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J Thorac Cardiovasc Surg 1995;109:716-720
© 1995 Mosby, Inc.
CARDIAC AND PULMONARY REPLACEMENT |
Los Angeles, Calif.
From the Division of Cardiothoracic Surgery, Department of Surgery, and Division of Cardiology, Department of Medicine, University of California at Los Angeles Medical Center, Los Angeles, Calif.
Address for reprints: Hillel Laks, MD, Division of Cardiothoracic Surgery, UCLA Medical Center, CHS 62-182A, 10833 LeConte Ave., Los Angeles, CA 90024.
Abstract
Access to the donor heart at the time of harvest provides a unique opportunity for genetic manipulation of this organ before transplantation. We sought to determine (1) if donor mouse hearts express a foreign gene administered at harvest and, (2) if so, what route of gene delivery is most effective. At harvest, 30µg of promoter cytomegalovirus-luciferase deoxyribonucleic plasmid in cationic liposomes was injected directly into the myocardial apex (group I), into the right atrium (group II), or into the coronary arteries (group III). The donor hearts were then transplanted into the abdomen of recipient mice of the same strain. The transplanted hearts were removed in 4 days and luciferase expression was assayed by immunohistochemistry. In group I, luciferase activity was localized to the apex. In group II, where plasmid was delivered into the right atrium, luciferase expression was detected in the right ventricle and sparsely in the coronary perivascular area. In group III, where plasmid was injected into the coronary arteries, the transplanted hearts demonstrated luciferase expression in (1) perivascular areas surrounding coronary arteries and veins, (2) coronary capillaries, and (3) the endocardia of both ventricles. This study suggests that (1) donor mouse hearts can be genetically modified at the time of harvest and (2) intracoronary infusion of plasmid yields the most effective method of delivery. Administration of plasmid in the coronary arteries localizes the expression to the endocardium and the coronary vasculature, both sites of immunologic interactions after heart transplantation. (J THORAC CARDIOVASC SURG 1995;109:716-20)
The concept of gene transfer refers to alteration of genetic content of target cells via addition of foreign gene. The development of technology to transfer genetic material into the heart and vasculature of experimental animal models has opened new frontiers in studying the pathogenesis and treatment of cardiovascular diseases. The objective of gene therapy is the efficient delivery of desired genetic material to target cells, resulting in stable and localized production of a recombinant protein. The feasibility of gene transfer to arterial wall cells has been demonstrated in several animal models with a variety of techniques. 
1-5 However, an optimal system for direct in vivo gene transfer that meets all of these criteria is yet to be developed.
The application of gene transfer to the field of transplantation is uniquely appealing because of access to the donor organ at the time of harvest. It is potentially feasible that a foreign gene may be transferred to the harvested organ, creating a "transgenic" allograft. The application of gene transfer techniques to experimental transplantation has been limited. 6 We therefore sought to determine (1) if donor mouse hearts express a foreign gene administered at harvest and (2) if so, what route of gene delivery is most effective.
METHODS
Construction of expression vector
The firefly luciferase gene was used as the marker gene for this study. The plasmid expression vector, pCMV-luciferase, consisted of a full-length firefly Photinus pyralis luciferase complementary deoxyribonucleic acid (cDNA) under the control of cytomegalovirus promoter/enhancer. 2 The vehicle for transfer was cationic liposome. Thirty µg of CMV-luciferase DNA plasmid and 50 µg of cationic liposomes (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethyl-ammonium chloride; Lipofectin; Bethesda Research Laboratories, Gaithersburg, Md.) were added to reduced serum medium (Opti-MEM; Gibco Laboratories, Grand Island, N.Y.) to a final volume of 200 µl. The DNA-liposome solution was prepared approximately 30 minutes before the transfection procedure.
Experimental groups
Both donor and recipient mice wer of C57BL/6 strains (Jackson Laboratories, Bar Harbor, Maine). Donor hearts were arrested with cold, diluted (Because of high viscosity) University of Wisconsin solution (Du Pont Pharmaceutical, Wilminton, Del.). In group I (n = 4), the DNA-lipsome solution was directly injected into the myocardioal apex on cardiac arrest. The plasmid-liposome solution in group II (n = 4), with the distal ascending aorta clamped, the plasmid-liposome solution was directly injected into the ascending aorta over 2 to 3 minutes.
Transplantation
Heterotopic intraabdominal transplantation was performed by means of the technique previously described by Corey, Winn, and Russell. 7 In brief, donor hearts were arrested with dilute, cold University of Wisconsin solution. The plasmid-liposome solution was administered according to the protocol described earlier. The donor hearts were excised in standard fashion and stored in the University of Wisconsin solution until the recipient was prepared. During this time, the transfection medium was in contact with the donor heart. The plasmid-liposome solution was not washed out of the donor organ before transplantation. The heterotopic cardiac transplantation as performed by anastomosing the donor aorta to the side of the recipient infrarenal aorta. An end-to-side anastomosis between the donor pulmonary artery and recipient infrarenal inferior vena cava was performed next. The total ischemic time was 45 minutes to 1 hour, which was also the duration of exposure of donor hearts to plasmid-liposime solution. The function of the transplanted hearts was assessed daily by abdominal palpation. 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" prepared by the Institute of Laboratory Animal Resources and published by the National Insitutes of Health (NIH Publication No. 86-23, revised 1985).
Analysis of luciferase expression
The transplanted and recipient hearts were harvested in 4 days when the mice were sacrificed. Miltiple 10 µm thick cross sections of the hearts at the apex and midportion of the ventricle were obtained. Luciferase expression was assayed immunohistochemically with a polyclonal rabbit immunoglobulin G (Cortex Biochem, Inc., San Leandro, Calif.). Immunohistochemical staining was performed in a three-step indirect avidin-biotin immunoperoxidase assay (Vector, ABC Kit, Burlingame, Calif.) 8 Luciferase expression was assessed by one of us (J. H. Q.) in a blind fashion.
RESULTS
All donor hearts were functioning well at the time of harvest; no acute cellular rejection was apparent on histologic evaluation. Immunohistochemical staining of the donor hearts at the mid-ventricular level for luciferase expression is illustrated in Table I. The mid-ventricular cross sections of the hearts in group I did not reveal any luciferase expression. However, when apical cross sections were examined, luciferase expression could be detected on the apical vasculature and myocytes. In group II, the plasmid-liposome solution was administered through the inferior vena cava into the right atrium; the donor hearts in this group revealed luciferase expression on the right ventricular endocardium and sparse expression around the coronary vasculature. No luciferase expression could be detected on the left ventricular endocardium in group II. In group III, plasmid-liposome solution was injected into the aortic root, perfusing the coronary arteries in an antegrade fashion. Luciferase expression was evident on the left ventricular endocardium, right ventricular endocardium, coronary capillaries, and around the coronary vessels (Figs. 1 and 2).
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DISCUSSION
The findings of this study suggest that donor mouse hearts can be transfected at the time of harvest. Intracoronary infusion of plasmid appeared to be an efficacious route of delivery of reporter plasmid, yielding detectable expression on the endocardia, coronary capillaries, and perivascular area. Expression of the reporter plasmid was localized to the transplanted hearts and did not appear to induce cellular rejection of the isografts.
The utility and importance of gene therapy for several disorders has recently been reviewed. 9,10 Direct in vivo gene transfer to vascular wall cells represents an ideal system for production of secreted proteins with locally therapeutic effects. Extension of the principles of experimental gene transfer to heart transplantation has been limited. Wang and associates 6 studied the expression of reporter genes in a rat cardiac isograft model after direct apical gene injection. They observed stable expression of the reporter gene locally for 2 months. The present study demonstrates that foreign genes can be introduced into the donor hearts at the time of harvest. Among the three tested routes of delivery, the intracoronary administration of expression vector yielded the most widespread and strategically important transfection. Luciferase expression was observed on the endothelial cells and in the perivascular area. The endocardium and perivascular area, the sites of initial host-recipient immunologic interaction, are the ideal sites for introduction of genes intended to alter the immune response. This study further documents that gene transfer to the donor hearts at the time of harvest is safe (no evidence of cellular rejection in transplanted hearts), localized (reporter plasmid expression confined to the donor heart), and feasible.
Application of direct gene transfer techniques to the field of heart transplantation offers several possibilities: (1) studying the role of individual genes separately in immunologic interactions, (2) the development of localized therapeutic measures limited to the donor heart, (3) evaluation of gene regulatory/promoter mechanisms responsible for expression of endogenous proteins such as histocompatibility antigens, and (4) alteration of donor organ "foreignness" by controlling human leukocyte antigen (HLA) expression via antisense oligonucleotide transfer. 11 The latter technique allows for transfer of recombinant genes coding for messenger ribonucleic acid (mRNA), which binds to HLA mRNA and effectively blocks HLA expression. Genetic modification of nonhuman hearts to allow xenotransplantation is an established area of investigation. 12 Application of gene transfer technology at the time of donor procurement to the field of xenotransplantation may further the goals of this area of research. Improvements in gene transfer methods to yield efficient delivery techniques, producing biologically effective recombinant protein levels, in specific cellular targets are needed to further the applications of gene therapy in transplantation.
In conclusion, donor mouse hearts can be transfected by a reporter plasmid at the time of harvest Intracoronary infusion of reporter plasmid yielded detectable expression in perivascular areas and endocardia, both sites of immunologic interaction between the donor and the host after transplantation. Intracoronary administration of plasmid may allow genetic alteration of donor organs in the near future.
Appendix: DISCUSSION
Dr. Robert A. Guyton (Atlanta, Ga.).
I am confused about the exposure of the plasmid to the donor heart. Was the plasmid-luciferase combination injected before arrest of the donor heart, that is, while the donor heart was metabolically active, and what was the duration of exposure of the donor heart to the plasmid? Was the donor heart at 4° C while the plasmid was exposed to the donor heart?
Dr. Ardehali.
The donor hearts were arrested with University of Wisconsin solution; the plasmid-liposome solution was then administered according to the described protocol. The allografts were not flushed thereafter. The hearts were then preserved in cold University of Wisconsin solution for 45 minutes to 1 hour until the recipients were prepared. Therefore, transfection occurred during the cold ischemic time (45 minutes to 1 hour).
Acknowledgments
We gratefully acknowledge the technical assistance of Thomas Drake, MD, in photomicrography.
Footnotes
Read at the Seventy-fourth Annual Meeting of The American Association for Thoracic Surgery, New York, N.Y., April 24-27, 1994. 
References
This article has been cited by other articles:
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