vein graft gene transfer, quantifying transgene expression, comparing gene transfer efficacy in vein grafts with that in the native (ungrafted) vein and carotid artery, identifying cellular targets of gene transfer, and establishing the time course of transgene persistence in vein grafts. These studies provide insights into the impact of early vessel injury on gene transfer efficacy in early vein grafts.

4.1 Therapeutic significance of vein graft gene transfer
Although moderately reduced compared with ungrafted veins, transgene expression in early vein grafts should be adequate to examine the effects of therapeutic transgenes on vein graft thrombosis, endothelial injury and subsequent intimal proliferation. Transgene expression is well maintained for at least 3 days, and it is during this early ‘window’ that key pathologic processes are initiated. Efficient adenoviral-mediated gene transfer therefore provides a potential tool to inhibit maladaptive responses to bypass grafting, or to enhance beneficial responses. Endothelial injury, sustained early after bypass grafting, is a key initiator of subsequent intimal hyperplasia [9, 21]. Consequently, an early therapeutic intervention aimed at minimizing endothelial injury, or enhancing endothelial repair, may potentially reduce intimal hyperplasia and graft thrombosis and may also enhance transgene expression in vein grafts.

Our findings indicate that the endothelium is the preferential target of adenoviral gene transfer in veins and vein grafts, with relatively infrequent transduction of smooth muscle cells. This observation is consistent with previous studies of gene transfer in uninjured arteries [20, 30]; medial smooth muscle cells can be transduced after arterial injury or with high-pressure delivery, which overcome the endothelial barrier to virus penetration [31, 32]. Thus, adenoviral gene transfer represents a feasible approach for intraoperative gene therapy in early venous bypass grafts and may have particular application to strategies aimed at targeting the endothelium.

4.2 Relative efficacy of vein graft gene transfer
We demonstrate high levels of transgene expression in vein grafts at 3 days, but note about a two-fold reduction when compared with the carotid artery and with the jugular vein which did not undergo bypass grafting. This reduced expression likely reflects the injury sustained by vein graft due to exposure to the arterial circulation [21]. Early vein grafts, whether infected with adenovirus or not, showed marked upregulation of VCAM-1 and ICAM-1 expression, and were heavily infiltrated with PMNs, whereas these features were not seen in ungrafted jugular veins exposed to the same virus solution. In vein grafts, the inflammatory cells adhered to the endothelium, formed aggregates in the subendothelial space, and were associated with endothelial disruption. This intense intimal inflammation injures the primary target of adenoviral gene transfer, the endothelium, and likely accounts for the observed reduction in transgene expression in vein grafts. Our findings of reduced transgene protein levels and reduced numbers of X-Gal staining endothelial cells in vein grafts raise the possibility that there may be a loss or increased turnover of endothelial cells in the early vein graft. Alternatively, reduced transgene protein levels in vein grafts may be a result of down-regulated transgene expression in activated endothelial cells.

The time course of transgene persistence in jugular veins and vein grafts provides further information on the impact of vessel injury and remodeling on vein graft gene transfer efficacy. Between 3 and 7 days, transgene expression in vein grafts declined more rapidly than in ungrafted veins, suggesing that exposure of vein grafts to the arterial circulation results in a progressive reduction in transgene expression. The subsequent loss of transgene expression in both jugular veins and vein grafts by 14 days is typical of first-generation adenoviral vectors and is similar to earlier studies of transgene persistence in arterial models [30, 33]. Second-generation adenoviral vectors are much more inert, and may provide much longer transgene persistence [34–37]. However, the findings of this study remain important, since the impact of bypass grafting on gene transfer efficacy is unlikely to be significantly improved by second-generation vectors.

4.3 Adenoviral-mediated inflammation in veins and vein grafts
Although adenovirus can cause acute vascular injury at very high titers [20, 32, 38], several key points suggest that adenovirus infection itself did not significantly contribute to the acute injury observed in vein grafts in this study:

(1) We used a titer of adenovirus (1x109 pfu·ml–1) that lies well below the threshold at which significant endothelial injury occurs. We and others [20]have shown that higher titers cause a progressive increase in PMN inflammation, accompanied by endothelial vasomotor dysfunction in rabbit arteries [K.M. Channon, S.E. George, manuscript submitted].

(2) The ungrafted control vein was infected with exactly the same adenovirus preparation, at the same titer, as the contralateral vein graft. In these ungrafted jugular veins, no increased VCAM-1 or ICAM-1 expression, nor any increase in endothelial inflammatory cell infiltrate was seen, despite their exposure to adenovirus, and a very high transduction efficiency.

(3) Infiltration with CD18+ polymorphonuclear leukocytes is a characteristic feature of early vein grafts [21, 22], and was not increased in infected vein grafts in comparison with control vein grafts which were not exposed to adenovirus.

Thus, these observations strongly suggest that, at the infecting titer used, endothelial injury in early vein grafts after gene transfer is a result of the vein graft procedure and its hemodynamic sequelae, and not due to infection with adenovirus.

Implanting a genetically modified vein into the arterial circulation subjects it to endothelial injury, platelet and fibrin deposition, inflammatory cell infiltration and subsequent remodeling. These processes have a significant impact on intraoperative vein graft gene transfer. Nevertheless, transgene expression in early vein grafts remains high for at least 3 days, despite these disruptive processes. Our study highlights the utility of intraoperative gene transfer in vein grafts, both as a tool to investigate endothelial biology after bypass grafting, and as a potential therapeutic strategy.

Time for primary review 32 days.


   Acknowledgements
 
K.M.C. is a British Heart Foundation Clinical Scientist Fellow. G.J.F. holds a Travelling Fellowship from Trinity College, Dublin. This work was supported by an American Heart Association–North Carolina Affiliate Grant in Aid (to K.M.C.) and USPHS Grants HL48662 (to S.E.G.) and HL15448 (to P.O.H.). M.A.B. is a Clinician Scientist of the American Heart Association and S.E.G. is an Established Investigator of the American Heart Association.


   References
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
 References
 

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S. L. Meyerson, C. L. Skelly, M. A. Curi, and L. B. Schwartz
Gene Therapy for Cardiovascular Disease
Seminars in Cardiothoracic and Vascular Anesthesia, November 1, 2000; 4(4): 289 - 300.
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