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Ann Thorac Surg 1999;68:1810-1814
© 1999 The Society of Thoracic Surgeons

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Original Articles

Transfection of pulmonary artery segments in lung isografts during storage

^a Division of Cardiothoracic Surgery, Washington University School of Medicine, St. Louis, Missouri, USA

Address reprint requests to Dr Patterson, Division of Cardiothoracic Surgery, Washington University School of Medicine, 3108 Queeny Tower, One Barnes Hospital Plaza, Saint Louis, MO 63110

	Abstract

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Background. Proximal pulmonary artery segment (PPAS) endothelial transfection of lung grafts may be useful in ameliorating ischemia-reperfusion injury and rejection and may provide beneficial downstream effects on the whole lung graft. Transfection immediately after lung transplantation may be efficacious in ameliorating allograft dysfunction after transplantation.

Methods. In F344 rats, the PPAS was isolated and injected with 0.03 mL of GL-67/DOPE–chloramphenicol acetyl transferase (CAT) plasmid DNA. The PPASs were exposed for 60 minutes at several temperatures. The lung grafts were stored in saline solution (group 1, n = 24) or LPDG solution (group 2, n = 27) for 12 or 24 hours at 4° to 37°C. In group 3 (n = 42), PPASs were stored in endothelial cell culture medium and incubated at 10° or 37°C in a carbon dioxide incubator for 3 to 72 hours. Group 4 (n = 18) served as transplanted controls; after 3 to 24 hours’ preservation at 4°C in LPDG solution, lung grafts were transplanted. Transgene expression of PPASs was assessed with two CAT activity assays, thin-layer chromatography enzyme-linked immunosorbent assay and immediately after the preservation period (groups 1 to 3) or 24 hours after transplantation (group 4).

Results. In group 1, transgene expression did not appear. In groups 2 and 3, transgene expression was apparent after any storage duration at 37°C. Transgene expression increased successively with longer storage periods. In group 4, transgene expression was detected after any storage duration. The enzyme-linked immunosorbent assay is able to quantify the expression of CAT activity, but thin-layer chromatography is more sensitive.

Conclusions. Transgene expression did not occur during conventional cold storage. Transgene expression in rat PPASs during storage is possible with warm storage (37°C) and appropriate storage solution.

	Introduction

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Ischemia-reperfusion injury and rejection remain major obstacles to successful transplantation [1]. Gene transfer to the lung graft has the potential to reduce these substantial problems. Recently, we [2, 3] demonstrated ex vivo gene transfer to whole-lung isografts using an adenoviral or liposomal vector. However, because of the vast surface area of the pulmonary microvasculature, a large amount of gene-vector construct is necessary to obtain an efficient vector to host cell ratio. High concentrations of deoxyribonucleic acid (DNA) can be associated with toxicity [4]. Gene transfer to the proximal pulmonary artery segment (PPAS) of the donor lung could avoid this difficulty and possibly provide a beneficial downstream effect to the entire graft. We [5] have demonstrated the feasibility of gene transfer to PPASs using both adenoviral and liposomal vectors. Further, [6] we have examined the efficacy of ex vivo transforming growth factor (TGF)-ß1 gene transfection using a rat lung ischemia-reperfusion injury model. In these experiments, ex vivo TGF-ß1 gene transfection of either the PPAS or the whole donor lung did not decrease subsequent ischemia-reperfusion injury. We speculated that early ischemia-reperfusion injury had already occurred before the expression of TGF-ß1 protein. Transgene expression during storage or immediately after reperfusion is likely necessary to reduce lung allograft dysfunction after transplantation. The aim of this study was to determine the feasibility of gene transfer to PPASs of lung grafts during storage.

	Material and methods

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Cationic lipid–plasmid DNA complexes
The plasmid pCF1-CAT (Genzyme Corporation, Framingham, MA) consists of the human cytomegalovirus immediate early gene promoter/enhancer, a hybrid intron, the chloramphenicol acetyl transferase (CAT) complementary DNA, the bovine growth hormone polyadenylation signal sequence, and the kanamycin resistance gene, as previously described [7]. Lipid 67 (GL-67; Genzyme Corporation) is an amphiphile consisting of a hydrophobic cholesterol lipid anchor linked to a spermine head group in a T-shaped configuration and was used in a 1:2 molar ratio with the neutral co-lipid, L-dioleoyl phosphatidylethanolamine (DOPE). Before use, dried lipid films were hydrated with sterile water or saline solution, vortexed, placed on ice for 10 minutes, and then vortexed again. Equal volumes of GL-67/DOPE and plasmid DNA were mixed and incubated at room temperature for 30 minutes. The final concentrations were 1 mmol/L cationic lipid and 4 mmol/L plasmid DNA.

Animals
Inbred male F344 rats (Harlan Sprague Dawley Inc, Indianapolis, IN) weighing 250 to 290 g were used in all experiments. All animal procedures were approved by the Animal Studies Committee at Washington University. 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).

Gene transfer to pulmonary artery segments
Transfection of pulmonary artery segments was performed as previously described [5]. With anesthesia, mechanical ventilation, and systemic heparinization, donor rat lungs were flushed through the main pulmonary artery with 20 mL of cold (4°C) saline solution or low-potassium–dextran–1% glucose (LPDG) solution. Heart-lung blocks were extracted, and the left pulmonary artery was isolated from the hilum to the proximal pulmonary trunk. A 24-gauge polyethylene catheter was inserted from the right ventricle into the left pulmonary artery, which was then clamped distally. The proximal end of the left pulmonary artery was ligated over the catheter just distal to the main pulmonary artery bifurcation. Lipid-gene construct, 0.03 mL was injected into the isolated left pulmonary artery segments (PPAS). After injection, lung grafts were stored in the storage solution at each of several temperatures to be described. One hour after injection, the ligature and the distal clamp were removed from the pulmonary artery.

Endothelial cell culture medium
Endothelial cell culture medium (ECCM) was prepared on the basis of RPMI (Roswell Park Memorial Institute) 1640 medium [8] and supplemented with 5 mmol/L HEPES buffer, 10 mmol/L sodium pyruvate, 2 mmol/L L-glutamine, 20% heat-inactivated fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin, 15 µg/mL endothelial cell growth supplement (Collaborative Biochemicals Inc, Bedford, MA), and 17.6 U/mL heparin.

Experimental design and groups
Lung grafts were allocated into study groups depending on the conditions of storage.

Saline storage group (group 1)
Lung grafts were flushed with 20 mL of cold saline solution (4°C), and PPASs were exposed to lipid-gene construct for 1 hour at 4°, 10°, 23°, or 37°C in saline solution. Subsequently the lung grafts were stored for 12 or 24 hours in saline solution at each temperature (n = 3 per group).

LPDG storage group (group 2)
Lung grafts were flushed with 20 mL of cold LPDG solution (4°C), and PPASs were exposed to lipid-gene construct for 1 hour. Lung grafts were stored for 12 hours in LPDG solution at 4°, 10°, 23°, or 37°C (n = 3 per group) or 24 hours at 4°, 10°, 23° (n = 3 per group), or 37°C (n = 6) in LPDG solution.

ECCM storage group (group 3)
Pulmonary artery segments were flushed with 20 mL of cold LPDG solution (4°C) and exposed to lipid-gene construct for 1 hour at 10° or 37°C in ECCM. Subsequently PPASs were stored and incubated for 3, 6, 12, 24, 48, or 72 hours (n = 6 per group) in ECCM at 37°C in a carbon dioxide incubator or stored for 12 or 24 hours in ECCM at 10°C (n = 3 per group).

Transplanted controls (group 4)
Transplanted controls were made to be compared with the other three groups. Pulmonary artery segments were flushed with 20 mL of cold LPDG solution (4°C), exposed to lipid-gene construct for 1 hour, and stored for 3 (n = 6), 6, 12 (n = 3 per group), or 24 hours (n = 6) in LPDG solution at 4°C. Subsequently, the lung graft was implanted using a modification of the previously described cuff technique [9].

Gene expression
Chloramphenicol acetyl transferase expression in the PPASs was determined immediately after the preservation period in groups 1, 2, and 3 and 24 hours after transplantation in group 4. The assessments of CAT expression were used as described here.

Chloramphenicol acetyl transferase activity assay
Three animals in each group noted in Table 1 were assessed by CAT activity assay using thin-layer chromatography (TLC). The CAT activity assay has been described previously [3]. In the presence of functional CAT enzyme, both monoacetylated and diacetylated forms of chloramphenicol are produced, which are distinct from the nonacetylated chloramphenicol by TLC. Combined densitometry of both monoacetylated and diacetylated chloramphenicol was determined using the NIH Image program for Macintosh, 1998. The density of expression in PPASs stored for 24 hours in group 4 was established as the standard (100%) by which to compare the other groups.

	Results

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View larger version (29K): [in this window] [in a new window]   Fig 1. Relative density of chloramphenicol acetyl transferase activity. Mean density of monoacetylated and diacetylated forms of chloramphenicol from thin-layer chromatography was measured using the NIH Image program. The density found in proximal pulmonary artery segments (PPASs) stored for 24 hours in group 4 was established as the standard (100%) against which to compare the other groups. The PPASs stored at 37°C in groups 2 and 3 showed gross transgene expression, and the expression increased successively. In group 4, similar and gross expression appeared in all PPASs.

LPDG storage group (group 2)
Transgene expression was not apparent on TLC after either storage period (12 and 24 hours) at 4° or 10°C. Using densitometry, transgene expression was not detected after either storage period at 4° or 10°C (1.0% ± 0.7% to 1.3% ± 0.2%). After 24 hours’ storage at 23°C, very faint transgene expression was apparent (5.6% ± 5.9%). Faint transgene expression appeared on TLC after 12 hours’ storage at 37°C (25.0% ± 10.8%), and this expression increased after 24 hours’ storage (78.8% ± 36.7%).

ECCM storage group (group 3)
In the group stored for 3 hours at 37°C, faint transgene expression was apparent (10.9% ± 15.3%). After 6 hours’ storage, density of CAT was decreased, but it then gradually increased with longer times to a maximum in the 72 hours’ storage group (12, 24, 48, and 72 hours: 27.3% ± 11.1%, 48.5% ± 25.1%, 83.9% ± 40.4%, and 103.5% ± 32.2%, respectively).

Transplanted controls (group 4)
In transplanted controls, significant levels of transgene expression were detected at all storage periods (3, 6, 12, and 24 hours: 94.2% ± 101.7%, 94.6% ± 23.3%, 102.9% ± 36.5%, and 100.0% ± 41.7%, respectively).

ELISA for chloramphenicol acetyl transferase
The ELISA was performed to quantify CAT and compare results with the CAT activity assay results (Fig 2). The groups assessed by CAT ELISA were selected on the basis of a positive signal on CAT activity assay. For example, group 1 was not assessed because CAT expression was not detected in the CAT activity assay. In group 4, the 3 and 24 hours’ storage groups were selected for CAT ELISA.

View larger version (24K): [in this window] [in a new window]   Fig 2. Quantification of chloramphenicol acetyl transferase (CAT) by enzyme-linked immunosorbent assay. The quantity of CAT enzyme was measured in nanograms per segment of pulmonary artery (ng/PA). The proximal pulmonary artery segments stored for 24 hours at 37°C in group 2 showed a high level of CAT. In group 3, CAT was detected and increased after 12 hours’ storage. In group 4, CAT was detected with a high standard deviation.

ECCM storage group (group 3)
In lung grafts stored for 3 and 6 hours, CAT enzyme was not detected by ELISA (0.00 ± 0.00 ng/PA), although transgene expression was apparent in the CAT activity assay (TLC). In the 12-hour storage group, CAT enzyme was detected, and levels successively increased with storage time (12, 24, 48, and 72 hours: 0.04 ± 0.01, 0.07 ± 0.04, 0.06 ± 0.01, and 0.16 ± 0.04 ng/PA).

Transplanted controls (group 4)
In group 4, grafts stored for 3 and 24 hours were assessed by CAT ELISA. The quantities of CAT enzyme were 0.13 ± 0.17 and 0.40 ± 0.59 ng/PA, respectively.

	Comment

Top Abstract Introduction Material and methods Results Comment References

 
Transfection of a PPAS and subsequent gene expression offers potential advantages over more widespread gene delivery strategies. First, it is possible to confine the cationic lipid–plasmid DNA complexes to the isolated arterial segments and not expose other organs to a gene product unnecessarily. Second, using high vector to endothelial cell ratios and ideal storage times, transgene expression efficiency might be maximized. Third, if a potential therapeutic transgene is used, downstream effects of the secreted protein may be beneficial to the whole graft.

We have demonstrated consistent and reproducible transgene expression when the cationic lipid GL-67 is used to transfect whole lung grafts [3] or proximal segments of the pulmonary artery [5]. Cationic lipids have been demonstrated to provide both a safe and an efficient method of gene transfection. Liposomes have no replication risk and do not activate the host immune-inflammatory response, which does occur with viral vectors. They also provide easier transfection protocols. No toxicity has been detected even with repeated transfection [10, 11].

Ischemia-reperfusion injury and rejection remain major obstacles to successful transplantation [12]. Ischemia-reperfusion injury begins immediately after reperfusion and is amplified during the first 60 minutes of reperfusion [13, 14]. It is probable that a transfection strategy aimed at decreasing reperfusion injury would have to result in successful transgene expression prior to or immediately after reperfusion. We [6] examined the efficacy of ex vivo TGF-ß1 gene transfection using a rat lung ischemia-reperfusion injury model. However, ex vivo TGF-ß1 gene transfection of either a PPAS or the whole lung did not affect ischemia-reperfusion injury in rat lung grafts. We speculated that ischemia-reperfusion injury had already occurred in this experimental model before the expression of TGF-ß1. Transgene expression immediately after transplantation or during storage may be the ideal requirement for ex vivo gene transfer. Transgene expression immediately after lung transplantation could potentially decrease allograft dysfunction after transplantation.

In these experiments, saline solution was used as storage solution at three different temperatures (4°, 10°, 23°, and 37°C) in group 1. Transgene expression was not detected after either 12- or 24-hour storage at any temperature. In group 2, the extracellular LPDG solution was selected because of its purported beneficial effects on lung grafts during storage. Transgene expression was not detected after either storage period at cold temperatures (4° or 10°C). Very faint expression appeared after 24 hours’ storage at 23°C. In the LPDG group, warm storage at 37°C was required to achieve significant levels of transfection during storage. In the transplanted controls (group 4), significant and similar levels of transgene expression were detected from all PPASs at all storage periods. This demonstrates that under conditions of cold storage, the ex vivo transfection strategy does not result in adequate gene expression until subsequent reperfusion of the PPASs.

The lack of transgene expression in the absence of reperfusion is intriguing. In liver transplant models, endothelium is more sensitive to ischemic damage than parenchymal cells [15]. Severe hypothermia for a prolonged period is harmful to cultured umbilical vein endothelial cell structure and viability in vitro [16]. To confirm the presence of endothelium in the PPAS, we extracted the endothelial cell monolayer from PPASs stored for 3 to 24 hours in LPDG solution at 4°C using the techniques reported by Hirsch and colleagues [17]. The integrity of the endothelial cell monolayer was maintained in all PPASs (data not shown). Speculating that the lack of transgene expression was due to the lack of endothelial cell viability, we used ECCM and warm temperature (37°C).

In group 3 grafts stored for 3 hours in ECCM, faint transgene expression was apparent. Almost successively, the density increased, with maximum expression in the 72-hour-storage group. The 48 and 72 hours’ storage groups showed expression and densities comparable to transplanted controls. However, transgene expression did not appear after cold storage in ECCM.

For the quantification of CAT, CAT ELISA was performed. The highest level of CAT was detected in the group stored for 24 in LPDG solution at 37°C (group 2). The transplanted controls (group 4) had a high standard deviation compared with these values in the other groups. The CAT activity assay is more sensitive than the CAT ELISA assay. In group 3, CAT was not detected by ELISA in either 3- or 6-hour-storage groups at 37°C, though CAT activity was apparent in the CAT activity assay. However, CAT ELISA did quantify CAT enzyme levels, and results were comparable among groups that had similar densities in the CAT activity assay.

In conclusion, transgene expression in PPASs during storage is possible. Expression successively increases during 72 hours under conditions of warm storage in appropriate storage solution.

	Acknowledgments

 
We thank Kathleen Grapperhaus for technical assistance and Dawn Schuessler for secretarial support. This work was supported by National Institutes of Health grant R01 HL41281.

	References

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