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Ann Thorac Surg 1998;66:814-819
© 1998 The Society of Thoracic Surgeons

Original articles: Cardiovascular

Gene transfer to vein graft wall by HVJ–liposome method: time course and localization of gene expression

Hong-zhi Bai, MDa, Yoshiki Sawa, MDa, Wei-da Zhang, MDa, Tomoyuki Yamakawa, MDa, Ryuichi Morishita, MDb, Yasufumi Kaneda, MDc, Hikaru Matsuda, MDa

a First Department of Surgery, Osaka University Medical School, Osaka, Japan
b Department of Geriatric Medicine, Osaka University Medical School, Osaka, Japan
c Institute for Molecular and Cellular Biology, Osaka University, Osaka, Japan

Accepted for publication April 30, 1998.

Address reprint requests to Dr Matsuda, First Department of Surgery, Osaka University Medical School, Yamada-Oka 2-2, Suita, Osaka 565, Japan


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Background. A novel gene transfer method using liposomes with a viral envelope of hemagglutinating virus of Japan (HVJ) has been reported to be very effective for gene transfection into somatic cells and might be applicable to improve the patency of vein grafts. The present study examined the time course and localization of gene expression to assess the feasibility of ex vivo gene transfer into the vein graft by the HVJ–liposome method.

Methods. The HVJ–liposome complex containing either ß-galactosidase plasmid DNA (deoxyribonucleic acid) or no genes (controls) (experiment 1) or fluorescein isothiocyanate–labeled oligonucleotides either with or without HVJ-liposomes (experiment 2) was infused into rabbit vein grafts and allowed to incubate before autologous transplantation to carotid arteries.

Results. In experiment 1, all grafts incubated with ß-galactosidase plasmid with HVJ-liposomes showed the blue staining of X-gal 7 days after operation, whereas the controls did not. The blue granules were present in the medial and adventitial tissue and were still present after 14 days. In experiment 2, many fluorescein isothiocyanate–labeled nuclei were observed in the graft wall 2 and 4 days after operation and remained present mainly in the media of HVJ-liposome–treated grafts after 7 and 14 days, when no fluorescein isothiocyanate activity was observed without HVJ–liposome treatment.

Conclusions. These results demonstrated the feasibility of ex vivo transfection to the medial and adventitial tissue of the vein graft by the HVJ–liposome method and suggest the possibility of its clinical application to prevent vein graft failure.


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Saphenous vein grafts remain the most commonly used conduits for bypass grafting, as usable arterial grafts such as internal mammary artery grafts are limited in number, and artificial substitutes for small-caliber grafts have not achieved satisfactory results [1–4]. Nevertheless, vein graft occlusion, associated with either thrombus formation or neointimal hyperplasia, remains an unsolved short-term and long-term problem [1–4].

Recent advances in cellular and molecular biology have not only elucidated the mechanisms of vein graft failure [5–8] but have also produced a potential strategy to solve this problem by means of gene transfection [9, 10]. That is, if some specific proteins that prevent thrombus formation, inhibit neointimal hyperplasia, or both could be produced by transfected genes [11–13], or if transcription of some specific molecules responsible for graft failure could be inhibited by antisense oligodeoxynucleotide (ODN) or transcription factor decoy therapy [14, 15], this might help to enhance graft patency. A gene transfer method using liposome with a viral envelope of hemagglutinating virus of Japan (HVJ) (HVJ–liposome method) has been reported to be highly efficacious for in vivo gene transfection [9, 16] and could be used to alter vein grafts [16].

To establish an appropriate gene/ODN therapy protocol for vein grafts, it is essential to define the area and the duration of the transfected gene expression. In this study, we examined the time course and localization of transfected gene/ODN expression to assess the feasibility of gene therapy for vein grafts when performed ex vivo with the HVJ–liposome method during operative procedures.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Comment
 Acknowledgments
 References
 
Plasmid construction
In the first experiment, we introduced complementary DNA (deoxyribonucleic acid) plasmids of ß-galactosidase (ß-gal) to vein grafts by the HVJ–liposome method to examine the transfected gene expression by histochemistry. The ß-gal plasmid was prepared as previously described [17]. Briefly, pAct-c-myb (a gift from Dr Ishii, Institute of Physical and Chemical Research, Tokyo, Japan) containing the 5'-promoter region (370 base pairs) and the first intron (900 base pairs) of the chicken ß-acting gene was restricted with XhoI/BamHI and cloned into the SalI/BamHI site of pUC19. This plasmid (pUC-Act-c-myb) was restricted with NcoI/XbaI to remove c-myb and ligated with SalI linker (8mer; Takara Syuzo Co, Tokyo, Japan). The Escherichia coli ß-gal gene (3.1 kb), isolated from pMC1871 by restriction with SalI, was cloned into this site.

Fluorescein isothiocyanate–labeled oligonucleotide
In the second experiment, we introduced fluorescein isothiocyanate (FITC)–labeled phosphorothioate ODNs (FITC–ODNs) (16 mer) to determine the localization of the transfected areas. The FITC–ODNs were provided by Clontech Inc (Palo Alto, CA). Fluorescein 18–ON phosphramidite was used to label ODN with FITC on the 3' and 5' ends of the ODN [18].

Preparation of HVJ-liposomes
The HVJ-liposomes containing plasmid DNA and high-mobility group 1, which enhances the mobility of genes to nuclei in the cytoplasm, were prepared as previously reported (Fig 1A) [9, 17]. Briefly, dried lipid (phosphatidylserine, phosphatidylcholine, and cholesterol combined at a weight ratio of 1:4.8:2) was mixed with plasmid DNA (200 µg) (previously incubated at 20°C for 1 hour with high-mobility group 1), shaken vigorously, and sonicated to form liposomes. Purified HVJ (Z strain) was inactivated by ultraviolet irradiation (110 erg · mm-2 · s-1) for 3 minutes just before use. The liposome suspension mixed with HVJ was incubated at 4°C for 10 minutes and at 37°C for 30 minutes. The HVJ–liposome complex was then collected for use after removal of free HVJ. As reported previously [9, 17], this preparation method has been optimized to achieve maximal transfection efficiency, and the final concentration of DNA was estimated to be 10 µg/mL in the liposomes.



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  		Fig 1. (A) Preparation method for hemagglutinating virus of Japan (HVJ)–liposome complex and (B) procedures to transfect vein graft ex vivo. chol/TF = cholesterol/; DNA = deoxyribonucleic acid; HMg-1 = high-mobility group 1; ODN = oligodeoxynucleotide; PL = phosphatidylcholine; PS = phosphatidylserine; TS = transfection.)

 
Animals
The study comprised 22 adult male New Zealand White rabbits weighing 2.5 to 2.9 kg (Kbl-NZW; Nihon-Animal Co, Osaka, Japan) and fed on a standard diet (ORC4; Oriental Yeast Corp, Osaka, Japan). Ten rabbits were used for transfection with the HVJ–liposome complex containing either ß-gal plasmid DNA (n = 6) or no genes (n = 4) (experiment 1), and the other 12 rabbits were treated either with FITC–ODNs with HVJ-liposomes (final FITC–ODN concentration 3 µmol/L (n = 8) or with FITC–ODNs alone (30 µmol/L) (n = 4) (experiment 2). Procedures were in accordance with the guidelines approved by the Institutional Animal Care and Use Committee, Osaka University Medical School. All animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH publication 85-23, revised 1985).

Surgical procedures
General anesthesia was induced with xylazine hydrochloride (5 mg/kg intramuscularly) and atropine sulfate (0.02 mg/kg intramuscularly) and maintained with inhalation of 1.2% sevoflurane. Through a midline incision in the ventral area of the neck, the second branch of the external jugular vein was dissected free with "no-touch" technique. After intravenous injection of heparin sodium (300 U/kg of body weight), approximately 30 mm of the vein was harvested and gently irrigated with heparinized isoosmotic sodium chloride solution (0.9 g/L) containing papaverine hydrochloride through a 22-gauge catheter cannulated at the end. After the other end had been clamped with a hemostatic clip, approximately 500 µL of HVJ–liposome complex was infused into the graft through the catheter and allowed to incubate on ice for 20 minutes according to the previous experimental protocol performed in rat carotid arteries (Fig 1B) [14, 15]. The incubation temperature was set at 4°C because it is reportedly suitable for the HVJ-liposomes to bind to the cell membrane [19] and because it is appropriate for graft preservation.

After the scheduled incubation period, the HVJ-liposomes were removed from the vein graft. The graft was interposed in the autologous carotid artery with maintenance of proper direction of flow. End-to-end anastomosis was performed using the cuff technique [20], which permits anastomosis without sutures and thus helps minimize surgical injury to the vessel wall. The anastomosis was completed by circumferential ligation with a 7-0 suture, and the neck wound was closed in layers.

Animal sacrifice and tissue preparation
The grafts were harvested on postoperative days 7 and 14 in experiment 1 and on postoperative days 2, 4, 7, and 14 in experiment 2. In experiment 1, the harvested grafts were fixed in 2% glutaraldehyde solution for 1 hour, permeated with 20% and 30% sucrose solutions for 1 hour each, and divided into two parts. One part was frozen in OCT compound (Miles Scientific, Elkhart, IN) in a cryomold in liquid nitrogen and cut into frozen sections 5 µm thick. The other part of the graft and the frozen sections were placed in a 5-bromo-4-chloro-3-indolyl-ß D-galactopyranoside (X-gal) staining solution for 24 hours at 4°C for identification of ß-gal expression. In experiment 2, the grafts were frozen directly in OCT compound and cut into frozen sections. After incubation in 0.3% eriochrome black T solution (Wako, Tokyo, Japan) to diminish the autofluorescence from elastin, cross sections were observed with a fluorescent microscope equipped with differential interference contrast apparatus (Olympus, Tokyo, Japan).


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Comment
 Acknowledgments
 References
 
None of the animals sustained any serious complications during the study period. All of the grafts were patent and processed for further analysis.

Experiment 1: grafts transfected with ß-Gal plasmid
The expression of transfected genes was evaluated by en bloc histochemical analysis of the vein grafts treated with ß-gal plasmid DNA with HVJ-liposomes. Figure 2 shows a graft harvested 7 days postoperatively and stained with X-gal solution. Gross observation revealed that the luminal surface of the grafts transfected with ß-gal plasmid by the HVJ–liposome method showed blue staining by the X-gal solution (see Fig 2A), whereas the control grafts treated with HVJ-liposomes with no genes had no staining (see Fig 2B). Microscopic investigation of the cross sections from grafts harvested 14 days after operation showed the blue granules localized in the medial and adventitial wall but not in the newly formed intima (Figs 3A, 3C). In control grafts, no blue granules were identified in the vein graft wall (Figs 3B, 3D).



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  		Fig 2. Gross examination of en bloc segments of vein grafts exposed to hemagglutinating virus of Japan-liposomes containing either (A) ß-galactosidase plasmid DNA or (B) no genes. (X-gal; x8 before 38% reduction.)

 


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  		Fig 3. Microscopic views of sections from vein grafts exposed to hemagglutinating virus of Japan-liposomes containing either (A, C) ß-galactosidase (ß-gal) plasmid DNA or (B, D) no genes. All sections were stained with X-gal to detect the ß-gal expression before sectioning, and counterstaining was done with hematoxylin in C and D. The arrows indicate borders between neointima and media. (x200 before 19% reduction.)

 
Experiment 2: distribution of FITC–labeled ODNs in vein graft wall
The distribution of ODNs by the HVJ–liposome method and the duration of their expression were evaluated histochemically with FITC–ODNs. Figure 4 shows cross sections of grafts treated with FITC–ODNs either with or without HVJ-liposomes. Four days postoperatively, the graft wall was clearly labeled with fluorescein transfected with HVJ-liposomes (see Figs 4A, 4C). The jugular vein of the rabbit was composed of a single layer of endothelial cells, a few layers of medial cells, and adventitial tissue. Four days after operation, the neointima had scarcely formed in the graft. Thus, FITC–labeled cells were observed mainly in the medial cells and in the adventitial cells (see Fig 4C). Without HVJ-liposomes, the graft wall was labeled only slightly with FITC. However, its intensity was much weaker than with HVJ-liposomes (see Figs 4B, 4D). Fourteen days postoperatively, when the neointima had formed around the luminal circumference, the FITC–labeled area was still present in the graft wall treated with HVJ-liposomes (see Fig 4E), whereas no fluorescein was observed in grafts treated with FITC–ODNs alone (see Fig 4F). In grafts transfected by the HVJ–liposome method, the FITC–labeled area was confined mainly to the medial and adventitial tissue and was hardly observed in the newly formed intima or in the endothelial cells (see Fig 4E). It is thought that the neointima is formed with proliferation and migration of medial smooth muscle cells (SMCs). Thus, our observation indicated the FITC–ODNs are preserved mainly in the medial and adventitial walls where the HVJ-liposomes are originally transfected and are hardly transferred to the newly formed intima.



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  		Fig 4. Microscopic views of sections from vein grafts exposed to fluorescein isothiocyanate–labeled phosphorothioate oligodeoxynucleotides either (A, C, E) with hemagglutinating virus of Japan-liposomes or (B, D, F) without them. The grafts were harvested on day 4 in A, B, C, and D and on day 14 in E and F. All sections were incubated in erichrome black T solution to diminish the autofluorescence from elastin and were observed with a fluorescent microscope equipped with differential interference contrast apparatus. The arrows indicate the borders between neointima and media. (A, B: x200 before 14% reduction; C, D, E, F: x400 before 14% reduction.)

 

   Comment
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
Acknowledgments
 References
 
In this study, we demonstrated that ex vivo gene/ODN transfection into the vein graft wall by means of the HVJ–liposome method during bypass grafting is feasible and that the expression of the transfected material is maintained more than 2 weeks after operation and is confined mainly to the medial and adventitial areas.

The HVJ is Paramyxovirus, a kind of RNA (ribonucleic acid) virus containing two characteristic glycoproteins on the envelope 19: HN protein, which takes part in binding to the cell surface and is most effective at 0°C, and F protein, which has a role in cell fusion most effectively at 37°C. Both proteins are resistant to ultraviolet irradiation and retain their features, though the HVJ is inactivated not to grow. The HVJ–liposome method was developed by using these features of HVJ and lipofection and has been reported to markedly enhance the efficacy of gene/ODN transfection by increased uptake, nuclear localization, and intracellular stability [9, 17]. Our study aimed not only to assess the feasibility but also to define the limitations of gene/ODN transfection into the vein graft by means of the HVJ–liposome method and to establish an appropriate gene therapy protocol for vein graft failure.

There have been some reports on gene transfer to the vein graft [16, 21, 22], but these studies did not describe the time course and localization of gene expression in detail, especially after neointimal formation in the vein graft wall. Using recombinant adenovirus carrying genes encoding a soluble form of vascular cell adhesion molecule, Chen and colleagues [21] demonstrated that the transfected genes were expressed in the cells lining the luminal surface and in the adventitia 3 days postoperatively. Kupfer and associates [22] tested the feasibility of gene transfer by means of adenovirus-transferrin/polylysine-DNA complexes with ß-gal reporter gene and showed that genes could be transfected to the endothelial cells and to the medial and adventitial cells 3 and 7 days after operation but did not refer to the neointima area. Mann and coauthors [16] performed antisense ODN transfer with the HVJ–liposome method and identified the transfected area just 1 hour after operation.

In a previous study, we investigated the process of remodeling in venous arterial grafts in the same animal model used in this study and observed that formation of the neointima begins after 7 days and continues to gradually increase in thickness up to 6 months after operation (unpublished observations). It is thought that many factors are involved and related to vein graft failure, such as thrombus formation, SMC proliferation, endothelial cell dysfunction, inflammatory cell adhesion, and extracellular accumulation [1, 7]. Each of these factors occurs at different sites in the vein graft and at different times. For gene therapy, it is important to deliver the genes exactly to the cells of interest. In other words, the success of this therapeutic strategy depends on the distribution and the duration of the transgene/ODN expression.

We used ß-gal plasmids and FITC–labeled ODNs as reporter genes and ODNs, respectively. The ODNs are reported to selectively block the expression of specific genes in an antisense or decoy manner [14, 15]. In this study, both were demonstrated to be distributed and remain mainly in the medial and adventitial walls of the vein graft, presumably in the SMCs and fibroblasts. Their expression was clearly observed even 2 weeks postoperatively. The neointima is composed mainly of SMCs that proliferate and migrate from the media, and atherosclerotic change is thought to superimpose on the neointima [1, 3, 4]. If SMC proliferation can be inhibited effectively in the medial tissue by this method, it may help reduce thickening of the neointima, prevent atherosclerotic change, and thus enhance graft patency. It is also interesting and maybe reasonable that the reporter genes/ODNs were scarcely observed in the neointima. This is probably because intimal SMCs divide or lose the transfected genes/ODNs during proliferation and migration. Therefore, it would hardly be expected that this method could transduce some specific genes/ODNs into the newly formed intima.

As for the endothelial cells, we could not clearly identify the expression of the transfected genes/ODNs in these cells 7 days and 2 weeks after operation when the intima was newly formed.

The rabbit jugular vein is composed of only a few layers of endothelial cells and SMCs, and the vein wall is so frail that this rabbit model of vein grafts may not be comparable to human vein grafts. In a previous study, we observed that most of the cells in the vascular wall are seriously injured and lost immediately 2 and 4 days after operation. This was probably the result of the operative procedure and the remarkable expansion caused by exposure to the arterial circulation even in animals that were not transfected and had a meticulous operation (unpublished observations). By 14 days postoperatively, the endothelial cells are fully regenerated through proliferation and migration, a finding consistent with the observations of Zwolak and associates [8]. Therefore it is reasonable to observe regenerated endothelial cells with little expression of transfected gene/ODNs. This also suggests that gene therapy targeting endothelial cells may be limited to treatment at an earlier period such as the inhibition of thrombus formation, unless the native endothelial cells are treated adequately to survive thereafter.

In conclusion, this study demonstrated the feasibility and the limitations of gene transfer to the vein graft by means of the HVJ–liposome method by examining the time course and localization of the transfected gene/ODN expression. This method appears to be a promising strategy for prevention of vein graft failure because it can be performed ex vivo and away from the patient and minimizes the adverse effects of gene/ODN transfection.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
References
 
This study was supported in part by research grants 0657071 and 07457293 from the Ministry of Education of Japan.

We thank Dr Junichi Masuda for useful advice on the histopathologic analysis.


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 

  1. Bryan A.J., Angelini G.D. The biology of saphenous vein graft occlusion: etiology and strategies for prevention. Curr Opin Cardiol 1994;9:641-649.[Medline]
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  6. Francis S.E., Hunter S., Holt C.M., et al. Release of platelet-derived growth factor activity from pig venous arterial grafts. J Thorac Cardiovasc Surg 1993;108:540-548.
  7. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 1993;362:801-809.[Medline]
  8. Zwolak R.M., Kirkman T.R., Clowes A.W. Atherosclerosis in rabbit vein grafts. Arteriosclerosis 1989;9:374-379.[Abstract/Free Full Text]
  9. Kaneda Y., Iwai K., Uchida T. Increased expression of DNA cointroduced with nuclear protein in adult rat liver. Science 1989;243:375-378.[Abstract/Free Full Text]
  10. Nabel E.G. Gene therapy for cardiovascular disease. Circulation 1995;91:541-548.[Free Full Text]
  11. Ohno T., Gordon D., Sna H., et al. Gene therapy for vascular smooth muscle cell proliferation after arterial injury. Science 1994;265:781-784.[Abstract/Free Full Text]
  12. Chang M.W., Barr E., Seltzer J., et al. Cytostatic gene therapy for vascular proliferative disorders using a constitutively active form of Rb. Science 1995;267:518-522.[Abstract/Free Full Text]
  13. Von der Leyen H.E., Gibbons G.H., Morishita R., et al. Gene therapy inhibiting neointimal vascular lesion: in vivo transfer of endothelial cell nitric oxide synthase gene. Proc Natl Acad Sci USA 1995;92:1137-1141.[Abstract/Free Full Text]
  14. Morishita R., Gibbons G.H., Ellison K.E., et al. Single intraluminal delivery of antisense cdc2 kinase and proliferating-cell nuclear antigen oligonucleotides results in chronic inhibition of neointimal hyperplasia. Proc Natl Acad Sci USA 1993;90:8474-8478.[Abstract/Free Full Text]
  15. Morishita R., Gibbons G.H., Horiuchi M.H., et al. A gene therapy strategy using a transcription factor decoy of the E2F binding site inhibits smooth muscle proliferation in vivo. Proc Natl Acad Sci USA 1995;92:5855-5859.[Abstract/Free Full Text]
  16. Mann J.M., Gibbons G.H., Kernof R.S., et al. Genetic engineering of vein grafts resistant to atherosclerosis. Proc Natl Acad Sci USA 1995;92:4502-4506.[Abstract/Free Full Text]
  17. Kato K., Nakanishi M., Kaneda Y., Uchida T., Okada Y. Expression of hepatitis B virus surface antigen in adult rat liver. J Biol Chem 1991;266:3361-3364.[Abstract/Free Full Text]
  18. Morishita R., Gibbons G.H., Dzau V.J. Analysis of cellular uptake of the fluorescein-labeled, antisense oligonucleotides. Oligotechniques 1994;2:1-3.
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  20. Mizuta T., Nakahara K., Shirakura R., et al. Total nonmicrosuture technique for rat lung transplantation. J Thorac Cardiovasc Surg 1991;102:159-160.[Medline]
  21. Chen S.J., Wilson J.M., Muller D.W.M. Adenovirus-mediated gene transfer of soluble vascular cell adhesion molecule to porcine interposition vein grafts. Circulation 1994;89:1922-1928.[Abstract/Free Full Text]
  22. Kupfer M.J., Ruan X.M., Liu G., Matloff J., Forrester J., Chaux A. High-efficacy gene transfer to autologous rabbit jugular vein grafts using adenovirus-transferrin/polylysine-DNA complexes. Hum Gene Ther 1994;5:1437-1443.[Medline]



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