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Ann Thorac Surg 1999;68:1805-1809
© 1999 The Society of Thoracic Surgeons

Original Articles

Ex vivo transfection of pulmonary artery segments in lung isografts

Motoki Yano, MDa, Masafumi Hiratsuka, MDa, Itaru Nagahiro, MDa, Bassem N. Mora, MDa, Ronald K. Scheule, PhDa, G. Alexander Patterson, MDa

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
Top
 Abstract
Introduction
 Material and methods
 Results
 Comment
 References
 
Background. Gene transfer to lung grafts may be useful in ameliorating ischemia-reperfusion injury and rejection. Proximal pulmonary artery endothelial transfection may provide beneficial downstream effects on the whole lung graft. We have already demonstrated the feasibility of in vivo and ex vivo transfection in proximal pulmonary artery segments of rat lung grafts. The aim of this study was to determine the optimal conditions for and duration of transfection.

Methods. Orthotopic left lung transplantation was performed in F344 rats after donor lung proximal pulmonary artery segments were isolated and injected with lipid 67/DOPE–chloramphenicol acetyl transferase (CAT) complementary deoxyribonucleic acid construct. Effect of exposure time was studied by exposing donor pulmonary artery segments to the construct for 0, 30, and 60 minutes prior to transplantation. In another series of experiments, pulmonary artery segments were exposed to the construct for 60 minutes prior to transplantation. Onset and duration of gene expression were determined after sacrificing animals at 3, 6, 12, and 24 hours and 3 days as well as 1 week, 2, 4, and 8 weeks after transplantation. Effect of exposure temperature was studied by exposing pulmonary artery segments to the construct for 60 minutes at 4°, 10°, and 23°C. These recipients were sacrificed on postoperative day 3. Effect of exposure pressure was studied by using two volumes of the construct (0.01 and 0.03 mL). These recipients were sacrificed on postoperative day 3. Transgene expression was assessed by chloramphenicol acetyl transferase activity assay.

Results. Transgene expression was similar after 30- and 60-minute exposure. Transgene expression was evident within 3 to 6 hours after operation and persisted at 8 weeks after operation. Expression was detected at all temperatures and was equivalent at both exposure pressures.

Conclusions. Gene transfection into graft pulmonary artery segments is possible under a range of conditions applicable to clinical lung transplantation.


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Comment
 References
 
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 vector or lipid. However, because of the vast surface area of the pulmonary microvasculature, a large amount of gene construct is necessary to obtain an efficient vector to host cell ratio. High concentrations of deoxyribonucleic acid (DNA) have also been a problem [4]. On the hypothesis that gene transfer to the proximal segment of pulmonary artery could avoid this disadvantage and allow a beneficial downstream effect to the whole graft, we [5] demonstrated the feasibility of gene transfer to pulmonary artery segments using both adenoviral and liposomal vectors. Our preliminary work suggested that this strategy might have a beneficial downstream effect.

Numerous studies have been undertaken to determine optimal conditions for gene transfer in vitro. Studies regarding optimum conditions for in vivo transfection are limited. In addition, the factors controlling ex vivo transfection are poorly understood. The aim of this study was to examine the influence of factors such as exposure time, temperature, and pressure that may affect efficiency and duration of transfection of graft proximal pulmonary artery segments.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Comment
 References
 
Cationic lipid–plasmid DNA complexes
In this study, cationic lipid chloramphenicol acetyl transferase (CAT) gene complex was selected because this complex is excellent as a reporter gene complex. It is easily prepared. We have developed substantial experience with the necessary assays described here. The plasmid pCF1-CAT (Genzyme Corporation, Framingham, MA) consists of the human cytomegalovirus immediate early gene promoter/enhancer, a hybrid intron, the CAT complementary DNA, the bovine growth hormone polyadenylation signal sequence, and the kanamycin resistance gene, as previously described [6]. 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, 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 concentration was 1.32 mg DNA/mL (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).

Ex vivo gene transfer to pulmonary artery segments
Orthotopic left lung transplantation was performed by means of a modification of the previously described cuff technique [7]. Ex vivo transfection of the pulmonary artery 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 to avoid precipitation of cationic lipid. Heart-lung blocks were extracted, and the donor 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 proximal left pulmonary artery segment. After injection, lung grafts were stored in saline solution. Subsequently, the ligature and distal clamp were removed from the pulmonary artery. The donor left lung grafts were implanted immediately after the various exposure periods to be described.

Experimental design and groups
Effect of exposure time
The effect of exposure time was studied by exposing donor pulmonary artery segments to lipid-gene construct for three different periods at 10°C. In group 1 (n = 3), the preparation of the recipient was finished before injection of lipid-gene construct to keep close to 0 minutes’ exposure time. In groups 2 and 3, the lipid-gene construct was confined in the isolated donor proximal pulmonary artery segments for 30 (n = 3) and 60 minutes (n = 6), respectively. Subsequently, the lung grafts were transplanted. Animals were sacrificed on POD 3.

Effect of exposure temperature
The effect of exposure temperature was studied by exposing pulmonary artery segments to lipid-gene construct for 60 minutes at three different temperatures. In groups 4, 5, and 6, lung grafts were stored in saline solution at 4°, 10°, and 23°C (n = 6 per group), respectively. Animals were sacrificed on POD 3.

Effect of exposure pressure
The effect of exposure pressure was studied by using two different injection volumes. To equalize the total amount of lipid-gene construct, 0.01 mL of lipid-gene construct (1.32 mg DNA/mL) was injected in group 7 (n = 6) and 0.03 mL of the lipid solution, which was diluted threefold with saline solution (0.44 mg DNA/mL), in group 8 (n = 6). Animals were sacrificed on POD 3.

Onset and duration of recombinant expression
Pulmonary artery segments were exposed to lipid-gene construct for 60 minutes prior to transplantation. Onset and duration of gene expression were determined after sacrificing animals at 3, 6, 12, and 24 hours and 3 days (n = 3 per group) as well as 1 week, 2, 4, and 8 weeks (n = 4 per group) after transplantation. In these experiments, the group exposed to 0.03 mL of lipid-gene construct (1.32 mg DNA/mL) for 60 minutes at 10°C and sacrificed on postoperative day (POD) 3 (group 3) was established as the standard.

Chloramphenicol acetyl transferase activity assay
Transgene expression was detected by a CAT activity assay as described previously [3, 8]. In the presence of a functional CAT enzyme, both monoacetylated and diacetylated forms of chloramphenicol, which are distinct from the nonacetylated chloramphenicol, are produced by thin-layer chromatography. Combined densitometry of both monoacetylated and diacetylated chroramphenicol was determined using the NIH Image program for Macintosh, 1998.

Graft function
In an effort to determine whether exposure temperature or exposure pressure would have an impact on subsequent graft function in groups 4 to 8, isolated function of the left lung isograft was assessed by arterial blood gas analysis during mechanical ventilation with 100% oxygen (tidal volume, 1.5 mL; respiratory rate, 100/min; and positive end-expiratory pressure, 1.0 cm H2O) as previously described [5].

Statistical analysis
All values are presented as the mean ± the standard deviation. One-way analysis of variance with pairwise comparison by the Fisher method and unpaired two-group t test were used to compare differences between corresponding groups. Differences were considered significant when the p value was less than 0.05.


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Comment
 References
 
Chloramphenicol acetyl transferase activity
Effect of exposure time
Total exposure times were 6.6 ± 0.2, 47.2 ± 2.1, and 84.8 ± 4.5 minutes in groups 1, 2, and 3, respectively. The total exposure times included both the periods of cuff attachment and graft lung implantation. Transgene expression was detected as the presence of monoacetylated and diacetylated forms of chloramphenicol in all groups (Fig 1A). The relative density is shown in Figure 1B. The density of transgene expression in group 3 (60 minutes) was established as the standard (100%). The densities of groups 1 (0 minutes) and 2 (30 minutes) were 115% ± 18% and 105% ± 4% compared with the density of the 60 minute-exposure group (group 3) (100% ± 23%). All pulmonary artery segments showed high levels of CAT activity irrespective of exposure time. There were no significant differences between groups.



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  		Fig 1. Chloramphenicol acetyl transferase (CAT) activity in effect-of-exposure-time assessment groups. (A) Gross transgene expression was detected in all groups. (B) Density of gene expression. The density of the 60 minute-exposure group (group 3) was established as the standard (100%). All pulmonary artery segments showed high levels of CAT activity irrespective of exposure time. There were no significant differences between groups (N.S.).

 
Effect of exposure temperature
The group exposed at 10°C (group 5) was established as the standard. In groups transfected for 60 minutes at different temperatures, significant and similar levels of transgene expression were detected irrespective of exposure temperature (groups 4 [4°C], 5, and 6 [23°C]: 97% ± 9%, 100% ± 23%, and 91% ± 16%, respectively) (Fig 2). There were no significant differences between groups.



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  		Fig 2. Chloramphenicol acetyl transferase activity in effect-of-temperature assessment groups. The group exposed at 10°C (group 5) was established as the standard. Significant and similar levels of transgene expression were detected irrespective of exposure temperature. There were no significant differences between groups (N.S.).

 
Effect of exposure pressure
In both groups, similar levels of transgene expression were detected irrespective of exposure pressure (for group 7 [pressured] and 8 [nonpressured]: 36% ± 9% and 39% ± 10%, respectively) (Fig 3). There were no significant differences between groups. The density of transgene expression in both groups was approximately three times lower than that in the standard group (100% ± 23%), which received three times higher lipid-gene construct.



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  		Fig 3. Chloramphenicol acetyl transferase activity in effect-of-pressure assessment groups. There were no significant differences between groups (N.S.). The density of both groups was approximately three times lower than the standard group with three times higher lipid-gene construct.

 
Onset and duration of recombinant expression
The density of transgene expression in the animals sacrificed on POD 3 was established as the standard. Three hours after reperfusion, faint transgene expression was apparent (26% ± 12%) (Fig 4). Gradually the density increased (30% ± 34% at 6 hours and 34% ± 20% at 12 hours). Twenty-four hours to 3 days after reperfusion, the expression was maximal (74% ± 20% at 24 hours and 100% ± 23% at 3 days). High expression continued for 1 week (51% ± 15%). Transgene expression became faint 2 weeks after transplantation (18% ± 8%) and remained faint in groups studied 4 to 8 weeks postoperatively (4 weeks, 21% ± 17%; and 8 weeks, 20% ± 32%).



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  		Fig 4. Chloramphenicol acetyl transferase activity at the various sacrifice times. Three hours after reperfusion, faint transgene expression was apparent. Gradually the density increased, and 24 hours to 3 days after reperfusion, the expression was maximal. High expression continued for 1 week, and then transgene expression became faint. Four weeks to 8 weeks postoperatively, individual differences were noted, but transgene expression was still apparent.

 
Arterial blood gas analysis
Graft function was assessed by mean arterial oxygen tension (Fig 5). The mean arterial oxygen tension in group 4 (4°C) was higher than that of groups 5 (10°C) and 6 (23°C) (groups 4, 5, and 6: 359.4 ± 41.1 mm Hg, 315.3 ± 62.2 mm Hg, and 249.7 ± 57.6 mm Hg, respectively). However, the differences were not significant. Also there were no significant differences between groups 7 (283.9 ± 41.1 mm Hg) and 8 (331.3 ± 41.6 mm Hg).



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  		Fig 5. Arterial oxygen tension in effect-of-temperature assessment and effect-of-pressure assessment groups. There were no differences between corresponding groups.

 

   Comment
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
References
 
Transfection of a proximal segment of the donor pulmonary artery and subsequent gene expression offers certain advantages over whole-lung gene delivery strategies. First, in the in vivo and ex vivo setting, 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, it may be possible to use a therapeutic transgene that might produce a downstream effect from the secreted protein beneficial to the whole graft. Third, using high vector to endothelial cell ratios and ideal storage times, transgene expression efficiency might be maximized.

In the present study, we tested a liposomal vector. Since the introduction of the first cationic lipid by Felgner and associates [9] in 1987, numerous new lipids have been reported [10–13]. Cationic lipids have been demonstrated to provide both a safe and an efficient method of gene transfection. Liposomes, unlike viral vectors, have no replication risk and do not activate the host immune-inflammatory response, which viral vectors do. Liposomes also provide easier transfection protocols. No toxicity has been detected even with repeated transfection [14, 15]. Liposomes have been regarded as less efficient delivery systems [16]. Despite this perception, we have demonstrated consistent and reproducible transgene expression when the cationic lipid GL-67 was used to transfect whole lung grafts [3] or proximal segments of the pulmonary artery [5].

Studies [17–20] on optimization of cationic lipid–mediated gene transfer have been performed, including the DNA dose, the ratio of DNA to lipid, and the importance of neutral co-lipids, cholesterol, or other substances. However, the factors controlling the efficiency of a cationic lipid transfection system are poorly understood, especially in the context of ex vivo transfection. The ex vivo approach allows manipulation of gene transfer conditions, such as exposure time and temperature.

In this study, we determined the efficiency of three different exposure periods at 10°C. These periods were considered short enough to be possible in the setting of clinical lung transplantation. The CAT activity assays did not show any effect of the exposure times on intensity of transgene expression. In the group with the shortest exposure time (group 1), about 6 minutes of exposure, lipid-gene construct was injected without a proximal clamp. After injection, the lipid-gene construct must be rapidly diluted and spread during the 6 minutes. However, even in group 1, gross expression was readily apparent. The mechanisms of the uptake of transgene into the target cells, transfer of transgene from the cytoplasm to the nucleus, and recombinant gene expression are still unclear, but these data suggest that lipid-gene construct bonds to the endothelium of the donor pulmonary artery segments within several minutes. In short, exposure time does not influence transgene expression when lipid-gene construct is injected into pulmonary artery segments with ex vivo techniques.

Similarly, exposure temperature did not significantly affect the magnitude of transgene expression. Gross expression was detected at all temperatures. This result indicates that this gene transfer system is applicable under the clinical conditions of cold lung preservation.

Exposure pressure did not affect transgene expression in this study. In group 3 with a three times higher dose, relative density of transgene expression was approximately three times higher than that in groups 7 and 8. In a previous study [5], an adenoviral vector traversed the endothelium and internal elastic lamina, and transgene expression was detected even in the smooth muscle cells. Transgene expression using cationic lipid GL-67 was detected only on the endothelium of the pulmonary artery segments, though Keogh and associates [15] reported that cationic lipid–DNA complexes could traverse the endothelium and internal elastic lamina. Under complete confinement of lipid-gene construct, the dose of this construct may be more important than exposure pressure.

The onset and the duration of recombinant gene expression have been reported in numerous in vitro and in vivo studies using cationic lipids. Onset of gene transfection within 5 minutes in vitro [15] and 1 hour after injection in vivo [4] and long-term transgene expression for up to 8 weeks [21, 22] have been noted. In this study, transgene expression was evident within 3 hours after transplantation, and maximal expression was detected 24 to 72 hours after transplantation. By POD 7, the expression decreased gradually but persisted at reduced levels for 8 weeks after transplantation. In this ex vivo model, transgene expression occurred rapidly after graft perfusion and, as expected, proved transient.

As might be expected, graft function in the group exposed to lipid-gene construct and stored at 4°C (group 4) appeared to be superior to that observed in groups stored at 10°C or 23°C (groups 5 and 6). However, there were no significant differences in graft function using oxygenation as an assessment variable measured at all temperatures with or without pressure.

Cationic lipid–mediated gene transfer to pulmonary artery segments may be a useful strategy that is not adversely affected by various conditions such as exposure time, exposure temperature, and exposure pressure that can occur under conditions of clinical preservation and transplantation. This transfection system may be useful in reducing ischemia-reperfusion injury and rejection, which remain major obstacles to successful transplantation. Gene encoding for a variety of proteins such as transforming growth factor-ß1, interleukin-10, superoxide dismutase, and nitric oxide synthase gene might be used in an effort to obtain a downstream effect on the whole graft.

In conclusion, gene transfection into graft pulmonary artery segments using GL-67:–plasmid DNA complexes is an excellent gene transfer system and is efficacious under a range of conditions that can occur in clinical lung transplantation.


   Acknowledgments
 
This work was supported by National Institutes of Health grant 1 R01 HL41281. We thank Kathleen Grapperhaus for technical assistance and Dawn Schuessler for secretarial support.


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 References
 

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  2. Boasquevisque C.H.R., Mora B.N., Schmid R.A., et al. Ex vivo adenoviral-mediated gene transfer to lung isografts during cold preservation. Ann Thorac Surg 1997;63:1556-1561.[Abstract/Free Full Text]
  3. Boasquevisque C.H., Lee T.C., Mora B.N., et al. Liposome-mediated gene transfer to lung isografts. J Thorac Cardiovasc Surg 1997;114:783-792.[Abstract/Free Full Text]
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  6. Lee E.R., Marshall J., Siegel C.S., et al. Detailed analysis of structures and formulations of cationic lipids for efficient gene transfer to the lung. Hum Gene Ther 1996;7:1701-1717.[Medline]
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  9. Felgner P.L., Gadek T.R., Holm M., et al. Lipifection. Proc Natl Acad Sci USA 1987;84:7413-7417.[Abstract/Free Full Text]
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Accepted for publication April 23, 1999.




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