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Delivery of Recombinant Adenoviruses to Human Saphenous Vein

  • Sarah J. George
  • Andrew H. Baker
Part of the Methods in Molecular Medicine™ book series (MIMM, volume 30)

Abstract

The human saphenous vein is the most commonly used conduit for coronary artery bypass grafting owing to its ready availability, ease of harvesting, and favorable surgical handling (1). However, it suffers from a progressive decline in patency, resulting in a graft failure rate of 50% after 10 yr (2,3). This high failure rate is caused by either early thrombosis occlusion, which occurs in the first year after graft implantation, or the later development of intimal thickening and superimposed atherosclerosis (1, 2, 3).

Keywords

Recombinant Adenovirus Intimal Thickening Vein Segment Sterile Forceps Human Saphenous Vein 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

1 Introduction

The human saphenous vein is the most commonly used conduit for coronary artery bypass grafting owing to its ready availability, ease of harvesting, and favorable surgical handling (1). However, it suffers from a progressive decline in patency, resulting in a graft failure rate of 50% after 10 yr (2,3). This high failure rate is caused by either early thrombosis occlusion, which occurs in the first year after graft implantation, or the later development of intimal thickening and superimposed atherosclerosis (1, 2, 3).

Genetic manipulation of cells in human saphenous vein grafts aimed at preventing graft failure is a promising application of gene therapy in cardiovascular disease. The surgical technique provides a unique opportunity for gene transfer, because the vein is removed from the patient’s leg and is subjected to surgical preparation. Surgical preparation involves adventitial stripping, side-branch ligation and distension with heparinized blood. The vein is then placed in heparinized blood for up to 45 min while the chest is prepared. It is therefore clinically feasible to use this time period to carry out gene transfer.

The human saphenous vein organ culture model provides a reproducible, well-characterized model of intimal thickening (4,5). A neointima of vascular smooth muscle cells (VSMC) forms during 14 d of culture due to migration and proliferation of VSMC (4). Therefore, using this model, the effect of gene transfer on the processes of VSMC migration and proliferation that lead to intimal thickening can be studied in human tissue in the absence of a deleterious immune response to the delivery vehicle.

Molecular techniques such as those described in previous chapters have identified candidate genes that may be useful for prevention of vein graft failure. These include antiproliferative genes, antithromobolytic genes, antimigratory genes, and suicide genes. We have used recombinant adenoviruses to deliver antimigratory genes to the vein wall since adenoviruses are highly efficient and infect both dividing and nondividing cells. Additionally, the viral DNA is not integrated, resulting in transient recombinant gene expression. The production of recombinant adenoviruses and other gene transfer vehicles is described in detail in other chapters (see  Chapters 22 and  25).

2 Materials

All solutions and equipment used should be sterile.

2.1 Vein Collection and Preparation for Adenoviral Infection

Collection medium: RPMI 1640 tissue culture medium containing 20 mM HEPES-buffered, 5 μg/mL amphotericin B, and 20 IU/mL sodium heparin. Store in 20 mL aliquots at −20°C. Prewarm to 37°C, immediately before use add 0.225 mg/mL papaverine hydrochloride (McCarthy Medical, Wrexham, UK).

2.2 Infection with Recombinant Adenovirus

  1. 1.

    Wash medium: RPMI 1640 tissue culture medium containing 20 mM HEPES, 2 mM l-glutamine, 8 μg/mL gentamicin, 100 IU/mL penicillin, and 100 μg/mL streptomycin. Store at 4°C. Prewarm to 37°C before use.

     
  2. 2.

    18-gauge×32 mm catheter needle (Abbocath®-T, Venisystems™, Abbott Ireland, Sligo, Republic of Ireland).

     
  3. 3.

    Vessel cannula with 3 mm beveled tip and one-way valve (DLP Inc, Grand Rapids, MI).

     
  4. 4.

    4/0 Mersilk™ braided silk suture (Ethicon Ltd, Edinburgh, UK).

     
  5. 5.

    Lockable three-way stopcock (Vygon, Ecouen, France).

     

2.3 Organ Culture

  1. 1.

    Serum-free incubation medium: RPMI 1640 tissue culture medium containing 2 g/L bicarbonate, 2 mM l-glutamine, 8 μg/mL gentamicin, 100 IU/mL penicillin, and 100 μg/mL streptomycin.

     
  2. 2.

    Incubation medium: To a 50-mL Falcon tube containing 35 mL of serum-free incubation medium add 15 mL of filtered foetal calf serum (FCS). Store at −20°C. Prewarm to 37°C before use.

     
  3. 3.

    Phosphate-buffered saline (PBS): 0.15 M NaCl, 7.5 mM Na2HPO4, and 1.9 mM NaH2PO4, pH 7.4.

     
  4. 4.

    10% buffered formal saline: Add 100 mL of 30% fomaldehyde solution to 900 mL of PBS.

     
  5. 5.

    Organ culture dishes: To a 45-mm glass Petri dish (BDH, Poole, UK) add 5 mL of Sylgard resin (BDH) mixed 1∶10, place at 37°C overnight to set. Sterilize in autoclave.

     
  6. 6.

    Polyester mesh (size P500, G. Bopp & Co. Ltd, Derbyshire, UK) cut into 15×15 mm squares and sterilize in an autoclave.

     
  7. 7.

    Minuten pins (size A1, Natkins and Doncaster, Kent, UK): Sterilize by autoclaving.

     

3 Methods

3.1 Vein Collection and Preparation for Adenoviral Infection

  1. 1.

    Obtain “surgically prepared” human saphenous vein segments (approx 8–10 cm long), from coronary artery bypass patients after adventitial stripping, side branch ligation, gentle manual distension, and storage in heparinized blood for 60–120 min, at the completion of the bypass procedure (see Notes 1 and 2 ).

     
  2. 2.

    Collect veins in 20 mL of prewarmed collection medium.

     
  3. 3.

    In a laminar flow hood, place vein segments in a 16-cm glass Petri dish containing 75 mL of wash medium. Using microscissors and forceps dissect the remaining adventitia and then bisect the vein transversely (see Note 3 ).

     
  4. 4.

    Place one piece into a small Petri dish containing 5 mL of wash medium to serve as the uninfected control.

     

3.2 Infection with Recombinant Adenovirus

  1. 1.

    Remove the sheath of an 18-gauge×32 mm catheter needle from the catheter needle and trimmed to 2 cm in length with scissors (see arrow in Fig. 1 B).

     
  2. 2.

    Insert the catheter sheath into the lumen of the piece of vein to be infected (see Notes 4 and 5 ).

     
  3. 3.

    Hold the catheter sheath in place with forceps and check for the presence of obstructing valves by injecting 0.5 mL of wash medium into the vein lumen via the catheter sheath using a 1 mL sterile syringe. If the wash medium does not flow through the vein segment, this process should be repeated at the other end of the vein (see Note 6 ).

     
  4. 4.

    When unobstructed flow of wash medium is observed, insert a vessel cannula with 3 mm beveled tip and one-way valve into the lumen of the other end of the vein.

     
  5. 5.

    Cut a 10 cm length of sterile 4/0 Mersilk™ braided silk suture with microscissors.

     
  6. 6.

    Place the suture around the end of the vein with the vessel cannula inserted.

     
  7. 7.

    Using two pairs of sterile forceps tie two knots in the silk suture to secure the vessel cannula ( Fig. 1 A) (see Note 7 ).

     
  8. 8.

    Insert the catheter cannula into the other end of the vein segment.

     
  9. 9.

    Cut another 10 cm length of sterile 4/0 Mersilk™ braided silk suture with microscissors.

     
  10. 10.

    Secure the catheter sheath in place with the silk suture in a similar fashion as described above for the vessel cannula ( Fig. 1 C).

     
  11. 11.

    While still in their sterile wrapping, twist the taps of two three-way stopcocks to close the stopcocks.

     
  12. 12.

    Remove both stopcocks from the wrapping and place into the Petri dish containing the vein and wash medium.

     
  13. 13.

    Attach one stopcock to the vessel cannula ( Fig. 2 A).

     
  14. 14.

    Using a 1 mL sterile syringe medium inject wash medium through the catheter sheath until it just enters the distal cannula.

     
  15. 15.

    Briefly microfuge the cryovial containing the adenovirus to ensure that the 100 μL is at the bottom of the tube.

     
  16. 16.

    Take up 100 μL of recombinant adenovirus solution at 1.2×1010 pfu/mL into another 1 mL sterile syringe (see Note 8 ).

     
  17. 17.

    Hold the catheter sheath vertically at the place of the Nylon suture knot with sterile forceps.

     
  18. 18.

    Insert the syringe containing the adenovirus into the catheter sheath and inject the contents into the lumen of the vein proximal cannula without increased pressure ( Fig. 2 B) (see Note 9 ).

     
  19. 19.

    Carefully remove the 1 mL syringe from the catheter sheath, and using forceps attach the other stopcock to the catheter sheath ( Fig. 2 C) (see Note 10 ).

     
  20. 20.

    Remove the wash medium and replace with fresh medium.

     
  21. 21.

    Incubate the infected and uninfected vein segments for 1 h at room temperature in the laminar flow hood (see Note 11 and 12 ).

     
Fig. 1.

Stages of adenoviral infection protocol I. (A) Vein segment with vessel cannula inserted and secured in one end. (B) Vein segment with vessel cannula secured, trimmed catheter sheath is placed in medium (see arrow). (C) Vein segment with vessel cannula and catheter sheath inserted into both ends. Scale bar in panel (A) applies to all panels and represents 1 cm.

Fig. 2.

Stages of adenoviral infection protocol II. (A) Vein segment with vessel cannula and catheter sheath inserted and one closed stop cock attached to the vessel cannulae. (B) Vein segment with a 1 mL syringe inserted into the catheter sheath. (C) Vein segment with stop cocks inserted into both ends. (D) Vein set up as an organ culture. Scale bars in panels (A) and (B) represent 1.25 cm, scale bar in (C) represents 2 cm, and scale bar in panel (D) represents 0.5 cm.

3.3 Organ Culture

  1. 1.

    After incubation, remove the cannulae by cutting the vein segment at the end of the cannula with microscissors.

     
  2. 2.

    Place the infected region of the vein into a small glass Petri dish containing set Sylgard resin and 10 mL of wash medium.

     
  3. 3.

    Using microscissors cut the vein longitudinally to open it out and then transversely into three 5–10 mm segments (see Note 13 ).

     
  4. 4.

    Divide the control vein similarly.

     
  5. 5.

    Pin each vein segment down, endothelial surface uppermost on a 15×15 mm square of polyester mesh resting on set Sylgard resin in glass Petri dishes with minuten pins ( Fig. 2 D).

     
  6. 6.

    Wash the segment twice with 5 mL of wash media.

     
  7. 7.

    Add 5 mL of incubation medium and then culture for up to 14 d at 37°C under 95% air/5% CO2.

     
  8. 8.

    Every 2 d remove incubation medium, wash vein segment twice with 5 mL aliquots of wash medium, and add 5 mL of fresh incubation medium.

     
  9. 9.

    At the required time point, remove vein segments (see Note 14 and 15 ).

     

4 Notes

  1. 1.

    Ensure that vein used is of good quality by collecting as soon as possible after the completion of the bypass surgery. Vein that is not fresh will have poor intimal formation.

     
  2. 2.

    Check that the vein has some remaining endothelial cells by immunocytochemistry as the presence of endothelial cells is essential for intimal thickening in this model.

     
  3. 3.

    When removing the adventitia be careful to avoid cutting small holes in the vessel wall.

     
  4. 4.

    When choosing the segment of vein for gene transfer, avoid areas of vein with side branches because these may be a source of leakage and because valves are commonly situated around the side branches.

     
  5. 5.

    Although this method describes the use of human saphenous vein, it could also be used to infect animal veins or arteries or human arteries. However, if other vessels are used, it may be necessary to change the size of vessel cannulae and catheter sheath.

     
  6. 6.

    Take care to avoid vein segments with valves, because these can obstruct the entry of adenovirus into the lumen of the entire length of the vein segment.

     
  7. 7.

    When tying the knots in the silk suture to secure the vessel cannula and catheter sheath, check that these are secure and that they are not too close to the end of the vein or the cannulae, as the injection of the adenovirus may dislodge them.

     
  8. 8.

    When taking the adenovirus up into the 1 mL syringe avoid bubbles.

     
  9. 9.

    Always ensure that a little air is taken into the 1 mL syringe before the adeno-virus (approx 200 μL of volume). This will propel the virus through the catheter sheath and into the lumen of the vein.

     
  10. 10.

    After insertion of the second stopcock and during the 1 h incubation period, the vein can be gently massaged with sterile forceps to mix the contents of the vein lumen.

     
  11. 11.

    During the 1 h incubation it must be ensured that the vein segment is completely immersed in wash medium so that the surface of the vein does not dry out.

     
  12. 12.

    If the vein appears deflated and the lumen contents have leaked out, the vein segment should be discarded because infection of nonlumenal cells may have occurred.

     
  13. 13.

    When cutting the infected vein segment into three 5–10 mm segments, the position from which the vein segment was taken should be noted. This will allow the identification of problematic regional infections.

     
  14. 14.

    After required culture period, wash the vein segments twice with PBS. To detect luminal reporter gene, expression, fix the vein segment in situ and stain. Typical luminal detection of β-galactosidase expression is shown in Fig. 3 A. Then paraffin wax embed the vein segment and cut transverse sections. Typical β-galactosidase expression in a transverse section is shown in Fig. 3 B. To detect specific protein expression and examine the effect of the gene transfer on neointimal thickening, fix the vein segment in 10% formal buffered saline and paraffin wax embed or freeze in isopentane, and store at −70°C. Cut paraffin or frozen sections and carry out histological staining and immunocytochemistry (5,6).

     
  15. 15.

    We have used this organ culture model to maintain vein segments for a maximum of 14 d without loss of cell viability. Prolonged incubation after 14 d or use of large vein segments in culture will lead to loss of cell viability and distortion of data.

     
Fig. 3.

Detection of the reporter gene β-galactosidase in vein segments after 7 d of culture. (A) Typical en face staining of infected vein segment, dark color indicates cells expressing β-galactosidase. (B) Control vein segment with no (β-galactosidase expression. (C) Transverse section of infected vein segment, dark color on luminal surface cells indicates cells expressing β-galactosidase. (D) Transverse section of control vein segment with no β-galactosidase expression. Scale bar in panel (A) applies to panels (A and B), and represents 0.1 cm and scale bar in panel (C) applies to panels (C and D) and represents 25 μm.

References

  1. 1.
    Bryan, A. J. and Angelini, G. D. (1994) The biology of saphenous vein graft occlusion: etiology and strategies for prevention. Curr. Opin. Cardiol. 9, 641–649.CrossRefPubMedGoogle Scholar
  2. 2.
    Campeau, L., Enjalbert, M., Lesperance, J., Bourassa, M. G., Kwiterovich, P., Jr., Wacholder, S., and Snideman, A. (1984) The relation of risk factors to the development of atherosclerosis in saphenous-vein bypass grafts and the progression of disease in the native circulation. N. Eng. J. Med. 311(21), 1329–1332.CrossRefGoogle Scholar
  3. 3.
    Lytle, B. W., Loop, F. D., Cosgrove, D. M., Ratliff, N.B., Easly, K., and Taylor, P. C. (1985) Long term (5 to 12 years) serial studies of internal mammary artery and saphenous vein coronary bypass grafts. J. Thorac. Cardiovasc. Surg. 89, 248–258.PubMedGoogle Scholar
  4. 4.
    Soyombo, A. A., Angelini, G. D., Bryan, A. J., Jasani, B., and Newby, A. C. (1990) Intimal proliferation in an organ culture of human saphenous vein. Am. J. Pathol. 137(6), 1401–1410.PubMedGoogle Scholar
  5. 5.
    George, S. J., Williams, A., and Newby, A. C. (1996) An essential role for platelet derived growth factor in neointima formation in human saphenous vein in vitro. Atherosclerosis 120, 227–240.CrossRefPubMedGoogle Scholar
  6. 6.
    George, S. J., Johnson, J. L., Angelini, G. D., Newby, A. C., and Baker, A. H. (1998) Adenovirus-mediated gene transfer of the human TIMP-1 gene inhibits SMC migration and neointima formation in human saphenous vein. Human Gene Ther. 9, 867–877.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 1999

Authors and Affiliations

  • Sarah J. George
    • 1
  • Andrew H. Baker
    • 1
  1. 1.Bristol Heart InstituteUniversity of BristolBristolUK

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